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Endodontic Microbiology

Second Edition

Edited by

Ashraf F. Fouad Freedland Distinguished Professor and Chair Department of Endodontics School of Dentistry, University of North Carolina Chapel Hill, NC, USA

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Library of Congress Cataloging-in-Publication Data Names: Fouad, Ashraf F., editor. Title: Endodontic microbiology / edited by Ashraf F. Fouad. Description: Second edition. | Hoboken, NJ : John Wiley & Sons Inc., 2017. | Includes bibliographical references and index. Identifiers: LCCN 2016042792 | ISBN 9781118758243 (cloth) | ISBN 9781118975497 (Adobe PDF) |

ISBN 9781118975503 (epub) Subjects: | MESH: Dental Pulp Diseases–microbiology | Dental Pulp Diseases–drug therapy | Periapical Diseases–microbiology |

Periapical Diseases–drug therapy | Anti-Infective Agents–therapeutic use | Root Canal Therapy Classification: LCC RK351 | NLM WU 230 | DDC 617.6/342–dc23 LC record available at https://lccn.loc.gov/2016042792

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Dedication

To Amal, Fikry, Lori, Amani, George, Anthony Gade, and Edward; thank you for providing me the opportunity, the inspiration, the motivation, and the love.

Ashraf F. Fouad

Contents

Contributors ix Preface xi Preface to the First Edition xiii

1 Microbial Perspectives in the Twenty-First Century 1 William Wade

2 Diagnosis, Epidemiology, and Global Impact of Endodontic Infections 11 Dag Ørstavik

3 Microbiology of Dental Caries and Dentinal Tubule Infection 25 Robert M. Love and Anne C.R. Tanner

4 Culture-Based Analysis of Endodontic Infections 51 Gunnar Dahlén

5 Molecular Analysis of Endodontic Infections 81 José F. Siqueira, Jr, and Isabela N. Rôças

6 Extraradicular Endodontic Infections 129 Brenda P. F. A. Gomes and Ericka T. Pinheiro

7 Virulence of Endodontic Bacterial Pathogens 149 Christine Sedgley

8 Viruses in Endodontic Pathosis 179 Mohamed Sabeti

9 Fungi in Endodontic Infections 197 Bilge Hakan Sen and B. Güniz Baksi

10 Severe Head and Neck Infections 231 Jaime S. Brahim and Robert A. Ord

11 Endodontic Infections and Pain 251 Anibal Diogenes and Ken M. Hargreaves

12 Systemic Antibiotics in Endodontic Infections 269 Ashraf F. Fouad

13 Topical Antimicrobials in Endodontics 287 Anil Kishen

14 Endodontic Infections in Incompletely Developed Teeth 311 George T.J. Huang, Domenico Ricucci, and Louis M. Lin

15 Prognosis of Healing in Treated Teeth with Endodontic Infections 341 Shimon Friedman

16 Endodontic Infections and Systemic Disease 385 Ashraf F. Fouad

Glossary 409 Index 413

vii

Contributors

Editor

Ashraf F. Fouad, DDS, MS Freedland Distinguished

Professor and Chair Department of Endodontics School of Dentistry,

University of North Carolina

Chapel Hill, NC, USA

Authors

B. Güniz Baksi, DDS, PhD Professor Department of Oral Diagnosis

and Radiology School of Dentistry Ege University Izmir, Turkey

Jaime S. Brahim, DDS, MS Professor, Undergraduate Director Oral and Maxillofacial Surgery

Department University of Maryland Dental

School and Hospital Baltimore, MD, USA

Gunnar Dahlén, DDS, PhD (Dr Odont)

Professor and Chairman Department of Oral Microbiology

and Immunology Institute of Odontology Sahlgrenska Academy, University

of Gothenburg Gothenburg, Sweden

Anibal Diogenes, DDS, PhD Diplomate, American Board of

Endodontics Director, Advanced Program in

Endodontics Department of Endodontics University of Texas Health

Science Center at San Antonio Dental School

San Antonio, TX, USA

Shimon Friedman, DMD Professor, MSc Endodontics

Program University of Toronto Faculty of

Dentistry Toronto, Ontario, Canada

Brenda P.F.A. Gomes, MSc, PhD, BDS

Professor, Endodontics Piracicaba Dental School State University of Campinas Piracicaba, SP, Brazil

Ken M. Hargreaves, DDS, PhD Professor and Chair Department of Endodontics University of Texas Health

Science Center at San Antonio Dental School

San Antonio, TX, USA

George T.J. Huang, DDS, MSD, DSc

Professor Director for Stem Cells and

Regenerative Therapies

Department of Bioscience Research

College of Dentistry University of Tennessee Health

Science Center Memphis, TN, USA

Anil Kishen, PhD, MDS, BDS Professor and Head Discipline of Endodontics Faculty of Dentistry, University of

Toronto Toronto, Ontario, Canada

Louis M. Lin, BDS, DMD, PhD Professor of Department of

Endodontics College of Dentistry New York University New York, NY, USA

Robert M. Love, BDS, MDS, PhD, FRACDS

Professor, Dean and Head of School

School of Dentistry and Oral Health

Griffith University Queensland, Australia

Robert A. Ord, DDS, MD, FRCS, FACS, MS

Chairman and Professor Department of Oral and

Maxillofacial Surgery University of Maryland Medical

Center

x Contributors

University of Maryland School of Dentistry

Baltimore, MD, USA

Dag Ørstavik, Cand Odont, Dr Odont

Professor and Head, Department of Endodontics

Institute for Clinical Dentistry University of Oslo Oslo, Norway

Ericka T. Pinheiro, MSc, PhD, BDS

Assistant Professor Department of Dentistry University of São Paulo São Paulo, Brazil

Domenico Ricucci, MD, DDS Laboratory Head Private practice Cetraro, CS, Italy

Isabela N. Rôças, DDS, MSc, PhD

Professor, Department of Endodontics;

Head, Molecular Microbiology Laboratory

Faculty of Dentistry, Estácio de Sá University

Rio de Janeiro, Brazil

Mohamed Sabeti, DDS, MA Diplomate, American Board of

Endodontics Associate Professor Loma Linda University Los Angeles, CA, USA

Christine Sedgley, MDS, MDSc, FRACDS, MRACDS(ENDO), PhD

Professor and Chair Department of Endodontology School of Dentistry, Oregon

Health & Science University Portland, OR, USA

Bilge Hakan Sen, DDS, PhD Ege University Izmir, Turkey

José F. Siqueira, Jr, DDS, MSc, PhD

Chairman and Director PostGraduate Program in

Endodontics

Faculty of Dentistry, Estácio de Sá University

Rio de Janeiro, Brazil

Anne C.R. Tanner, BDS, PhD Senior Member of Staff Department of Microbiology The Forsyth Institute Cambridge, MA, USA; Associate Clinical Professor Harvard School of Dental

Medicine Boston, MA, USA

William Wade, BSc, PhD Professor of Oral Microbiology Centre for Immunology and

Infectious Disease Blizard Institute Barts and The London School of

Medicine and Dentistry Queen Mary University of

London London, UK

Preface

Much has happened in endodontic microbiology since the publication of the first edition of this book. Hun- dreds of important research studies and reviews have been added to the literature in this important field. We now have many better epidemiologic studies on the prevalence of endodontic disease, its association with systemic disease, and its potential contributions to major morbidity and mortality of patients. The area of polymicrobial infections is now recognized as a major public health problem. In the last decade it has seen innovations in research methodologies as well as the conceptual descriptions of how these infections can produce disease. In the field of endodontic micro- biology, next generation sequencing is now commonly used in research, revealing hundreds if not thousands of microbial taxa that are involved in endodontic patho- sis. The study of microbial virulence has also seen major advances. These include the interplay of differ- ent pathogens, such as bacteria, viruses, and fungi, to increase the pathogenicity of either, the host–microbial interactions, the differences in clinical presentations, and responses to treatment observed with different genomic and epigenetic variations in the host, bac- terial load issues, quorum sensing, and the keystone pathogen concept that describes how a pathogen can induce host changes that converts a microbial commu- nity to become dysbiotic.

Endodontic microbiology research still has many frontiers that have not been adequately studied. These include the reasons why chronic infections can exac- erbate to produce severe and spreading infections, the degree to which endodontic microflora travel to distant

sites in acute and chronic infections, the exact relation- ship between residual bacteria and healing, and the effects of residual bacteria on the success of regener- ative therapies. The interaction of the microbial com- munity in the deep carious lesion or the necrotic pulp with the host response can produce chronic asymp- tomatic disease or severe pain. The degree to which the composition of the microflora, the expressed vir- ulence factors, and the host’s innate susceptibility to disease interact to produce the resultant clinical mani- festation needs further elucidation.

The ability to eliminate microbial irritants is paramount to adequate healing in endodontics. There are still no clinical markers that can predict the long- term responses to vital pulp therapy or to endodon- tic treatment. Scientific explorations that utilize cutting edge technologies, such as shotgun sequenc- ing or metagenomics, transcriptomics, and proteomics have not been sufficiently incorporated in endodontic research. The degree to which microbial elimination is required to mediate the regeneration of the dental pulp, or even just revitalization in the pulp space, is not clear. Finally, we still do not have rapid molecu- lar methods of identifying antibiotic resistance, which would allow the efficient and effective selection of the right antibiotic. These and many other questions will continue to inspire many studies and insights that would allow us to improve the success of our treat- ment modalities, save more teeth from extraction, and improve the patients’ quality of life.

Ashraf F. Fouad

Preface to the First Edition

Endodontic infections are very prevalent, because they mostly represent complications of dental caries and its treatment, as well as traumatic injuries to teeth, which are all very prevalent occurrences. Collectively, they represent the majority of dental infections that present with significantly acute local and systemic signs and symptoms. This is the first textbook devoted to the study of endodontic infections, which hitherto has been limited to isolated single chapters in endodontic text- books. This textbook is intended to provide a collec- tion of work showing the state of the knowledge in this field. It is also intended to provide some research questions and hypotheses that, hopefully, will stimu- late more efforts to understand the disease process and identify effective treatment methods.

The study of endodontic microbiology has been complicated by difficulty in epidemiological data in obtaining adequate endodontic diagnosis on large numbers of nonpatient populations. In addition, sam- pling is a major challenge in endodontics. Contami- nation from the tooth surface, caries, or saliva must first be avoided. Access to the potentially very com- plex root canal anatomy and disruption of biofilm on the majority of canal walls in these areas is neces- sary. It is almost impossible to differentiate specimens obtained from the apical and coronal portions of the root canals; thus, the effect of location of microflora within the canal is poorly understood, and can only be studied in teeth that are extracted. Finally, sampling after completion of treatment to assess effectiveness of treatment and determine the long-term outcome risk is complicated by the fact that only the areas that could be reached could be sampled.

The difference in sensitivity between tradi- tional culturing and modern molecular methods are

especially important in endodontic microbiology, because the endodontic specimen has so little material, and sensitivity, therefore, has a major role in microbial identification. The description of traditional bacterial pathogens and their virulence factors represents most of the available literature today. The contributions of the not-yet-cultivated bacteria and the bacteria ren- dered temporarily uncultivable by traditional treatment methods have not been adequately studied. Likewise, we are just beginning to understand some of the con- tributions of fungi and viruses to the pathogenesis of endodontic infections.

The debate on viable versus dead microorgan- isms that are detected by molecular techniques must be resolved by using more accurate technologies that assess microbial counts, their viability, and their pathogenicity. Likewise, consistent and stringent methodologies, including sequencing of amplification products, are essential for assuring accurate results and enabling comparisons among studies.

Persistent endodontic pathosis may be due to per- sistent infection or new infection after treatment. Sam- pling of apical lesions during periapical surgery is complicated by the lack of sterility of the surgical field. Therefore, the microbiology of nonhealing endodontic cases is still in its infancy at this time.

It is clear that in order to determine effective treat- ment modalities, better sampling and identification techniques must be employed and more adequately designed outcome studies need to be performed.

Finally, the relationship between endodontic patho- sis and systemic disease must be more comprehen- sively studied. Endodontic infections were historically thought to contribute to numerous systemic diseases. While the potential for systemic spread of an acute

xiii

xiv Preface to the First Edition

endodontic infection is well-known and documented, earlier studies have failed to demonstrate that chronic endodontic infections contribute to systemic diseases. However, these hypotheses must be reexamined now that we have more accurate research tools. In addition, the creation of large patient databases for longitudinal

analysis of treatment outcomes, and their relationships with systemic disease will be imperative in future stud- ies that address this issue.

Ashraf F. Fouad

Chapter 1 Microbial Perspectives in the Twenty-First Century William Wade

1.1 Introduction 1.2 Genomics 1.3 Molecular microbial ecology and the

study of uncultivable bacteria 1.4 Intraspecies variation 1.5 Metagenomics and metatranscriptomics

1.6 Bacterial–bacterial communication 1.7 Host–bacterial interactions 1.8 Complex infectious diseases 1.9 The future

1.10 References

1.1 Introduction

The final quarter of the nineteenth century was arguably the golden age of medical microbiology. The ground-breaking work of Pasteur, Koch, and others led to the development of broth and agar media that were able to support the growth in the laboratory of the major bacterial pathogens affecting humans. The abil- ity to grow these organisms in pure culture led to the production of vaccines for many of the diseases they caused. These advances, and the subsequent discovery and development of antimicrobials, led to the mistaken belief that infectious disease had been beaten.

Of course, it is now realized that this optimistic viewpoint is not justified, not least because of the rapid emergence of bacterial resistance to antimicrobials. Indeed, the consensus view is that the battle against bacterial resistance is currently being lost, because of both the difficulty and costs associated with devel- oping new antimicrobials and indiscriminate use of those currently available. The predicted ultimate fail- ure of antimicrobial strategies has led to renewed inter- est in elucidating the pathogenic mechanisms used by

bacteria to cause disease, with the ultimate aim of devising new methods of disease prevention and treatment.

At the same time, interest in the microbial popula- tions of the Earth has been intense and new techniques have become available to characterize the bacterial communities found in every ecosystem on the planet. These have revealed the quite astonishing diversity of microbial life on Earth and the extreme complexity of most bacterial communities. Furthermore, the extent of subspecific diversity is only now being fully appre- ciated. Bacterial readily exchange DNA and can “shuf- fle” their own genomes to generate diversity with the ultimate aim of responding and adapting to environ- mental change. As discussed later, bacteria in commu- nities communicate with each other and, in the case of commensals living with plants and animals, their hosts. These interactions operate at various levels and can be remarkably sophisticated. The twenty-first cen- tury will be a period of tremendous advances in our understanding of the microbial world.

The aim of this chapter is to review recent deve- lopments in microbiology and to highlight selected

2 Endodontic Microbiology

areas that are likely to change our conceptual view of infectious disease as a whole, and oral and endodon- tic infections in particular. Inevitably, a single short chapter cannot provide a comprehensive overview of an entire discipline, but the interlinked topics cov- ered are those that will undoubtedly change our view of the microbial world and its relationship with the human host.

1.2 Genomics

The sequence of the human genome was published in 2001. The benefits of this outstanding achievement are now being realized with the identification of genes responsible for or causing a predisposition for a large number of diseases (Wellcome Trust Case Control Consortium 2007). At the same time, and largely pos- sible because of the technical advances made as part of the human genome sequencing effort, genomes of other organisms are being sequenced, including those of bacteria.

As of February 2015, the sequencing of the genomes of 26 522 bacteria and 647 archaea had been com- pleted, while 15 800 and 424, respectively, were in progress or available as a draft (for more information see www.genomesonline.org). As expected, the data obtained have revealed the enormous genetic potential contained within bacterial genomes; in each genome sequenced, around one-third of the genes present have been novel and the function of a significant proportion remains unknown.

The availability of genome sequence data is allow- ing a far more robust bacterial classification to be con- structed than previously possible. Bacterial taxonomy was once based purely on phenotypic characters and was very inexact because of the difficulties involved in obtaining and interpreting such data compared to plants and animals where differences in phenotype are far more obvious. In recent years, genetic informa- tion has been increasingly used, but on a limited scale, and typically only the sequences of the 16S riboso- mal RNA (rRNA) and other housekeeping genes have been used. New methods are now being introduced to make use of the sequence data available for complete genomes (Konstantinidis and Tiedje 2005). In gen- eral, the results of using such methods have supported the 16S rRNA gene taxonomy at species and genus level but, in addition, have provided improved clar- ity of the relationships among the higher taxonomic

ranks, where substantial overlap between ranks has been observed.

The results of the analysis of some genomic data have been extremely surprising. A Gram-positive coccus found in amoebae could not be identified by the conventional molecular analysis of 16S rRNA gene sequencing because no ribosomal genes could be amplified for sequencing. Genomic data explained this difficulty by revealing that the organism was actually a virus, the largest yet discovered. Now named Mimivirus, the large virus particles are up to 0.8 μm in diameter, the size of many bacteria. It primarily infects amoebae but has been implicated as a cause of pneumonia on serologic grounds and has caused a laboratory-acquired pneumonia in a researcher (Raoult et al. 2007). At the other end of the bacterial scale, members of the genus Epulopiscium, found in the intestine of certain surgeonfish (Angert et al. 1993), have been discovered that are visible with the naked eye.

In addition to correctly identifying evolutionary oddities, genomic data have identified numerous novel biochemical pathways with the potential for exploita- tion. Among these are some novel antimicrobials although the range of targets within bacterial cells that has arisen by natural evolution is rather narrow. A more promising avenue to the development of novel antimicrobials is to use genomic data to identify novel targets for antimicrobial treatments (Pucci 2006). Pre- dictions can be made from genome data as to how essential a given gene is to an organism and there- fore how disrupting the gene would affect the vitality of the organism. These predictions can then be tested in an appropriate manner experimentally using a wide range of methods that have been developed in response to the availability of genomic data. These include ran- dom mutagenesis mediated by transposons or inser- tion of plasmids, targeted gene disruption or in vivo techniques such as signature-tagged mutagenesis and in vivo expression technology. Structural genomics, where sequence data is used to predict the structure of essential bacterial proteins, is also being used to identify potential targets for antimicrobials. Finally, comparative genomics can be used to identify com- mon features of pathogens affecting a particular body site to custom design antimicrobials for specific pur- poses, for example, respiratory tract infection.

Next generation sequencing technologies such as the Roche 454 and Illumina systems have been introduced and have brought the ability to sequence

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Microbial Perspectives in the Twenty-First Century 3

bacterial genomes within the reach of individual laboratories. Accurate interpretation of the data remains a challenge, however, although a number of useful software programs are now available (Edwards and Holt 2013). The information obtained thus far has been of extraordinary value in understanding the role of pathogenic bacteria in disease and is the fundamental basis of other new technologies such as transcriptomics and proteomics. The next task will be to understand how gene products interact both within a bacterial cell and in response to external stimuli from the environment and other organisms.

1.3 Molecular microbial ecology and the study of uncultivable bacteria

Almost without exception, oral infections are polymi- crobial in nature and difficult to study because around half of the bacteria present in the oral cavity cannot be grown using conventional culture media. It has long been recognized that not all bacteria from a given habi- tat can be cultured on artificial media in the laboratory. Indeed, it has been estimated that less than 2% of bac- teria on Earth can be cultured.

Methods for the characterization of complex bac- terial communities were developed as a consequence of the use of DNA sequence data for the construction of evolutionary trees. This was done by comparing the sequences of genes encoding essential functions, the so-called housekeeping genes that are found in all cellular organisms. The gene most commonly used to date has been encoding the small subunit (16S) rRNA molecule. Ribosomes have the essential function of translating messenger RNA (mRNA) into amino acid chains and, because of the need to preserve function, have evolved slowly. Some of the regions of the gene have changed very little over time and are therefore vir- tually identical in all bacteria. These regions are very useful for the design of universal polymerase chain reaction (PCR) primers that can amplify the gene from a wide range of different bacteria. Other regions are more variable and can be used to discriminate between organisms, almost to species level. Woese and col- leagues used small subunit rRNA comparisons to con- struct a tree of life (Figure 1.1), which showed that bacteria had evolved into two domains, the Archaea and Bacteria, while eukaryotic organisms fell into a single third domain, the Eukarya (Woese 1987). It was

originally thought that organisms found in the domain Bacteria were those found in normal environments while the Archaea were present in extreme environ- ments such as the deep sea and associated with volca- noes and so on. However, these associations have since been shown not to be true and members of Archaea are now known to be widely distributed, and an archaeal genus, Methanobrevibacter, can be found in the human mouth.

A major consequence of the availability of this tree is that unknown organisms of any type can be identi- fied simply by sequencing their rRNA gene and adding the sequence to the tree or by directly comparing the sequence with the hundreds of thousands of bacte- rial sequences held in the sequence databases. Com- plex bacterial communities can be characterized by the PCR, cloning, and sequencing of 16S rRNA. Such studies have been performed with samples from the human mouth in health and disease and are described in more detail in Chapter 5 and throughout the text- book. A common finding of every study to date has been to confirm that around half of the oral micro- biota is uncultivable. Around 700 species have been detected, 95% of which belong to the phyla Fir- micutes, Bacteroidetes, Proteobacteria, Actinobacte- ria, Fusobacteria, and Spirochaetes (Dewhirst et al. 2010). Other phyla consistently detected are Syn- ergistetes, Chloroflexi, and the un-named phylum- level divisions GN02, SR1, and TM7 (Camanocha and Dewhirst 2014). The Human Oral Microbiome Database (www.homd.org) lists the bacterial taxa found in the mouth and provides descriptions of their phenotypes, where available, with links to genome sequence data as well as a 16S rRNA gene sequence identification tool (Chen et al. 2010).

Major efforts are now being made to improve our understanding of currently unculturable bacteria. These include the development of new culture media that better mimic the natural environment. Very often, laboratory culture media are far richer in nutrients than the natural habitat and the use of dilute media or filtered natural substrate has been successful in culturing pre- viously uncultured organisms. This approach has not been applied systematically to the study of oral uncul- turable bacteria, but should be possible. Around half of oral bacteria cannot be cultivated in vitro. There appears to be no single reason for this but it has been shown that because oral bacteria naturally live in a multispecies community, some species require the presence of other bacteria to grow. The development

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4 Endodontic Microbiology

Fig. 1.1 Phylogenetic tree showing representatives of the domains Eukarya, Archaea, and Bacteria.

of coculture methods linked with enrichment of tar- get organisms by in situ hybridization has enabled the cultivation of members of a previously uncultivated lineage of the phylum Synergistetes (Vartoukian et al. 2010). The commonly occurring division TM7 has long been a target for cultivation and it has been found that it can be detected readily in mixed laboratory cultures but not cultured independently (Hugenholtz et al. 2001). The reason for this observation has been determined recently in that a TM7 phylotype has been revealed to be an obligate episymbiont of other bacte- ria, and also capable of entering the host bacterial cells (He et al. 2015). It has a small genome (approximately 0.7 MB) and lacks genes for the synthesis of essential amino acids and so presumably needs to obtain amino acids from its associated bacterial host. TM7 is a large phylum widely distributed in the environment as well as being a component of mammalian microbiomes and it will be interesting to determine if all representatives

of the division are episymbionts or if some have other lifestyles.

The potential benefit of growing previously uncul- tivable organisms has been demonstrated recently in the successful attempts to cultivate soil bacteria by providing a natural community in contact with the cul- ture system via a permeable membrane which has led to the discovery of a new antibiotic, teixobactin (Ling et al. 2015).

1.4 Intraspecies variation

The majority of microbiological diagnostic methods identify the target organism to species level. However, it is now recognized that individual strains within a species often vary markedly in their virulence. Within a species, some strains may be pathogenic while others are harmless. The extent of the genetic variation within

Microbial Perspectives in the Twenty-First Century 5

species, however, has only been fully realized by the sequencing of the genomes of multiple representatives of the same species.

In one such study, three strains of Escherichia coli were compared: the well-known harmless lab- oratory strain K12, an enterohaemorrhagic serotype O157 strain of the group associated with beef prod- ucts, and a uropathogenic strain. It was found that they had only 39% of their genes in common, a surpris- ingly small number (Welch et al. 2002). These com- mon genes encoded the functions that gave the strains their identity as members of the species E. coli, while the remaining genes gave them the ability to colonize particular body sites and/or damage the host by means of a specialized set of virulence factors appropriate for their natural habitat and lifestyle. Genes acquired from other organisms by horizontal gene transfer can be critical to that organism’s behavior, and in the past may have been the reason a species was given a partic- ular name. For example, if the mainly harmless envi- ronmental organism Bacillus cereus acquires plasmids pXO1 and pXO2, which carry genes coding for four toxins and the enzymes required to make a capsule, it becomes Bacillus anthracis, the causative agent of anthrax.

This work has given rise to some new genomic con- cepts. The core genome is that shared by all strains of the species, while the peripheral or accessory genome includes genes found in some strains but not others, but which nonetheless may be important in pathogenesis. Some bacteria go further and have two chromosomes; in this case, one normally encodes housekeeping genes and the second genes that confer fitness for competi- tion in the environment.

The range of genes encoded by the peripheral genome can be extensive. In a study of the genome sequences of eight Streptococcus agalactiae strains, the authors calculated the number of strains of the species that would have to be sequenced to reveal the full genetic diversity of the species (Tettelin et al. 2005). The result was infinity. In other words, S. agalactiae can incorporate DNA into its genome from such a wide range of sources that all the possible genes that could be found within this species will never be known.

The implications of these findings are significant. Although much work has been invested in the develop- ment of rapid assays to detect the presence of specific organisms in clinical samples, including those col- lected from oral diseases, the association of a species

with disease may be insufficient for diagnostic pur- poses. Detection of the presence (and expression) of specific virulence genes may be required to provide a meaningful microbiologic diagnosis. This will clearly be difficult for diseases where the virulence determi- nants important in disease are currently unknown or where multiple virulence mechanisms are operating.

1.5 Metagenomics and metatranscriptomics

As individual bacterial strains can vary greatly in their genetic composition and the assignation of an isolate to a species alone is likely to give a poor indication of its pathogenicity, alternative methods of analysis need to be developed to determine the role of bacterial com- munities in human disease. New methods are particu- larly needed for complex diseases because the bacterial communities associated with the mucous membranes, where these diseases primarily occur, are so diverse that their routine characterization is not practicable. A novel approach does not attempt to isolate and purify all of the component species and strains, but, rather it considers the whole community and all of its con- stituent genes as a whole. The bacterial community found at a habitat is termed the microbiome and all the genetic material of the community members is the metagenome (Rondon et al. 2000).

The first stage in any such analysis is to extract DNA from all of the bacteria present in the sample. In early metagenomics studies, the DNA was cloned into either small plasmid vectors for ease of sequenc- ing or into systems such as bacterial artificial chro- mosomes (BAC), which allowed DNA fragments up to 100 kb in length to be stably maintained in an E. coli host and genes expressed to seek functions of interest. These approaches have now been superseded by the use of next generation sequencing, which does not involve a cloning step. The cloning approach has allowed a number of new antibiotics to be discov- ered from metagenomic analyses of soil and marine environments. For example, turbomycin A and B were discovered in this way (Gillespie et al. 2002). Inter- estingly, a single gene was responsible for the activity which was mediated by an interaction between indole, normally produced by E. coli, and the gene product. The success rate in identifying novel antimicrobials in metagenomic libraries has been low; typically, several

6 Endodontic Microbiology

hundred thousand clones have to be screened to find one new active compound. This relative lack of suc- cess partly reflects the methodology where E. coli is used as the host. Successful expression may require the presence of specific promoters or other accessory molecules and factors such as the G+C content of the insert and codon usage may adversely affect expres- sion. Thus, in general, cloned fragments of DNA are expressed most easily in their natural host and the more phylogenetically distant the clone host, the less likely that expression will be successful. To overcome this, new vector– host systems are being introduced for the expression of metagenomic libraries to enable a bet- ter match between the insert and the host in which it is being expressed. These include Streptomyces and Pseudomonas, two genera members of which natu- rally produce secondary metabolites with properties of interest. An alternative approach is to use bioin- formatic methods to screen for gene clusters encod- ing potentially useful compounds within metagenomic data. This approach has been successfully applied to environmental samples (Owen et al. 2013).

Metagenomic sequence data are being used to assemble genomes of bacteria in natural communi- ties. This has been successfully performed for the rel- atively restricted microbiota found in a subterranean acid mine drainage biofilm where near-complete bac- terial genomes were reconstructed for two previously uncultured bacteria (Tyson et al. 2004). More ambi- tiously, Venter et al. (2004) randomly sequenced the metagenome of water specimens collected from the Sargasso Sea. Over one trillion base pairs of DNA were sequenced which were found to be derived from 1800 species including 148 not previously character- ized. More than one million novel genes were found. However, it proved difficult to reconstruct complete genomes from these data because of the number of closely related species present and the large num- bers of mobile elements with high levels of sequence similarity. A number of software tools are now avail- able for genome assembly from metagenomic data, both as stand-alone programs or as online resources (Hunter et al. 2014) although it remains a challenging task.

Metagenome analyses allow an appreciation of the genetic potential of a microbial community but no indication of actual activity. Analysis of the mRNAs present in a sample shows which genes are currently being expressed and thus the functional activity of the

microbiota. RNA is extracted directly from the sample and then enriched for mRNA and reverse transcribed. The DNA is then fragmented and sequenced using next generation methods. After removal of contaminating and repetitive sequences, the reads are then mapped to reference sequences in nucleotide databases in order to identify genes and operons which have been expressed (Carvalhais et al. 2012). This approach has been suc- cessfully used with oral samples from subjects with periodontitis and caries (Duran-Pinedo et al. 2014; Simon-Soro et al. 2014).

1.6 Bacterial–bacterial communication

Originally thought of as simple dumb solitary crea- tures, it is now known that bacteria live together in communities with a number of features in common with multicellular organisms. The basis for the cooper- ation of individual bacterial cells within a community is communication. Communication is mediated by the production of signaling molecules often generically described as quorum-sensing molecules, after the first bacterial signaling system to be described.

In many circumstances, the total number of bac- terial cells in a community is important to the over- all health of the community. In the environment, the availability of nutrients and external stresses are fac- tors that cause the community to behave in a particular way. This may increase or decrease its overall rate of growth, become more motile to move to a new habitat to obtain nutrients and, once there, switch to a biofilm mode of growth to colonize the new environment. For pathogenic bacteria of exogenous source, bacterial– bacterial communication is particular important. The first bacteria to colonize the host will necessarily be present in small numbers and will want to multiply without alerting the host to their presence in order to avoid the host’s immune system. As virulence factors such as protein toxins are typically highly antigenic, the pathogen will not produce them until the com- munity is sufficiently numerous to resist the host’s defenses. Once this “quorum” has been achieved, the members of the community will turn on the production of their virulence genes in order to damage the host and cause disease.

The molecular basis for quorum sensing was first elucidated for the bioluminescent marine bacterium Vibrio fischeri. This organism is commonly found

Microbial Perspectives in the Twenty-First Century 7

as a symbiont in the light-producing organs of luminescent fish or squid. The signaling mechanism is a two-component system; the luxI gene produces autoinducer 1 (AI-1), an acyl homoserine lactone. This is produced constitutively, but when sufficient numbers of V. fischeri are present, the high con- centration of AI-1 enables binding to the receptor, the product of luxR, which activates transcription of the luciferase operon and leads to production of the light-emitting compounds.

Following its discovery in V. fischeri, AI-1 analogs have been found in a wide range of Gram-negative bacteria. The AI-1 of a particular species is normally specific to that species so that cross-talk is avoided within multispecies communities.

Gram-positive bacteria also produce signaling molecules, but those so far described have all been peptides derived from larger precursors by posttrans- lation modification. An important group of signaling molecules in Gram-positives is of those that induce competence. Competence is the ability of bacteria to take up DNA present in the environment. This is an important mechanism of genetic exchange and is par- ticularly common among members of the oral micro- biota such as the oral streptococci. In many strepto- cocci, the precursor molecule is Com C which is mod- ified as it is transported out of the cell by the ComAB transporter. The signaling molecule itself is the C- terminal end of Com C and is termed the competence- stimulating peptide (CSP). When the bacterial num- bers reach their quorum, CSP binds to the receptor, the histidine kinase Com D, which then stimulates the response regulator Com E.

In addition to the species-specific quorum-sensing mechanisms described above, bacteria also make use of nonspecific systems that allow general communica- tion between bacteria in communities, across species barriers. One such molecule, AI-2, is produced by, and can be detected by, a wide range of both Gram-negative and Gram-positive bacteria. AI-2 forms spontaneously from 4,5-dihydroxy-2,3-pentanedione (DPD). which is a product of the Lux S enzyme in the catabolism of S- ribosylhomocysteine. A number of oral bacteria have been shown to produce AI-2 and it has been found to have an important role in dental plaque formation, For example, Streptococcus oralis and Actinomyces naes- lundii are known to coaggregate early in the develop- ment of dental plaque biofilms and grow together in in vitro models, forming a profuse plaque with physical

interaction between cells of the two species. A luxS mutant of S. oralis that did not produce AI-2, how- ever, did not form such biofilms with A. naeslundii, while the mutualistic activity was restored by luxS complementation (Rickard et al. 2006). Quorum sensing is thus a central mechanism in bacterial metabolism, with particular importance in biofilm for- mation. The use of quorum-sensing inhibitors has potential for use as antibiofilm agents (Brackman and Coenye 2015).

Another group of bacterial cell-signaling molecules are the family proteins related to resuscitation- promoting factors (Rpf). Originally discovered in Micrococcus luteus where they were able to revive M. luteus cells that had entered a dormant phase, they were subsequently found to be widespread among members of the phylum Actinobacteria, the High G+C Gram-positives (Mukamolova et al. 1998). Inter- estingly, the growth of Mycobacterium tuberculosis, which is normally extremely slow in vitro, is greatly stimulated by Rpf. Rpf is a protein, structurally similar to lysozyme, which can exert its effects at extremely low concentrations. It has therefore been termed a bac- terial cytokine because of its resemblance to mam- malian cytokines that have similar properties. The molecular basis of its action has yet to be determined but it would appear to cleave the peptidoglycan of dor- mant cells and either release a second messenger or physically allow the cells to resume growth. Peptido- glycan fragments, muropeptides, have recently been recognized to be important mediators of communica- tion both between bacteria and between bacteria and eukaryotes (Dworkin 2014).

Novel mechanisms of bacterial communication are being discovered all the time, and it is extremely likely that a network of sophisticated interactions exists among the bacterial community in dental plaque. Many of these are clearly relevant to endodontic infection and the survival of bacteria under restorations and in the treated root canal. The realization that vege- tative bacterial cells can go into a dormant state, dis- tinct from endospore production, and survive for many years may explain how bacteria survive under restora- tions or despite calcium hydroxide treatment in the root canal. Furthermore, a change in the environment may stimulate the production of broad-range growth stim- ulation factors that cause the community to undergo rapid growth, causing damage to the affected tooth and pain to the patient.

8 Endodontic Microbiology

1.7 Host–bacterial interactions

All plants and animals are colonized by bacteria. Mam- mals are born sterile but extremely quickly become colonized with the microbiota characteristic for their species. The commensal microbiota associated with mammals has evolved over millions of years, and it is possible to reconstruct the evolution of the commensal microbiota in parallel with each mammalian host, the phenomenon of cospeciation. Thus, for the majority of bacteria found in the human mouth, there are versions of that organism found in other animals. For example, among the mutans group streptococci, associated with dental caries, S. mutans and S. sobrinus are found in humans, S. ferus and S. rattus in rats, S. cricetus in hamsters, and so on.

The recognition that human cells make up only around one-third of all cells in the body, with the major- ity of the remainder Bacteria (American Academy of Microbiology 2014), led to initiatives to describe the microbial populations and their genomes at var- ious body sites, principally the National Institutes of Health-funded Human Microbiome Project (HMP) (Human Microbiome Project Consortium 2012a). The principal findings of the HMP to date have been that each body site has its own characteristic micro- biota and that individuals have their own microbiome, which is relatively stable over time (Human Micro- biome Project Consortium 2012b; Ding and Schloss 2014).

Our normal microbiota protects us from exogenous infection via the phenomenon of colonization resis- tance. All external surfaces of the body are normally covered in bacteria and thus potential binding sites for exogenous pathogens are blocked. In addition, mem- bers of the normal microbiota can produce antimi- crobial substances that inhibit the growth of other organisms. However, if the commensal microbiota is disturbed then infection can result. For example, it is well known that treatment with antibiotics can disrupt the normal microbiota to such a degree that oppor- tunistic infection with other organisms such as col- iform bacteria or the yeast Candida albicans can occur. Vaginal thrush and antibiotic sore tongue are examples of such conditions.

The presence of the normal microbiota is essential for the proper development of the gut. The intesti- nal microbiota is highly diverse, with over 1000 bac- terial species present. A commonly found species, Bacteroides thetaiotaomicron, has profound effects on

the development of the blood supply to the gut. In germ-free mice, introduction of B. thetaiotaomicron induced intestinal angiogenesis (Stappenbeck et al. 2002). Interestingly, the genome of B. thetaiotaomi- cron includes an unusually high number of genes encoding signaling molecules of both the one- and two-component types (Xu et al. 2003). The mucin- degrading bacterial species Akkermansia muciniphila is associated with gut health and its numbers are depleted in inflammatory bowel disease (Belzer and de Vos 2012). It is one of a number of species that have found to be health associated and regarded as beneficial.

1.8 Complex infectious diseases

“Classic” infectious diseases normally occur when a pathogen infects a susceptible host and produces a spe- cific virulence factor that damages the host in a char- acteristic way, causing the signs and symptoms of the disease. For many diseases, particularly those associ- ated with the mucous membranes, no single pathogen has been identified, but instead the disease appears to be the result of an aberrant interaction between the host and its normal resident microbiota. These so-called complex infectious diseases include the inflammatory bowel diseases and oral infections such as chronic peri- odontitis and, to some extent, endodontic infections. It has been suggested that a change in composition of the microbiome to one that is in a state of dysbiosis may have a role in obesity, diabetes, mental health, and other conditions (Devaraj et al. 2013; Clarke et al. 2014). The question that remains to be answered is whether a dysbiotic microbiome is a primary driver of disease or whether it is a result of the disease process.

Host susceptibility is of primary importance in these diseases, but typically the susceptibility is conferred by multiple genes with no single genotype responsi- ble. For oral diseases such as periodontitis, the genes responsible have yet to be discovered although there is growing evidence that increased susceptibility is caused by subtle differences in the immune and inflam- matory responses. For example, genetic polymor- phisms associated with cytokines such as interleukin- 1 have been identified, which are associated with increased cytokine secretion and severity of chronic inflammatory disease (Brett et al. 2005). Another key factor is the environment in its widest sense. Host

Microbial Perspectives in the Twenty-First Century 9

factors such as stress are known to contribute to the severity of complex diseases, presumably by adversely affecting the immune system. Diet and social factors such as smoking can also be important, particularly in the principal oral diseases such as dental caries and the periodontal diseases.

It is likely that in the investigation of oral infections and diseases, we have clung too long to the classic infectious disease model and have sought single infec- tious causes for them in the hope that antimicrobials could be used in a targeted way to treat them. It must be remembered, however, that these diseases are bac- terial diseases and the presence of the normal micro- biota is required. By mechanisms as yet unknown, it appears that the communication and cooperation between the host and its commensal microbiota breaks down, resulting in damage to the host. Much work on these diseases is therefore currently being focused on better understanding health, the question being that if the human gut is colonized by so many bacteria with the potential to cause disease, how do the majority of individuals remain healthy? Better understanding of how this healthy balance is maintained will permit insights into how disease arises when the homeostasis breaks down. It may also be possible to influence the host–microbiome interaction with probiotic bacteria or by the administration of prebiotics to increase the relative proportions of beneficial bacteria (Claes et al. 2014). A more extreme treatment for patients with severe dysbiosis such as that seen in pseudomembra- nous colitis due to Clostridium difficile is a fecal trans- plant from a healthy donor, which has been shown to result in an excellent clinical response (Rao and Young 2015).

1.9 The future

We are still in the early years of the twenty-first cen- tury, so what do we have to look forward to in terms of how microbiology will impact on our understanding of infectious disease, including oral and endodontic infections? The Human Microbiome Project is pro- viding an enormous bank of data on the composition of the human-associated microbiota and its genetic potential. The next challenge is to exploit these data to devise novel preventive and therapeutic strategies. It may be possible, for example, to construct a mix- ture of beneficial bacteria as an alternative to fecal

transplants, to be used to prevent, or reverse, dysbio- sis. From the host perspective, advances in genetics will undoubtedly give us a far better understanding of individuals’ susceptibility to disease and the challenge will be put this into context with new knowledge of the genetic potential of the commensal microbiota in order to predict and influence interactions among the host, its microbiome, and the environment.

1.10 References

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Brett PM, Zygogianni P, Griffiths GS, et al. 2005. Functional gene polymorphisms in aggressive and chronic periodon- titis. J Dental Res 84: 1149–1153.

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Edwards DJ, Holt KE. 2013. Beginner’s guide to comparative bacterial genome analysis using next-generation sequence data. Microb Inform Exp 3: 2.

Gillespie DE, Brady SF, Bettermann AD, et al. 2002. Isola- tion of antibiotics turbomycin a and B from a metagenomic library of soil microbial DNA. Appl Environ Microbiol 68: 4301–4306.

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Human Microbiome Project Consortium. 2012b. Structure, function and diversity of the healthy human microbiome. Nature 486: 207–214.

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Chapter 2 Diagnosis, Epidemiology, and Global Impact of Endodontic Infections Dag Ørstavik

2.1 Endodontic disease: irritation, inflammation, and infection of the pulp and periapical tissues

2.2 Primary diagnostic criteria: subjective symptoms and radiographic changes 2.2.1 Pulpal involvement 2.2.2 Periapical diagnosis

2.3 Pulpal inflammation and infection: public health consequences

2.4 Epidemiology of endodontic diseases 2.4.1 Basic principles of epidemiologic

approaches to dental disease 2.4.2 Infections with pulpal involvement 2.4.3 Infections with periapical

involvement

2.4.4 Radiographic surveys of asymptomatic apical periodontitis: methodology

2.4.5 Results of epidemiologic surveys of asymptomatic apical periodontitis

2.5 Quality of root canal treatment and the development and persistence of apical periodontitis

2.6 Treatment strategies: prevention, treatment, and extraction

2.7 General oral health, oral health strategies, and tooth preservation as risk factors for oral infections

2.8 Conclusions 2.9 References

2.1 Endodontic disease: irritation, inflammation, and infection of the pulp and periapical tissues

Endodontics deals with diseases of the pulp–dentin organ and the periapical tissues. For practical pur- poses, these are infectious processes. Noninfectious conditions affecting the pulp or apical periodontium are much rarer and are seldom dealt with by specific endodontic treatment; however, they represent impor- tant differential diagnostic challenges.

The sources of pulpal and apical periodontal infec- tions are numerous. Traditionally, endodontic disease has been seen as a sequel to dental caries; however,

bacteria find their way to a vulnerable pulp in many other instances as well. Dental trauma is one well- known situation, so is pulp damage and infection fol- lowing preparation and restoration of teeth. Low-grade irritation of pulpal nervous elements can occur follow- ing attrition and erosion, sometimes developing into pulpal necrosis and infection.

Historically, the focus has been on the inflamma- tory reactions of the pulp and periapical tissues, asso- ciating clinical disease with the tissue response. The inflammatory reactions have been related to infection, but at times they have been related to tissue damage during treatment and to the toxic effects of medica- ments and materials. It is clearly an improvement in the

12 Endodontic Microbiology

concept of diagnosis and treatment planning that there has been a shift towards stressing the level and extent of the infectious process, rather than wild-guessing the type and degree of the inflammatory reaction. Inflam- mation of the pulp and periapical tissues is a sign of infection; clinically progressing disease is hardly ever caused by trauma or materials. This concept has been productive because virtually all successful therapeutic measures are directed towards combating or prevent- ing infection, with reduced or eliminated inflammation following as a consequence. Moreover, the concept of endodontic diseases as infections has implications for public oral health assessment in general, and places the association of local infectious disease in perspec- tive relative to local and regional (Ricucci and Bergen- holtz 2003) systemic health issues, particularly cardio- vascular disease (Caplan et al. 2006; Joshipura et al. 2006; Cotti and Mercuro 2015). In this context, it is important to relate epidemiologic aspects of pulpal and periapical disease to endodontic microbiology.

2.2 Primary diagnostic criteria: subjective symptoms and radiographic changes

2.2.1 Pulpal involvement

Initial pulpal infection is recognized primarily by clin- ical symptoms or through explorative excavation of involved dentin. While conventional radiography may suggest that a resorptive or carious process is imping- ing on the pulp, such methods do not allow definitive assessment of pulpal involvement.

Traditionally, it has been held that there is only a weak association between clinical features and the histologic characteristics of pulpitis (Cisneros-Cabello and Segura-Egea 2005; Giuroiu et al. 2015). However, in a recent study, Ricucci et al. (2014) were quite suc- cessful in correlating pulpal history and symptomatol- ogy with histology and histobacteriology in 95 human teeth. The diagnosis of pulpal infection/inflammation is therefore largely an operational one: based on expe- rience, and on knowledge of the underlying biologic processes, pulpitis is categorized as either reversible or irreversible. This scheme sidesteps the need to give a precise description of the extent and severity of inflammation in the pulp, but it allows for treat- ment decisions based on the extent of microbial con- tamination or infection. It is assumed that in the case

of reversible pulpitis, pulp vitality may be preserved with proper treatment; irreversible pulpitis implies that no treatment short of pulp extirpation and root fill- ing can eliminate the disease. Briefly, reversible pul- pitis causes clinical symptoms of short duration (sec- onds) and only when irritated by external stimuli, and the pulp proper is either not exposed or traumatically exposed for a short period only (< 2 days) (Heide and Kerekes 1987). By contrast, irreversible pulpitis gives rise to symptoms of longer duration (minutes) that may also occur spontaneously, and an exposure of the pulp to the oral environment through caries, fractures, or cracks is suspected or confirmed. This concept is sup- ported by clinical experience and experiments (Rodd and Boissonade 2000; Sigurdsson 2003; Iqbal et al. 2007; Ricucci et al. 2014) and by experimental studies on the effects of pulpal inflammation on nerve activ- ity (Rodd and Boissonade 2000; Bletsa et al. 2006; Kokkas et al. 2007). Box 2.1 lists the salient clinical signs of irreversible pulpitis (i.e., infection of the pulp necessitating endodontic treatment by root filling).

Box 2.1 Clinical characteristics of irreversible pulpitis

� Severe pain necessitating dental emergency treatment

� A history of repeated pain episodes � Self-medication with analgesics � Pain lingering after end of stimulus � Sleep or work affected � Supporting findings: positive, sometimes exag-

gerated pain on thermal or electrical stimula- tion; tooth localization difficult; percussion test largely negative

Sometimes, a carious process may have reached the pulp without any symptoms. Traditionally, and in most settings in a dental office, this is considered an irre- versible pulpitis (i.e., the tooth will need endodon- tic treatment; Bjørndal et al. 2010). However, mod- ern materials and aseptic techniques may also provide predictable results from pulp-preserving approaches in these clinical situations (Bogen et al. 2008; Mar- ques et al. 2015), but such approaches can be highly operator-sensitive (Miles et al. 2010) and therefore may not yet be recommended as standard practice.

Diagnosis, Epidemiology, and Global Impact of Endodontic Infections 13

Sensitivity testing by temperature or electrical pulses can give reasonably accurate assessment of nerve tissue activity in the pulp, but relating such recordings to the degree of pulpal inflammation is dif- ficult considering the large variation in such measure- ments and their dependence on other clinical param- eters associated with the tooth (Fischer et al. 1991; Chen and Abbott 2009; Alomari et al. 2011; Mejare et al. 2012).

Radiography, including cone-beam radiographic techniques, is useful in special circumstances, such as for detection of internal and external cervical resorp- tion (Celikten et al. 2014; Kalender et al. 2014; Ven- skutonis et al. 2014; Creanga et al. 2015; Dogramaci et al. 2015; Mavridou et al. 2016), which often affects the dental pulp. Pulp calcifications (diffuse and globu- lar) and obliteration as seen radiographically may give indications of the physiologic state of the pulp, but lit- tle information about pulpal infection or inflammation.

In summary, a clinical pulpal diagnosis is most often made based on anamnestic and subjective data sup- ported by sensitivity testing and caries excavation.

2.2.2 Periapical diagnosis

When the infection of the dental pulp affects the peri- odontium, apical periodontitis occurs. Pathologically, the inflammation is organized as a granuloma that may or may not develop a radicular cyst as a sequela (Nair 2008). Periapical disease also has a significant clinical component. In comparison with symptomatic pulpitis, symptomatic apical periodontitis is typically charac- terized by dull rather than sharp pain, and positive percussion and palpation tests (Iqbal et al. 2007; Sig- urdsson 2008). Total infection of the pulp with virulent organisms can give rise to acute apical abscess, a very painful and potentially harmful condition (Antunes et al. 2013; Chunduri et al. 2013; Moazzam et al. 2015), exemplifying a disease that historically defined the dental profession. Longstanding pulp infections with chronic apical lesions can exacerbate with the same symptomatic apical periodontitis or acute api- cal abscess. Apart from distinguishing such conditions from marginal periodontal inflammation, and in par- ticular a periodontal abscess, they are seldom difficult to diagnose.

Asymptomatic apical periodontitis is, on the other hand, dependent on radiographic signs for diagnosis.

In its early stages and during healing, this may be very difficult, whereas a well-established, asymptomatic periapical lesion is a simple condition to identify on radiographs (Ørstavik and Pitt Ford 2008). In teleo- logic terms, an infected root canal of a tooth is prob- ably perceived by the body as a risk zone for inva- sion by (life-threatening) microbes. A defense region is then established in which the tissue architecture is changed to prepare for the containment of invading microorganisms (Ørstavik and Larheim 2008). Bone is gradually replaced by granulomatous tissue with vascular and cellular components mobilized for host defense. These initial events produce changes in bone structure at the apex, which may be very hard to detect by periapical radiography (Brynolf 1967), and they may occur with teeth that still have vitality or at least neural activity in the pulp (Figure 2.1). When periapi- cal tissue remodeling has reached a state of complete granulomatous transformation, the lesion is very char- acteristic and easily diagnosed on the radiograph, par- ticularly when a cortical plate is affected (Figure 2.2). If such a tooth does not respond to sensitivity testing, a diagnosis of pulpal infection and apical periodontitis is certain, and treatment options instantly available. On the other hand, there may be total pulp necrosis and no infection or associated inflammation at the apex, such as when the pulp is devitalized by traumatic injury (Sundqvist 1976).

The chronic development of apical periodontitis may be totally without symptoms, in which case the term asymptomatic apical periodontitis is appropriate. However, symptoms may occur at any stage during the process, ranging from barely perceptible tenderness to the acute symptoms described earlier.

In summary, chronic asympomatic apical periodon- titis needs radiography for detection; symptomatic and acute phases are diagnosed by clinical symptoms and signs.

2.3 Pulpal inflammation and infection: public health consequences

The clinical aspects of endodontic diseases may be serious and with some consequences for individual and public health. Pulpitis and apical periodontitis are traditionally categorized under “caries and its seque- lae,” and it is certainly true that deep carious lesions

14 Endodontic Microbiology

(a) (b)

Fig. 2.1 Minimal bone structural changes at the apex in conjunction with chronic pulpitis (a), necessitating endodontic treatment (b).

are indicators of pulpal and periapical inflammation. This is a confounding factor for assessments of the relative importance of these conditions in the overall incidence and prevalence of orofacial pain. Pulpitis may be very painful and lead to loss of quality of life (Constante et al. 2012). It may also cause absence from work and loss of income (Miotto et al. 2012).

It is unfortunate that pulpal pain is pooled with other tooth-related pain and often with the whole specter of orofacial pain conditions in surveys and screening studies. If one assumes that emergency treatment of dental caries is initiated by pulpal pain and thus falls under the category of endodontic disease, symp- tomatic irreversible pulpitis and apical periodontitis

Diagnosis, Epidemiology, and Global Impact of Endodontic Infections 15

Fig. 2.2 Chronic apical periodontitis: incipient at mesial root, established at distal root of mandibular left first molar.

dominate as sources for acute dental pain in children and adults (Zeng et al. 1994; Lygidakis et al. 1998; Tulip and Palmer 2008). This may be debilitating to the patient and lead to absence from work and involve- ment of costly health services. While it is known that emergency dental services are in great demand in most countries, in urban as well as rural areas, there is scant information on the actual incidence and prevalence of acute pulpal and apical periodontal disease. Therefore, one can only speculate that there is still, even in communities with well-developed dental services, a significant impact on the general well-being by acute pulpal and periodontal conditions (Sindet-Pedersen et al. 1985; Richardson 2005; Cope et al. 2014).

It seems that psychologic factors influence the inci- dence and severity of orofacial pain including pulpal and periapical pain (Aggarwal et al. 2010). Therefore it is especially important for susceptible individuals to have conditions that cause acute dental pain treated quickly and efficiently.

A frequently overlooked situation is the association of pulpal and apical disease with tooth loss in the

elderly and in highly restored dentitions (Dikbas et al. 2013). Whereas marginal periodontal disease is gener- ally accepted as a significant cause of tooth loss, pulpal and apical diseases are important causes for extraction (Eckerbom et al. 1992; Lee et al. 2015) and may dom- inate after the age of approximately 50 years (Eriksen 1991).

The tooth with pulpitis is obviously in danger of becoming infected and developing apical periodonti- tis. Correct and prompt treatment of the acute situation is therefore important, not only to curb the pain and to re-establish a functional tooth, but also to reduce or eliminate the risk for the insidious spreading of the infection and the emergence of a periapical lesion. It has been known for a very long time that the prognosis for treatment of apical periodontitis is much poorer than expected treatment outcome after vital pulpec- tomy (see Chapter 15). Early detection and root canal treatment of teeth at definitive risk of developing root canal infection are therefore essential. Failure to pro- vide adequate treatment early will facilitate the devel- opment of an infection (Figure 2.3), which will reduce the prognosis.

16 Endodontic Microbiology

Fig. 2.3 Chronic apical periodontitis developing in 6 months after inadequate emergency treatment. The prognosis is reduced from > 95% to < 85%.

2.4 Epidemiology of endodontic diseases

2.4.1 Basic principles of epidemiologic approaches to dental disease

Longstanding diseases like asymptomatic apical peri- odontitis are well-suited for epidemiologic studies of prevalence; any untreated lesion may be viewed as per- manent and will be picked up if visible on radiographic cross-sectional surveys. On the other hand, acute den- tal pain must be expected to be short-lived in most cases as it is treated by medication or intervention. As a disease entity in epidemiologic studies, the incidence of endodontic disease is best assessed with a longitu- dinal study design, which picks up the peaks of pain that would not be found in a cross-sectional study.

An ecologic study focuses on the comparison of groups, rather than individuals. Dental pain and pul- pal infections may be viewed also in this context; age groups, ethnic groups, socioeconomic groups may

experience the diseases differently (Constante et al. 2012). The fact that dental pain conditions adversely affects the quality of life is no surprise (Shueb et al. 2015); the treatment and prevention of pain remain important aspects of dentistry.

2.4.2 Infections with pulpal involvement

Information in the literature about the incidence of dental and oral pain is scarce in itself (Lipton et al. 1993; Pau et al. 2003), and the separation of the pulpal or periapical component from the inclusive diagnosis is difficult. Nevertheless, given that irreversible pulpal inflammation is associated with severe and/or lingering pain, it seems reasonable to conclude that reports list- ing dental caries as a source of acute or severe pain in effect have pulpal inflammation as the source of pain. The few targeted epidemiologic data that do exist point to a limited, but significant, occurrence of acute pain of

Diagnosis, Epidemiology, and Global Impact of Endodontic Infections 17

pulpal origin (Sindet-Pedersen et al. 1985; Zeng et al. 1994; Lygidakis et al. 1998; Areai et al. 2011). This is an area in need of continued and extensive research. The incidence and prevalence of symptomatic pulpitis and apical periodontitis are obviously important for the targeting of dental services, and form important back- ground knowledge for the design of dental curricula and for public health measures. Comparative studies may be lacking, but it seems reasonable to assume that as the general dental health varies widely among populations within and across countries, so will the incidence and severity of pulpal and periapical pain.

2.4.3 Infections with periapical involvement

Asymptomatic apical periodontitis constitutes a dif- ferent challenge from pulpitis and symptomatic api- cal periodontitis. The insidious nature and frequently pain-free course of this disease makes it evasive to detection outside of the dental treatment situation. The fact that asymptomatic apical periodontitis relies on radiography for detection poses limitations on the pos- sibilities for screenings and population surveys. More- over, when radiographic data have been made available for analysis, lack of standardization in scoring makes comparisons across studies difficult.

The radiographic technique may also influence the ability to detect with certainty the occurrence of asymptomatic apical periodontitis. For population sur- veys, panoramic radiography provides information at far lower radiation dosage than full-mouth periapi- cal examinations, but the possibilities of detection of apical lesions may be diminished. Comparisons of

panoramic and periapical films for diagnosis of api- cal periodontitis suggest that there is some, but not a dramatic, reduction in the detectability of periapical lesions (Sameshima and Asgarifar 2001; Ridao-Sacie et al. 2007; Rios-Santos et al. 2010). Newer methods, such as tomography (Tammisalo et al. 1996), com- puted tomography (Huumonen et al. 2006) and cone- beam computed tomography (Lofthag-Hansen et al. 2007; Patel et al. 2007, 2015; Estrela et al. 2008; Kruse et al. 2015), are more sensitive and possibly more spe- cific than periapical radiographs, but the radiation dose limits their application for use in surveys.

2.4.4 Radiographic surveys of asymptomatic apical periodontitis: methodology

Verbal descriptors of the radiographic characteristics of asymptomatic apical periodontitis have included a widened periodontal space; interruptions of the lam- ina dura, and/or the presence of a radiolucent area at the site of exit of the pulp to the periodontal membrane. Only when there is an overt radiolucency associated with the root tip and a concomitant find- ing of a necrotic pulp, are the signs pathognomonic (Ørstavik and Larheim 2008). While it is possible to make assumptions from different studies with simi- lar descriptions of the criteria used for detection of asymptomatic apical periodontitis, the lack of stan- dardization makes it impossible to draw conclusions with any certainty.

The periapical index (PAI) was developed with the aim of overcoming this difficulty (Figure 2.4). It makes

Fig. 2.4 The periapical index. The periapical condition is scored by comparison with a series of reference radiographs of teeth with known histology. Reproduced with permission from Ørstavik et al. (1986).

18 Endodontic Microbiology

100

0 1

Scored as healthy Scored as diseased

Healthy Diseased

2N o.

o f te

e th

, p e r

ce n t o f ca

se s

3 4 5

Fig. 2.5 Dichotomization of PAI scores applied to epidemiology. Blue line, teeth without apical periodontitis; red line, teeth with apical periodontitis. A minimum of false positives (healthy apical periodontium scored as diseased; blue cases in red sector) is acceptable at the expense of some false negatives (diseased teeth registered as healthy; red cases in blue sector).

use of an ordinal scale with five steps indicating increasing severity of apical periodontitis (Ørstavik et al. 1986). The steps are represented by radio- graphs that have histologic verification from an exten- sive study on human cadavers (Brynolf 1967). This makes possible a visual reference scale that reduces the risk of personal bias otherwise associated with subjective radiographic assessments. Also, the system is used after extensive and standardized calibration of the observers, which facilitates comparisons of differ- ent studies and pooling of data. While developed for clinical follow-up studies of endodontic treatment in prospective studies, the PAI scoring system can easily be modified for use in epidemiologic surveys (Eriksen and Bjertness 1991). A general principle in epidemiol- ogy is to avoid scoring a healthy condition wrongly as disease. This is accomplished by restricting the catego- rization as “diseased” (i.e., with apical periodontitis) to teeth with scores 3–5 (Figure 2.5). In this way, some cases of asymptomatic chronic apical periodontitis will go undetected, but only a minimal number of healthy teeth will be scored as diseased.

Irrespective of the radiographic method of detection, it is apparent that radiographic assessments of apical periodontitis on the whole will underestimate its true incidence or prevalence (Brynolf 1967). Even with all these provisos, it may still be prudent to review and compare results from different areas and cohorts, as long as the shortcomings of the radiographic methods are kept in mind.

2.4.5 Results of epidemiologic surveys of asymptomatic apical periodontitis

When periapical disease was seen only as an extension of caries, epidemiologic studies paid little if any atten- tion to the incidence and prevalence of apical periodon- titis. On the basis of numerous institutional studies on the outcome of endodontic treatment, the notion that endodontic treatment was predictable and generally successful was accepted (Strindberg 1956; Grossman et al. 1964; Kerekes and Tronstad 1979; Ørstavik et al. 1987; Ng et al. 2011), and the extent and importance of apical periodontitis in the general population was largely overlooked.

In a series of studies, Eriksen and coworkers (Eriksen and Bjertness 1991; Eriksen et al. 1995; Marques et al. 1998; Sidaravicius et al. 1999; Alek- sejuniene et al. 2000; Skudutyte-Rysstad and Eriksen 2006) examined the general prevalence of apical peri- odontitis and placed it in its proper perspective. The PAI scoring system was used together with simple cri- teria for the assessment of root-filling quality. A pri- mary aim was to reassess the association of the qual- ity of the root filling as seen on the radiograph with the periapical status of the teeth. Similar to what had been documented in the institutional follow-up stud- ies, there was a clear association between poor root- filling quality and the presence of apical periodontitis, emphasizing the need for focus on high-quality tech- nical performance during the endodontic procedures.

However, there was also an unexpectedly high prevalence of apical periodontitis in most populations and age groups. This was a source of concern and had to be considered in oral health assessments in general. Moreover, the finding that pulpal and periapical dis- eases were major reasons for extractions in adults, sur- passing marginal periodontitis around the fifth decade of life, emphasized the impact of periapical health for retention of the dentition into old age (Eriksen 1991; Eriksen and Bjertness 1991; Eckerbom et al. 1992).

These studies have later been supplemented by sev- eral others from many countries and, with few excep- tions, the results are quite disheartening in different countries and populations, regardless of the degree and perceived quality of the dental services offered. Many studies have made use of the PAI scoring system; oth- ers rely on a simple assessment on the presence or absence of a radiolucent area indicating periodontitis. Figure 2.6 shows the prevalence of apical periodon- titis in populations from 18 different populations in

Diagnosis, Epidemiology, and Global Impact of Endodontic Infections 19

100

P re

va le

n ce

, p e r

ce n t

a b c d e

f g h i j k

l n

o p q

r s

Fig. 2.6 The prevalence of apical periodontitis in different populations. (a) Dugas et al. 2003; (b) Marques et al. 1998; (c) Frisk and Hakeberg 2005; (d) Loftus et al. 2005; (e) Buckley and Spangberg 1995; (f) DeCleen et al. 1993; (g) Eriksen 1991; (h) Dugas et al. 2003; (i) Kirkevang et al. 2001; (j) Frisk and Hakeberg 2005; (k) Chen et al. 2007; (l) Jiménez-Pinzón et al. 2004; (n) De Moor et al. 2000; (o) Saunders et al. 1997; (p) Sidaravicius et al. 1999; (q) Tsuneishi et al. 2005; (r) Kabak and Abbott 2005; (s) Segura-Egea et al. 2005.

different countries. Apical periodontitis occurs with a prevalence of 30–80% in different populations, gener- ally increasing in older age groups (Chen et al. 2007) and in populations at high risk of infectious disease, such as diabetics (Britto et al. 2003; Segura-Egea et al. 2005; Marotta et al. 2012).

Figures produced by this kind of surveys gener- ally do not account for alternative ways of dealing with apical periodontitis in different environments. It is tempting to speculate that populations with low prevalence have had teeth with apical periodontitis extracted: indeed, for the Portuguese population stud- ied by Marques et al. (1998), which showed the lowest prevalence, it was found that they had a lower mean number of remaining teeth than a comparable Nor- wegian population with higher prevalence of apical periodontitis (Eriksen and Bjertness 1991).

2.5 Quality of root canal treatment and the development and persistence of apical periodontitis

Institutional follow-up studies and epidemiologic sur- veys have all documented that there is a very clear correlation between presence of apical periodontitis and inadequate technical quality of the root filling as it

appears in the radiograph. The association is strongest for teeth that are diagnosed with apical periodontitis at the start of treatment, and far less dominant when the root filling is placed in teeth with no lesion prior to treatment (Sjögren et al. 1990). In the latter situa- tion, typically less than 10% of treated cases develop apical periodontitis; contrarily, teeth treated for pri- mary apical periodontitis show persistence of lesions in 20–25% of cases in institutional studies. In all like- lihood, there is a poorer outcome for both preoperative diagnoses in practice compared to the institutional set- ting. By inference, when epidemiologic surveys indi- cate that 30–40% of root-filled teeth have apical peri- odontitis, it seems fair to assume that less than 50% of teeth with apical periodontitis are cured in the average treatment setting in practice.

This should not be placed in a context to advo- cate more radical treatment or prophylaxis of apical periodontitis. The preservation of teeth by endodon- tic procedures is, after all, a clinically very successful and predictable procedure. The sequels to extractions and various prosthetic procedures, as alternative treat- ments, are numerous and often of greater consequence. However, these epidemiologic findings clearly point to a need for improvements in the quality of endodontic care.

2.6 Treatment strategies: prevention, treatment, and extraction

Despite efforts at preventing caries, at improving fill- ing therapies, and at protecting the tooth from mechan- ical trauma, endodontic disease remains an impor- tant issue in dental practice. Cost, functional needs, and aesthetics are considerations in deciding optimal therapy for a tooth with endodontic infection. Com- plete elimination of infection is assured if the tooth is extracted and, if functional or aesthetic needs do not mandate retention of the tooth or its replacement, this may be a preferred modality. However, if a functional tooth or its replacement is necessary, other sequels to treatment must be balanced in the equation.

A tooth with an endodontic infection may be extracted and replaced by a bridge. This will inevitably involve preparation and trauma to neighboring teeth and, unless they are in need of crown therapy irre- spective of the tooth under consideration, this weighs heavily against its replacement in a fixed bridge

20 Endodontic Microbiology

prosthesis. Furthermore, all teeth with restorations, and bridge abutment teeth especially, are susceptible to secondary caries and subsequent endodontic problems (Goodacre et al. 2003). The cure of one endodontic infection may thus easily lead to another.

The placement of an implant is often promoted as an alternative to endodontic treatment, and enthusiasm for implants have led some to suggest that indications for endodontic treatment should be more limited than has traditionally been held. Because of the differences in functional measures of performance, it is difficult to compare the suitability of the two approaches. By measuring tooth or implant retention and adding repair and replacement into a compound measure of success, some studies have been performed comparing implants with endodontic treatment (Doyle et al. 2007; Hanna- han and Eleazer 2008; Setzer and Kim 2014). Most studies conclude that performance is similar in the two situations. Furthermore, bacterial colonization around implants leading to peri-implantitis is a rather com- mon occurrence, introducing a new infection also in this alternative to endodontic treatment.

Extensive follow-up studies of endodontically treated teeth have demonstrated remarkably high reten- tion rates. It seems prudent to maintain a skeptical attitude to alternatives that rely on extraction of func- tioning teeth even if they need endodontic treatment.

2.7 General oral health, oral health strategies, and tooth preservation as risk factors for oral infections

Endodontic infections are mixed infections, involving a multitude of different microorganisms. The micro- bial communities vary within an affected tooth, among affected teeth in the same mouth, among different sub- jects, and over geographic regions. The primary tis- sue responses are directed at eliminating the tooth as the physical source of this type of infection. When effective, these responses reestablish an intact muco- cutaneous barrier which protects from new microbial attacks.

When the initial responses (pulpitis and apical peri- odontitis) fail to contain and eliminate the infection, subsequent events depends largely on the microbial composition of the infection and on the general resis- tance of the patient. The microorganisms associated with endodontic infections in most cases will have little

pathogenicity and low virulence. However, commonly found microorganisms like enterococci, Candida albi- cans, Burkholderia cepacia (Li et al. 2013), strepto- cocci, and staphylocci can cause infections in suscep- tible hosts (e.g., diabetics, immunocompromised and debilitated patients).

On rare occasions, life-threatening infections of endodontic origin can occur (Allareddy et al. 2012). Necrotizing fasciitis is an example of a major com- plication of endodontic infections (Leyva et al. 2013). These cases underscore the need for and add to the local indications for prompt and effective treatment of pulpitis and apical periodontitis.

The concept of endodontic diseases primarily as infections with the potential to spread and thereby to affect organs at distant sites may be important also for patients’ systemic health, particularly the risk of car- diovascular events (see Chapter 16). On the one hand, this affects the decision whether to provide antibiotic coverage prior to surgery in patients at risk of infec- tive endocarditis or infection of implants. On the other hand, the possible association of pulp and periapical infections with the risk of developing cardiovascu- lar disease has a major impact on the rationale and case selection for endodontic treatment, and especially on prophylactic efforts to prevent pulpal infection in the first place. Marginal periodontitis seems to have a definitive, albeit limited, association with cardiovascu- lar disease, and data are emerging indicating that this may be the case also for apical periodontitis (Caplan et al. 2006; Cotti et al. 2011; Pasqualini et al. 2012; Cotti and Mercuro 2015); however, others have failed to establish such an association (Frisk et al. 2003).

2.8 Conclusions

The notion that severe pulpitis or apical granulomas may be sterile or caused by medicaments or materials has been abandoned. Periapical lesions are virtually all apical periodontitis, and apical periodontitis is caused by microbial infection of the root canal system. Immi- nent or established infections of the pulp and periapi- cal tissues need to be contained or eliminated. Early and appropriate endodontic intervention is necessary in such cases, with emphasis on proper case selection and skilled technical performance of treatment.

The provision of high-quality endodontic care at all levels of dental service to the individual patient as well as to populations is therefore crucial for optimum

Diagnosis, Epidemiology, and Global Impact of Endodontic Infections 21

long-term oral health. The goals for these services are several: to prevent pulpal infection by effective caries prevention, by protection against dental trauma, and by appropriate dentin treatment under restorations; to limit pulpal pain as a source of discomfort and loss of work; and to eliminate dental infection and prevent its recurrence by root filling and surgical endodontic procedures.

Prognosis is clearly better for root fillings following vital pulp extirpation than for root fillings after treat- ment of established apical periodontitis. Early inter- vention in established pulpitis is therefore conducive to preventing pain, spread of infection, and tooth loss. However, this principle of case selection for treatment is often in conflict with the concept of the need to preserve the pulp itself. While desirable in theory and frequently successful, protection by capping of pulps exposed to caries in adults is less predictable that endodontic treatment of a tooth with vital pulp. Waiting to see if apical periodontitis develops may lead to an even less predictable situation, and requires that the patient adheres to a good follow-up sched- ule. Given the uncertain outcomes of pulpal protection techniques and of disinfection techniques for apical periodontitis, endodontic infections are best curtailed by early intervention: vital extirpation of the compro- mised pulp followed by root filling.

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Chapter 3 Microbiology of Dental Caries and Dentinal Tubule Infection Robert M. Love and Anne C.R. Tanner

3.1 Introduction 3.2 Oral biofilms associated with dental

caries 3.3 Microbiota of dental caries

3.3.1 Microbiota of initial caries including white spot lesions

3.3.2 Microbiota of early childhood caries

3.3.3 Microbiota of root caries 3.3.4 Microbiota of dentinal caries and

dentinal tubules 3.4 Microbial invasion of dentinal tubules

3.4.1 Colonization of dentinal tubules

3.5 Clinical aspects of dental caries microbiota and dentinal tubule infection 3.5.1 Invasion of coronal dentin:

influence on the progression and management of pulp disease

3.5.2 Invasion of radicular dentin: influence on the progression and management of periapical disease

3.5.3 Invasion of radicular dentin: influence on the progression and management of periodontal disease

3.6 Conclusions 3.7 References

3.1 Introduction

It is well established that bacteria are the prime etio- logic factor in the development and progression of den- tal caries, and pulp and periapical diseases. In the late nineteenth and early twentieth centuries, W.D. Miller demonstrated bacterial invasion of dentinal tubules of both carious and noncarious dentin and reported that the tubule microflora consisted of cocci and rods (Miller 1890). Sound experimental evidence in the 1960s established the fundamental role of bacteria in dental diseases. Keyes (1960) demonstrated that dental caries did not develop in germ-free animals fed a cariogenic diet, while Kakehashi et al. (1965) showed that pulp and periapical disease occurred in

surgically exposed rat molar pulps only when bacteria were present in the oral cavity. Indeed, in germ-free rats, exposed pulps remained healthy and were able to initiate repair by dentin bridging of the exposure, demonstrating the innate regenerative capacity of the dental pulp.

Dental caries is a complex polymicrobial biofilm disease process that primarily breaks down the dental hard tissues by producing an acid environment. Most individuals harbor the oral microbiota associated with caries and a shift to a cariogenic biofilm is possible in any individual, for example by consumption of a high sugar diet. Whenever dentin is exposed in the oral cav- ity through carious lesions, restorative or periodontal procedures, tooth wear, enamel or dentin cracks, or

26 Endodontic Microbiology

(a)

(c)

(d)

(e)

(b)

Fig. 3.1 Potential routes of infection of coronal and radicular dentin. Bacterial invasion of coronal dentinal tubules toward the pulp space (a) occurs as a result of a breach in the integrity of the enamel from dental caries, enamel cracks/fractures, or restorative procedures. Invasion of tubules toward the pulp also occurs when the cementum is breached as a consequence of periodontal disease or procedures. If unchecked, bacteria within dentinal tubules will enter and infect the pulp chamber and root canal space, and bacterial biofilms (b) will develop. Subsequently, bacterial invasion of radicular dentin occurs from the pulpal surface toward the dentinocemental junction. Invasion in cervical and mid-root radicular dentin readily occurs (heavy invasion shown in c), while the amount and depth of invasion in apical dentin is low (d). Inflammatory periradicular disease (e) results from the bacterial infection. Source: Love 2004. Reproduced with permission of John Wiley and Sons.

dental trauma, invasion of dentinal tubules by bacte- ria from supra- or subgingival dental plaque (biofilm) occurs (Tronstad and Langeland 1971; Pashley 1990; Peters et al. 1995; Love 1996a). Bacteria invad- ing coronal dentinal tubules (Figure 3.1) may cause pulpal disease (Brännström and Nyborg 1971) and subsequently take part in infection of the root canal system (Figure 3.1). As infection of the pulp space progresses, bacteria invade the radicular dentinal tubules (Figure 3.1c,d). If these bacteria are not removed or killed during endodontic treatment, the presence of vital bacteria within radicular dentin may be responsible for continued root canal infection (Haapasalo and Ørstavik 1987) and persistent apical periodontitis (Figure 3.1e).

The study and practice of endodontics aims to pre- vent and treat pulp and periapical disease and an under- standing of the mechanisms of biofilm formation, dental caries development, and bacterial invasion of dentinal tubules are central to these aims.

3.2 Oral biofilms associated with dental caries

Although specific bacterial species have been associ- ated with the development and progression of dental caries, and with pulp and periapical disease, these con- ditions are polymicrobial biofilm diseases. A biofilm is a complex consortium of microorganisms existing

Microbiology of Dental Caries and Dentinal Tubule Infection 27

as communities that exhibit a wide range of physical, metabolic, and molecular interactions. These interac- tions are important for the attachment, growth, and survival of species, enabling biofilms to develop and persist in what often appear to be hostile environ- ments such as the oral cavity, dentinal tubule, or root canal. This community lifestyle provides bene- fits to the microorganisms such as a broader habi- tat range for growth, increased metabolic diversity and efficiency, and enhanced resistance to environ- mental stress, antimicrobial agents, or host defenses (Caldwell et al. 1997; Shapiro 1998; Marsh and Bow- den 2000). Much of the work underlying oral biofilms has been undertaken on dental plaque, which behaves as a classic biofilm (Marsh 2004) characterized by surface attachment, structural heterogeneity, complex interspecies interactions, and production of an extra- cellular matrix of polymeric substances. They act as microbial high-density microniches that differ from the surrounding conditions. Oral biofilms colonize the surfaces of teeth, prostheses (biomaterials), gums, tongue (Marsh 2004), and other mucous membranes in both health and disease.

The underlying principle of dental biofilm formation is bacterial adhesion to, and subsequent colonization of tooth surfaces. Bacteria do not form strong attachments to mineralized tissue and require a conditioning sur- face on the tooth tissue to adhere to. The acquired pel- licle on tooth surfaces is derived from saliva and forms after a tooth surface is cleansed. This complex pro- teinaceous pellicle acts as an adhesive substratum for primary bacterial colonizers in the oral cavity existing as unattached planktonic cells. Initial bacterial adhe- sion involves a variety of mechanisms including ionic or hydrophobic bonds between bacterial surface com- ponents and the adhesion substratum (Figure 3.2) and occurs over a short time period (seconds to minutes) as the planktonic cells approach the substratum. In the second phase, bacterial adhesion molecules inter- act with the substratum (Figure 3.2) and the reactions (e.g., protein–protein interactions) may extend over hours to days (Jenkinson and Lamont 1997).

Streptococcus species are the major primary col- onizers (Nyvad and Kilian 1987; Dı́az et al. 2006) and express a large number of adhesins that can inter- act with many salivary constituents including alpha- amylase, proline-rich proteins, proline-rich glycopro- teins, statherin, salivary mucins and agglutinins, and α2-macroglobulin (Gibbons 1984). Some of these con- stituents including immunoglobulins, fibronectin, and

lactoferrin are found in serum, crevicular fluid, and dentinal tubule fluid and can act as adhesion molecules for primary bacterial colonization of the gingival crevice and dentin (Love 2002). Other early colo- nizers include Actinomyces, Veillonella, and Neisseria (Dı́az et al. 2006) and initial communities are unique in bacterial diversity and composition between subjects (Dı́az et al. 2006) and may differ between different areas or structures in the oral cavity; however, func- tional relationships derived from intermicrobial con- tact primarily determines how biofilm communities develop.

Interbacterial coaggregation is a cell–cell reaction that occurs between bacteria cells; it is an important aspect in early biofilm development. Coaggregation allows planktonic bacterial cells that cannot directly interact with surface substrate to colonize by binding to surface-bound early colonizers. Kolenbrander et al. (2002) proposed a spatial–temporal biofilm model starting with early colonizers forming the bottom layer and later colonizers such as Porphyromonas gingivalis and Bacteroides forsythus (now Tannerella forsythia) attaching via coaggregation reactions. Binding reac- tions between adjacent coaggregating bacteria allows bridging between three or more bacterial species (Figure 3.2c). This important process allows connec- tions between species that do not form coaggregations; for example, Fusobacterium nucleatum can coaggre- gate with numerous oral species and can act as a coor- dinator that bridges the early and late colonizers in oral biofilms (Kolenbrander et al. 2002).

Complex microbial communities develop by pro- cesses involving recognition of other species, metabolic signals or attractants, and the availability of usable substrates, adherent substrates, and host molecules. Early colonizers are typically oxygen- consuming species which create environmental con- ditions in the biofilm suitable for colonization by obli- gate anaerobes. Additionally, biofilm heterogeneity of pH, oxygen tension, and redox potential enables species with a wide range of growth requirements to coexist. Oral bacteria can obtain nutrients from foods ingested by the host (e.g., fermentable carbo- hydrates), while amino acids, peptides, proteins, and glycoproteins (which act as a source of sugars and amino-sugars) are mainly derived from saliva, gingi- val crevicular fluid, host tissue (e.g., necrotic dental pulp), or other bacteria. Similarly, periradicular fluid and inflammatory exudate are important nutritional sources for intraradicular and dentinal tubule bacterial

28 Endodontic Microbiology

Fig. 3.2 The sequence of adherence and colonization of tooth surfaces by bacteria. (a) Primary colonizing bacteria existing as planktonic cells interact with the conditioning film (e.g., acquired pellicle, dentinal tubule fluid, serum) on the tooth surface using longer range interactions (e.g., pili) or shorter range molecular interactions. (b) The early colonizers form strong bonds with the surface molecules in the conditioning film or components of the tooth substrate (e.g., collagen) by a variety of mechanisms and multiple adhesins. In conjunction with adhesion, the bacteria perform other functions such as adapting to the available nutrition, intermicrobial signaling, and production of an extracellular matrix. (c) Late colonizing bacteria enter the community by coaggregation reactions contributing to sequential binding and colonization of the developing biofilm. In this regard, Fusobacterium has been shown to be an important bridging organism allowing interactions between nonbinding bacteria. Within the biofilm intricate processes and interactions, such as quorum sensing, metabolic communication, genetic exchange, and competitive interactions, further shape the membership of the complex community, ensuring efficient utilization of nutrients and reduced susceptibility to host defences or therapeutic methods (e.g., antimicrobials). Source: Love 2004. Reproduced with permission of John Wiley and Sons.

biofilms. Communication between bacteria, mediated by secreted molecules, regulates several physiologic and virulence related properties, including biofilm for- mation. Density-dependent or quorum sensing systems are important gene regulated functions in response to cell density which influence a number of organ- ism functions including virulence, acid tolerance, and biofilm formation. Cooperative bacterial interactions are a feature of biofilm development; however, com- petition between bacteria (e.g., by bacteriocin produc- tion) also occurs. Numerous bacteriocins have been identified that are produced by bacteria to inhibit the growth of closely related bacterial strains.

These complex interactions are involved in the eco- logic balance of the oral ecosystem in health and disease. As examples, enhanced growth and tooth demineralization was observed when Lactobacillus

acidophilus was cultured with either Actinomyces israelii and/or Streptococcus mutans (Shen et al. 2004) while it has been established that low pH conditions in plaque select for mutans streptococci and lactobacilli (Marsh and Percival 2006). Studies using open-ended methods report a higher microbial diversity in health than that found in caries (Gross et al. 2012).

3.3 Microbiota of dental caries

Dental caries is caused by complex highly active biofilm microbiotas, which in the presence of dietary carbohydrates act as a consortium to produce an acidic environment that demineralizes teeth. Dental caries developed in humans following the transition from the predominantly meat-based diet of hunter-gatherers to

Microbiology of Dental Caries and Dentinal Tubule Infection 29

a diet that included grains and corn as observed in ancient populations (Cucina et al. 2011), and in diverse Native Indigenous populations (Schroth et al. 2010) with the introduction of high carbohydrate and sugar- based diets. Clinically, initial dental caries of enamel surfaces presents as white spot lesions (WSL), whereas dentinal caries either an extension of enamel caries or initiated on root surfaces presents with darker colors and softening of the mineralized tissues. Of particular interest to endodontics is the caries microbiota affect- ing dentin and dentinal tubules because deep dentinal caries can extend to infect the pulp and root canal system.

The microbiota of all stages in the carious pro- cess has been studied using culture and molecular approaches. Earlier culture studies of dental caries reported the presence of and caries-associations of Streptococcus mutans and other streptococci, Lac- tobacillus species, and Gram-negative Veillonella species (Loesche and Syed 1973; Milnes and Bow- den 1985). The cariogenic potential of the acidogenic and acid-tolerant S. mutans and Lactobacillus species led to substantial research focusing on a prime etio- logic role in caries by these taxa. Their importance was questioned, however, following the observation of a lack of caries in the presence of these species yet active caries when these taxa were not found (Boyar et al. 1989; Beighton 2005; Gross et al. 2012).

Molecular-based studies including cloning and sequencing strategies and 16S rRNA probe studies revealed a wide diversity of species in plaque of young children with early childhood caries (ECC), including in clinically healthy oral sites (Becker et al. 2002). Clonal analyses of ECC revealed 134 species/taxa when 72 children were characterized (Gross et al. 2012) and with over 200 taxa cultured using strict anaerobic techniques on samples from 80 children (Tanner et al. 2011a). Comparison between molecular and cultural analyses of the same samples from deep dentinal caries (Munson et al. 2004) and ECC (Kanasi et al. 2010; Tanner et al. 2011a) suggests that the major species in dental caries are cultivable, although in-depth sequencing studies indicate that additional species are present in low levels. Culture-based studies have added to our understanding of the microbiota of dental caries by facilitating detection of Actinomyces, Bifidobacterium, and Scardovia species that have been underestimated using molecular methods, and from the ability to test plaques (Lingström et al. 2000) and iso- lates (Van Houte et al. 1996) for acidogenicity. Overall,

the complexity of the bacteria associated with dental caries parallels the diverse microbiotas of endodontic lesions observed using similar molecular approaches (Li et al. 2010).

Of the multiple species detected in plaque associ- ated with ECC, caries-associated taxa are principally in the phylum Firmicutes which includes the gen- era Streptococcus, Lactobacillus, and Granulicatella, or Gram-positive rods in the phylum Actinobacteria which includes the genera Actinomyces, Actinobac- ulum, Corynebacterium, Rothia, Propionibacterium, Bifidobacterium, Allscardovia, Parascardovia, and Scardovia, all taxa that ferment carbohydrates to pro- duce acid.

3.3.1 Microbiota of initial caries including white spot lesions

The observation that S. mutans does not colonize the oral cavity until teeth erupted was dispelled by detec- tion of this species in predentate 6-month-old Aus- tralian Aborigine infants (Wan et al. 2001), and in infants in Micronesia (Tanner et al. 2002), indicating that the microbiologic conditions for caries develop- ment occur in early childhood. The microbiota of ini- tial WSL in young adults was found to comprise higher proportions of S. mutans and lactobacilli than sound enamel surfaces and caries-free subjects, although the proportions of total streptococci were similar in all groups (Van Houte et al. 1996) (Table 3.1). Simi- larly, van Ruyven et al. (2000) demonstrated a higher proportion of S. mutans and non-mutans streptococci isolates from WSL samples that lowered the pH of a glucose broth further than from control sites. In subjects with multiple lesions, S. mutans comprised 1.5% of the total microbial counts whereas other acidogenic non-mutans streptococci were detected more frequently at over 25% cultivable microbiota. Lactobacilli were detected at higher levels in initial carious lesions than non-lesion and caries-free young adults but Lactobacillus levels were much lower than other species (Van Houte et al. 1996), suggesting they were less important in WSL etiology. The acid-tolerant microbiota of initial lesions in young adults indicated that while streptococci were the dominant species detected at pH 5, less than 50% were identified as S. mutans (Svensater et al. 2003). Other acid-tolerant taxa detected included lactobacilli and, at lower frequencies, veillonellae, yeasts, and bifidobacteria. However, only S. mutans was associated with caries.

30 Endodontic Microbiology

Table 3.1 Major species associated with dental caries

White spot lesion Early childhood Root Dentinal caries and Phylum/genus initial caries caries caries tubule infection

Firmicutes Streptococcus S. mutans S. mutans S. mutans S. mutans

S. sobrinus S. sobrinus S. mitis S. sanguinis S. intermedius S. cristatus S. intermedius S. salivarius S. salivarius S. gordonii S. sanguinis

S. intermedius S. parasanguinis S. salivarius

Granulicatella G. elegans G. elegans Lactobacillus L. crispatus L. fermentum Lactobacillus sp. L. acidophilus

L. fermentum L. gasseri L. casei L. gasseri L. oris L. fermentum L. paracasei L. paracasei L. gasseri/johnsonii L. salivarius L. salivarius L. paracasei

L. plantarum L. rhamnosus

Pseudoramibacter P. alactolyticus P. alactolyticus Dialister D. invisus D. invisus Solobacterium S. moreii Enterococcus E. faecalis Selenomonas S. sputigena Selenomonas sp Veillonella V. atypica V. dispar

V. parvula Veillonella sp.

Actinobacteria Actinomyces A. gerensceriae A. gerensceriae A. israelii A. israelii

A. israelii Actinomyces sp A. naeslundii A. timonensis A. odontolyticus

Olsenella Olsenella sp O. profusa Olsenella sp

Atopobium A. parvulum Atopobium genomosp. C1 Atopobium sp. Atopobium sp. Rothia R. dentocariosa R. dentocariosa Propionibacterium Propionebacterium FMA5 Propionibacterium sp. P. acidifaciens Bifidobacterium Bifidobacterium sp. B. dentium B. breve Bifidobacterium sp.

Bifidobacterium sp. Bifidobacterium sp. Parascardovia P. denticolens P. denticolens Scardovia S. inopinata S. wiggsiae S. inopinata

S. wiggsiae S. wiggsiae Bacteroidetes Prevotella P. denticola Prevotella sp. P. multisaccharivorax P. melaninogenica

Prevotella sp. Fusobacteria F. nucleatum Yeasts Candida albicans

Microbial samples from caries-free sites of subjects with caries had a more similar composition to caries than to samples from caries-free subjects, suggesting that the caries microbiota spreads around the denti- tion to infect multiple sites. Thomas et al. (2012)

monitored the microbiota of initial caries development on restored and unrestored enamel and dentine sections worn in the mouth for 20 weeks using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Species that were detected

Microbiology of Dental Caries and Dentinal Tubule Infection 31

in initial caries-associated biofilms included S. mutans, S. sobrinus, Lactobacillus paracasei, L. fermentum, L. salivarius, L. crispatus, L. reuteri, L. gasseri, Scar- dovia inopinata, and Rothia dentocariosa (Table 3.1), expanding the range of acid-tolerant and acidogenic species detected in initial caries lesions.

WSL can develop quite rapidly in association with fixed orthodontic appliances, although most of these lesions remineralize when appliances are removed. The microbiota of these lesions in adolescents has been examined as a model of initial caries. Several studies confirmed the presence of S. mutans in WSL using culture (Boyer et al. 1996), microarray (Torlakovic et al. 2012), and quantitative PCR (Tanner et al. 2012). DNA probes based on 16S rRNA specific to differ- ent oral taxa have been developed and using these in a microarray analysis demonstrated that non-mutans WSL-associated taxa included Atopobium parvu- lum, Dialister invisus, Prevotella spp., Scardovia spp., Granulicatella elegans, Veillonellaceae spp., Selenomonas sputigena, and Actinomyces HOT 448 (Tanner et al. 2012; Torlakovic et al. 2012) (Table 3.1). Higher levels of S. mutans and Scardovia wiggsiae, a newly named species in Actinobacteria detected in severe childhood caries, were observed using quan- titative PCR (qPCR) in WSL than sites in ado- lescents without WSL. S. wiggsiae and S. mutans were associated with gingivitis in addition to WSL. Other gingivitis-associated species detected in the WSL samples included non-mutans streptococci, Pre- votella species, Streptococcus parasanguinis, Gemella hemolysans, Gemella sanguinis, and Actinomyces HOT 448. This suggested that some of the species detected in carious plaques have a primary associ- ation with gingival inflammation, another biofilm- associated clinical condition (Tanner et al. 2012).

These studies indicate that microbiota of WSL is complex, and need not be dominated by S. mutans. Other species important in WSL include non-mutans streptococci, several Gram-positive rod species includ- ing Actinomyces and Scardovia wiggsiae, as well as the Gram-negative rod Prevotella species.

3.3.2 Microbiota of early childhood caries

Dental caries in very young children can progress rapidly by affecting newly erupted, partially miner- alized deciduous teeth. While caries in the primary dentition was previously associated with the use of nursing bottles, giving rise to the name nursing bottle

caries, the use of bottles alone did not lead to caries in many children (O’Sullivan and Tinanoff 1993). These findings led to the reclassification of caries in the pri- mary dentition as early childhood caries (ECC), or in advanced cases as severe ECC. In the most aggressive cases, cavities advance through dentin and these chil- dren often undergo pulp or root canal treatments or tooth extraction to preserve the underlying permanent tooth. Early childhood caries is recognized as a sig- nificant public health problem with renewed interest in studying the etiology from both dietary and socioe- conomic perspectives (Gao et al. 2010; Fontana et al. 2011) as well as the associated microbiota (Marchant et al. 2001; Becker et al. 2002; Corby et al. 2005; Aas et al. 2008; Tanner et al. 2011b). Despite strong associations of early childhood caries with S. mutans by selective culture (Tanzer et al. 2001; Barsamian- Wunsch et al. 2004), and S. mutans and S. sobrinus using PCR methods (Okada et al. 2005), these species have not universally proved reliable in risk assessment (Fontana et al. 2011), indicating the need to study the ECC microbiota for additional microbial risk markers (Beighton 2005).

Considerable heterogeneity was observed in S. mutans (Alaluusua et al. 1996; Marchant et al. 2001) and Lactobacillus strains cultured from severe ECC even within individual children (Marchant et al. 2001). This indicates that more than a single strain infec- tion is responsible for severe ECC. The mutans strep- tococci consist of two related but distinct species, S. mutans and the more acidogenic and aciduric Streptococcus sobrinus, although S. sobrinus is cul- tured less frequently than S. mutans. Studies using PCR and qPCR (Nurelhuda et al. 2010) report that detection of S. mutans and S. sobrinus together can be associated with development of more new cari- ous lesions than either species alone (Okada et al. 2012). Using anaerobic culture, the major acid-tolerant species associated with severe ECC were S. mutans and S. wiggsiae (Tanner et al. 2011a) (Figure 3.3) as observed for WSL. This was confirmed using species- specific PCR, indicating that these bacteria in addi- tion to S. sobrinus and total bifidobacteria are asso- ciated with severe ECC (Mantzourani et al. 2009a; Palmer et al. 2010). Additionally S. mutans, S. sobri- nus, Veillonella species, and the non-mutans strepto- cocci Streptococcus vestibularis/salivarius and Strep- tococcus parasanguinis were shown to be elevated in ECC caries progression (Gross et al. 2012). A positive correlation between increased numbers of veillonellae

32 Endodontic Microbiology

Acid Agar Isolation

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Severe-ECC (n = 41) Caries-Free (n = 40) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 Chi Square Test

Fig. 3.3 Major species detected on acidic agar from severe early childhood caries (ECC) and caries-free children. Note the relationship of Streptococcus mutans and S. wiggsiae in ECC and the microbiota profile between health and disease.

and acidogenic streptococci (S. mutans, S. sobrinus, and Streptococcus vestibularis/salivarius) adds to the hypothesis that the acid-tolerant veillonellae flourish in the acidic niche, and could facilitate increased growth and acid production of the acidogenic bacteria (Becker et al. 2002).

Major species associated with severe ECC other than S. mutans included S. salivarius, Streptococcus parasanguinis, Streptococcus cristatus, Lactobacil- lus species, Actinomyces israelii, Actinomyces geren- sceriae, Actinomyces timonensis, Candida albicans, S. wiggsiae, Atopobium, Propionebacterium, and Veil- lonella species (Marchant et al. 2001; Becker et al. 2002; Corby et al. 2005; Aas et al. 2008; Tanner et al. 2011a) (Table 3.1). Additional bifidobacteria detected in childhood caries were Bifidobacterium dentium, Parascardovia denticolens, and Scardovia inopinata (Mantzourani et al. 2009a; Gross et al. 2010). Species observed in ECC in the absence of S. mutans include B. dentium, low pH non-mutans streptococci, Veil- lonella spp., Lactobacillus spp., and S. wiggsiae (Aas et al. 2008; Gross et al. 2012; Tanner et al. 2011a).

Similarly, Gram-positive rod species including Atopo- bium, Propionebacterium and Lactobacillus have been detected at higher levels than S. mutans in rampant caries (Aas et al. 2008). These studies confirmed the complexity of the microbiota of childhood caries, and that caries is not universally associated with S. mutans.

Most of the caries-associated species notably Bifidobacterium, Scardovia, Parascardovia, certain Streptococcus, including S. mutans, and Veillonella are acid-tolerant, and thus reflect their ability to survive the environment of actively progressing caries (Tanner et al. 2011a) (Table 3.2). In general, acid-tolerant species can lower the pH in glucose broth more than acid-sensitive species, consistent with the model of caries proposed by Takahashi and Nyvad (2011) (Fig- ure 3.4). While acid-tolerant bacteria may characterize progressing caries, not all aciduric or acidogenic species are caries associated. Similarly, many of the Gram-negative rod taxa including Selenomonas, Fusobacterium, Leptotrichia, Cardiobacterium, Neisseria, Campylobacter, Haemophilus, Capnocy- tophaga, Porphyromonas and Prevotella species were

Microbiology of Dental Caries and Dentinal Tubule Infection 33

Table 3.2 Acid tolerant caries species cultured on blood (pH 7) and/or acidic (pH 5) agar

Genus

Species/Taxa detected more frequently on blood agar

Species/Taxa no difference in detection on acid or blood agars

Species/Taxa detected more frequently on acid agar

Streptococcus S. mitis, S. mitis II, S. cristatu, S. sanguinis, S. gordonii

S. parasanguinis 1, S. constellatus, S. oralis, S. sobrinus, Streptococcus sp. HOT 058, 064, 071

S. mutans, S. parasanguinis II, S. salivarius, S. anginosus, S. intermedius, S. thermophilus

Granulicatella G. adiacens, G. elegans Abiotrophia A. defectiva Enterococcus E. faecalis Lactobacillus L. fermentum Gemella G. morbillorum, G.

haemolysans Staphylococcus S. epidermidis Parvimonas Parvimonas sp. HOT 107 Lachnospiraceae [G-1] Lachnospiraceae [G-1] sp.

HOT 110 Lachnoanaerobaculum L. saburreum Veillonella V. parvula V. dispar, V atypica Megasphaera M. micronuciformis Dialister D. invisus Selenomonas S. sputigena, S. artemidis, S.

flueggei, S. noxia, S. dianae, S, infelix, Selenomonas sp. HOT 137, 138, 139, 140, 149, 479, 639

Fusobacterium F. nucleatum ss. polymorphum, F. periodonticum, F. nucleatum ss. animalis, F. nucleatum ss. vincentii

Leptotrichia L. wadei, L. shahii, L. buccalis, L, hofstadii, Leptotrichia sp. 498

Actinomyces A. naeslundii, A. gerencseriae, A. israelii, A. georgiae, A. massilensis, Actinomyces sp. HOT 169, 170, 171, 175, 178

A. odontolyticus, A. dentalis, Actinomyces sp. HOT 177, 180

Rothia Rothia sp. HOT 188 Propionibacterium P. acnes Bifidobacterium B. dentium Scardovia/Parascardovia S. wiggsiae, P.

denticolens Atopobium A. parvulum Campylobacter C. showae, C. gracilis, C.

concisus

(continued)

34 Endodontic Microbiology

Table 3.2 (Continued)

Genus

Species/Taxa detected more frequently on blood agar

Species/Taxa no difference in detection on acid or blood agars

Species/Taxa detected more frequently on acid agar

Cardiobacterium C. hominis Eikenella E. corrodens Neisseria Neisseria sp. HOT 016 Kingella K. oralis Capnocytophaga Capnocytophaga sp. HOT

312, 325, 335, 336, 380, 412

Porphyromonas P. catoniae Prevotella P. nigrescens, P.

melaninogenica, Prevotella sp. HOT 317

P. denticola, P. maculosa, P. histicola, P. veroralis, Prevotella sp. HOT 314, 472

Terrahaemophilus T. aromaticivorans

Dynamic stability stage

Dominance of non-MS and actinomyces Net m

ineral gain

(lesion regression/arrest)

Net m ineral loss

(lesion initiation/progression)

Surface feature

shiny/sm ooth (enam

el)

dull/rough (enam el)

dull/soft (dentin)

shiny/hard (dentin)

{

“low-pH” non-MS and actinomyces

Acid-induced adaptation/selection

Increase in MS and non-mutans

aciduric bacteria

Mild/infrequent acidification

Moderate/frequent acidification

Severe/prolonged acidification

Acidogenic stage

Aciduric stage

Fig. 3.4 The caries process according to an extended caries ecologic hypothesis. This model proposes that dental plaque has a dynamic stability stage characterized by non-mutans streptococci (non-MS) and Actinomyces which maintain a stable plaque pH. This is disrupted when the plaque pH is lowered from bacterial acid production from a dietary change leading to an acidogenic stage accompanied by the possibility of tooth demineralization. An acidic plaque selects for acid-tolerant bacteria in the aciduric stage and conditions of tooth demineralization and dental caries. Our understanding of dental plaque composition suggests that the species in this model should be increased to include more Streptococcus species and more Gram-positive rod species than Actinomyces in the dynamic stability and acidogenic phases. The aciduric phase would include Bifidobacterium, Scardovia, and Lactobacillus species and mutans streptococci as in Table 3.1. Source: Takahashi and Nyvad 2011. Reproduced with permission of Sage Publications.

Microbiology of Dental Caries and Dentinal Tubule Infection 35

detected only on pH neutral, enriched blood agar suggesting that these species, while present in the complex biofilm community, may only have a minor or supporting role in the pathogenesis of caries.

Treatment studies of ECC that monitor caries micro- biota have generally focused on levels of mutans strep- tococci, which or may not be changed after interven- tions (Zhan et al. 2006). By monitoring the microbiota using a 300 taxon microarray (http://mim.forsyth.org/ homim.html) in children pre and post therapy, the microbial changes in several taxa were noted only in the children without new lesion development after ther- apy (Tanner et al. 2011b). This suggests that successful therapy may require changing the plaque composition as a whole, which is best monitored using rapid assays that target multiple species.

Bacterial invasion from deep caries into the pulp leads to pulpitis and endodontic infection. In children with severe ECC the microbiota cultured from newly exposed pulps comprised a subset of bacteria identified from caries including S. mutans, Parascardovia denti- colens, Bifidobacterium longum, Lactobacillus para- casei, and several additional Lactobacillus and Acti- nomyces species (Chalmers et al. 2015). A culture study of necrotic pulps of primary teeth also identi- fied a dominant Gram-positive microbiota (Ledezma et al. 2010). These findings differ from other reports of a dominant Gram-negative microbiota of root canal infections of deciduous teeth (Triches et al. 2014), sug- gesting that the microbiota can mature from that of ini- tial pulp infection to that associated with symptomatic root canals.

3.3.3 Microbiota of root caries

In adults with gingival recession, or periodontitis (Sao- tome et al. 2006) that exposes root dentin, caries can progress rapidly in the presence of reduced sali- vary flow, or individuals with grazing habits (frequent intakes of cariogenic foods). A dominance of acido- genic Gram-positive rod species has been associated with root caries, particularly Actinomyces and Lac- tobacillus species, in addition to S. mutans (Brails- ford et al. 2001; Saotome et al. 2006; Ikebe et al. 2008) (Table 3.1). A recent study (Hashimoto et al. 2011) reported that the dominant taxa in root caries detected by anaerobic culture, however, were Gram- positive rods in the genera Propionibacterium and Bifidobacterium in addition to Actinomyces, Lacto- bacillus, and Streptococcus species. While this was

a pilot study of six subjects, these findings are con- sistent with other recent reports. Examination of the microbiota of root caries using selective media in 30 subjects by lesion severity reported significant asso- ciations of mutans streptococci, lactobacilli, yeasts, and Bifidobacteriaceae with lesion severity (Mant- zourani et al. 2009a). Bifidobacteriaceae were detected in soft active lesions at 8% total anaerobic micro- biota compared with 4% for S. mutans and 31% for lactobacilli. Bifidobacterium dentium was the domi- nant Bifidobacteriaceae, followed by Parascardovia denticolens, Scardovia inopinata, and S. wiggsiae (Scardovia genomosp.C1), and, less frequently, Bifi- dobacterium breve and Bifidobacterium subtile. Bifi- dobacterium isolates from advanced root caries that lowered the pH in glucose culture <4.2 were second in detection frequency to S. mutans and above Lacto- bacillus species (Van Houte et al. 1996), further sug- gesting a role of bifidobacteria in root caries. Other species lowering the pH to <4.2 included the non- mutans streptococci S. milleri (mitis), S. intermedius, S. sanguinis and Actinomyces israelii (Van Houte et al. 1996).

Findings from molecular studies demonstrate that microbial complexity in caries extends to root caries. By sequencing and clonal analyses, the dominant species in root caries were Actinomyces and Lac- tobacillus species, S. mutans, Enterococcus faecalis (Table 3.1) with other Gram-positive rod species in Atopobium, Olsenella, Pseudoramibacter, and Propionibacterium and with Gram-negative saccha- rolytic Selenomonas and Prevotella species (Preza et al. 2008). By Human Oral Microbe Identifica- tion Microarray (HOMIM; Preza et al. 2009), the dominant root caries species included Lactobacillus species in the casei/paracasei/rhamnosus group (the 16S rRNA probes did not differentiate these taxa), Pseudoramibacter (Eubacterium) alactolyticus (a fer- mentative Gram-positive rod; Willems and Collins 1996), and S. mutans in infected dentin (Preza et al. 2009). A frequent observation was the difference in microbial composition of carious lesions among sub- jects, with no defined microbiota consistently observed for root caries.

3.3.4 Microbiota of dentinal caries and dentinal tubules

Carious lesions that have extended through the enamel to dentin have been studied as dentinal caries. The

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36 Endodontic Microbiology

interest in studying dentinal lesions is in part to improve our understanding of the etiology of caries in dentine, and partly, of interest to endodontics, to understand the microbiota of deep lesions and denti- nal tubule infection as a precursor of pulpal infection. As observed from caries from other sites, the dom- inant bacteria that have been cultured from dentinal caries comprised Streptococcus mutans, Lactobacil- lus, Actinomyces, and Veillonella species (Table 3.1). However, a wide diversity of species was detected, using a combination of molecular and anaerobic cul- tural methods to compare the microbiota of the mid- dle with advancing front of dentin lesions in adults (Munson et al. 2004). In this study, no significant differences were observed in the microbiota in the middle and deep parts of lesions of the five teeth that underwent this very comprehensive analysis. S. mutans, Rothia dentocariosa, and Propionibacterium acidifaciens were detected in all teeth sampled. The predominant cultivable microbiota comprised three Gram-positive rod species, Propionibacterium acidi- faciens, Olsenella profusa, and Lactobacillus rhamno- sus, whereas the dominant taxa identified from the par- allel molecular analysis were S. mutans, Lactobacillus gasseri/johnsonii, and Lactobacillus rhamnosus. Veil- lonella species, mainly V. dispar and several Prevotella and Fusobacterium species, were also detected. Strik- ing differences were observed in taxa detected from anaerobic culture and those using two different uni- versal primer sets for the molecular analysis; notable were the higher proportions of species in the phylum Actinobacteria from culture compared to molecular analysis (Munson et al. 2004), a similar observation seen in severe ECC (Kanasi et al. 2010; Tanner et al. 2011a). Munson et al. (2004) noted that unnamed taxa detected only by clonal analysis were all in genera with cultured species so it is likely they were cultivable. This is in contrast to subgingival, periodontal, and endodon- tic samples that contain taxa in uncultivated genera and likely not yet cultured (Munson et al. 2002).

Comparisons of the microbiota of shallower and deeper layers in dentinal caries have been studied. The species present in superficial middle and deep layers of advanced occlusal caries were similar when assayed using a set of 16S rRNA probes (Lima et al. 2011) as observed above from culture and clonal analysis (Munson et al. 2004). Of the 24 species probed, the highest concentrations detected included F. nucleatum, Atopobium genomospecies C1, Lac- tobacillus casei, Veillonella species, Streptococcus

species including S. mutans, Bifidobacterium species, and Rothia dentocariosa. Although streptococci were detected in 82% of the deep-dentinal lesions, S. mutans was identified in only 44%, consistent with other reports of caries being associated with taxa other than S. mutans (Lima et al. 2011). The microbiota of deep dentinal caries detected using a combination of selective culture and qPCR noted a dominance of Gram-positive species, particularly Lactobacillus aci- dophilus (five types), L. rhamnosus, L. paracasei, L. fermentum, and L. plantarum (Martin et al. 2002). Other species detected included A. israelii, A. odon- tolyticus, A. naeslundii and bifidobacteria, S. mutans, S. sanguinis, S. salivarius, and S. anginosus. The most frequently detected taxa were F. nucleatum and streptococci (97%), Gram-positive lactobacilli (95%), Prevotella and Actinomyces (91%), and P. melanino- genica (88%). A wide species diversity of 75 differ- ent taxa in deep dentinal caries was also observed from a cloning/sequencing analysis by Chhour et al. (2005). Additionally, there was considerable variabil- ity amongst samples: four samples had high Lacto- bacillus at over 95% clones, three samples had high proportions of Prevotella with other species including Olsenella and Pseudoramibacter alactolyticus, two samples had mid Lactobacillus/Prevotella levels, and one sample had low Lactobacillus/Prevotella counts. Other species detected included Selenomonas, Dial- ister, F. nucleatum, Eubacterium, Lachnospiraceae, Olsenella, Bifidobacterium/Scardovia, Propionebac- terium, and Pseudoramibacter alactolyticus.

The microbiota and acidity (pH) of active and inac- tive dentine lesions show a lower pH for active lesions than inactive lesions and increased levels of lactobacil- lus, but not S. mutans and S. sobrinus (Kuribayashi et al. 2012). In extensive dentinal caries, positive cor- relations between Lactobacillus levels at lower pH, and Prevotella species at higher pH levels were observed (Kianoush et al. 2014). The highest total bacterial loads were in plaque and superficial dentinal layers com- pared to deeper dentinal layers, with pH levels being most acidic in plaque and at shallow and mid-lesion depths. Increased caries activity at lower pH values, and differences in the composition based on local pH, fits the caries process model described by Takahashi and Nyvad (2011) (Figure 3.4).

In conclusion, studies have shown variation in the microbiota associated with caries and that it is not caused by one specific species nor is it a mono- species infection. In general, selective culture favors

Microbiology of Dental Caries and Dentinal Tubule Infection 37

detection of streptococci, lactobacilli, actinomyces, and bifidobacteria, whereas some molecular analyses show lower detection of actinomyces and bifidobac- teria. However, next generation sequencing studies that increase the depth of coverage from the micro- biota compared to clonal, probe, and cultural analy- ses (Siqueira et al. 2012) have the potential to bypass method differences. Recently, a metagenome study using 454 pyrosequencing (Belda-Ferre et al. 2012) showed clear differences between the microbiota of caries and caries-free subjects although identifications were mainly only to genus level. Similarly, metatran- scriptomic analysis of the microbial community of dental caries has provided new insight into the dynam- ics between bacteria and bacterial products that lead to tooth demineralization and cavitation (Burne et al. 2012; Zaura 2012; Nyvad et al. 2013). For exam- ple, sequence-based data showed an overrepresen- tation of mixed-acid fermentation and DNA uptake and competence in caries plaques, compared with an underrepresentation of different genes in samples from caries-free subjects (Belda-Ferre et al. 2012). Although gene activity was not linked to individual taxa, this approach has great potential in determin- ing caries-associated activity of the caries community compared with assaying individual species or limited combinations of species.

3.4 Microbial invasion of dentinal tubules

Several studies have demonstrated that only a subset of species can enter dentinal tubules, infect the root canal system, and invade radicular dentinal tubules (Kantz and Henry 1974; Sundqvist 1976; Dahlén and Bergenholtz 1980). In vivo studies showed that bacteria penetrated into the tubules of noncarious coronal dentin exposed to the oral environment and that invasion of tubules occurs readily within a week of exposure (Lundy and Stanley 1969; Olgart et al. 1974). With time, the numbers of tubules infected and the depth of infection increases (Lundy and Stanley 1969). The pattern of invasion is characterized by variable numbers of tubules penetrated and variable depths of penetration among different areas of dentin (Tronstad and Langeland 1971; Olgart et al. 1974). Inflammatory changes within the pulp are commonly observed and can be seen within a week of exposure (Olgart et al. 1974). The composition of the microflora invading exposed noncarious dentin has not been fully

elucidated but was dominated by Gram-positive taxa (Lundy and Stanley 1969; Brännström and Nyborg 1971; Tronstad and Langeland 1971; Vojinovic et al. 1973; Olgart et al. 1974) and probably resembles the composition of the deep dentinal caries (Table 3.1). Similarly, the microflora of radicular dentinal tubules infected from the root canal resembles that of deep dentinal caries with species of the genera Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus, Actinomyces, Propionibacterium dominanting the microflora (Peters et al. 2001).

3.4.1 Colonization of dentinal tubules

When caries, trauma, or restorative or periodontal pro- cedures breach the integrity of enamel or cementum the underlying dentinal tubules are prone to bacte- rial invasion. This process follows the principles of colonization and recent evidence demonstrates these principles.

Adhesion to dentin usually requires the cell to attach to some proteinaceous portion of the dentin matrix such as deposited salivary or tissue proteins or glycoproteins (conditioning film) or to components within the matrix. For example, dentinal tubules con- tain unmineralized collagen (Dai et al. 1991) and oral streptococci bind to collagen type I when adsorbed onto hydroxyapatite surfaces (Liu et al. 1990a,b), to unmineralized collagen, and to root dentin (Swital- ski et al. 1993). Binding to collagen was shown to be mediated by expression of oral streptococci antigen I/II polypeptide adhesins on the surface of the bacteria (Love et al. 1997), and it has also been shown that these polypeptides are necessary for bacterial invasion of dentin (Love et al. 1997). Similarly, experiments using Enterococcus faecalis mutants deficient in ser- ine protease and the collagen-binding protein (Ace) demonstrated that these molecules contribute to cell adhesion to radicular dentin (Hubble et al. 2003). Thus, evidence suggests that bacterial cell recognition of collagen may facilitate bacterial adhesion to exposed dentin or cementum (Figure 3.5), upregulate produc- tion of antigen I/II polypeptide, and induce a morpho- logic growth response, manifested by long-chaining of streptococcal cells, facilitating tubule invasion (Love et al. 1997) (Figure 3.6).

Nutritional supply within a dentinal tubule may influence the depth of bacterial penetration. This is partly dependent upon the patency of the tubule as diffusion of substances into dentinal tubules from the

38 Endodontic Microbiology

Fig. 3.5 Transmission electron micrograph demonstrating a colony of bacterial cells invading a radicular dentinal tubule. Note the close approximation of peripheral cells of the colony with the wall of the tubule indicative of cell attachment to tubule structure, an essential step in colonization. Source: Love 2004. Reproduced with permission of John Wiley and Sons.

oral cavity, pulpal fluid, or periradicular tissues is pro- portional to tubule diameter. This may account for the higher numbers of cariogenic bacteria present within superficial dentin (Edwardsson 1987), where the lev- els of fermentable carbohydrates and oxygen from the oral cavity are likely to be higher than in deeper dentin. Similarly, the anaerobic environment and pos- sible presence of tissue components (e.g., hemin) within coronal or radicular dentin close to the pulp space is likely to favor growth and survival of fas- tidious organisms such as Prevotella intermedia and Peptostreptococcus micros (now Parvimonas micra) (Love 2007).

Studies demonstrate that bacteria may compete or cooperate in invasion of dentinal tubules. This may be the result of a number of factors such as bacterial-induced alteration of the tubule environ- ment (e.g., oxygen tension facilitating or inhibiting growth of other organisms), bacterial production of bacteriocins that will inhibit growth of other bacte- ria, or cooperative bacterial coaggregation reactions. Nagaoka et al. (1995) showed that invasion of dentin by Streptococcus sobrinus or Actinomyces naeslundii was inhibited in the presence of Lactobacillus casei while invasion by L. casei was enhanced. Similarly, it has been shown that dentinal tubule invasion by Porphy- romonas gingivalis was promoted when cocultivated with S. gordonii but not in the presence of S. mutans. This invasion pattern was related to a specific coag- gregation interaction between S. gordonii and P. gin- givalis cells mediated by the streptococcal antigen I/II polypeptides promoting tubule invasion by P. gingi- valis (Figure 3.7), while the antigen I/II polypeptide SpaP of S. mutans did not have the same binding capacity or invasive affect on P. gingivalis, indicating a species-specific function of antigen I/II polypeptides (Love et al. 2000).

The pulp–dentin complex defensive processes can effectively inhibit bacterial invasion of dentin; these are particularly active when a vital pulp is present. However, as the pulp loses structure and function as a result of the bacterial insult, such as from the effect of bacterial products like lipopolysaccharide, bacteria are able to invade more readily. The contents of a dentinal tubule have an important role in defense functions and colonization of a tubule. Although the

Fig. 3.6 A model for tubule invasion by primary colonizing streptococci. Cell surface adhesins on S. gordonii attach the cells to unmineralized collagen within a tubule and the cells undergo a series of reactions resulting in chaining growth and invasion of the tubule. Source: Love 2002. Reproduced with permission of John Wiley and Sons.

Microbiology of Dental Caries and Dentinal Tubule Infection 39

Fig. 3.7 A model for tubule invasion by secondary colonizers. Secondary colonizers (P. gingivalis) may be able to attach to components of the dentine matrix but this does not allow them to invade a tubule (1, 2). The cells instead attach to primary colonizing bacteria (S. gordonii), which allows them to invade a tubule (3, 4). Source: Love 2002. Reproduced with permission of John Wiley and Sons.

composition of dentinal tubule fluid in vital dentin is not fully known, it resembles serum with proteins such as albumin and immunoglobulin G (IgG) being present (Knutsson et al. 1994), additionally other blood pro- teins, such as fibrinogen, may be found in dentinal tubules after cavity preparation (Knutsson et al. 1994; Izumi et al. 1998). Similar molecules, derived from fluid originating from alveolar bone and periodontal ligament and saliva, are present in nonvital radicu- lar and coronal dentinal tubules. It has been shown that albumin, fibrinogen, and IgG present within denti- nal tubules decrease fluid flow through dentin in vitro (Pashley et al. 1982; Hahn and Overton 1997) and inhibit bacterial invasion of radicular dentinal tubules (Love 2002) by interacting with bacterial cells or physically occluding tubules and reducing dentin per- meability. Bacterial cells within dentinal tubules also reduce dentin permeability (Michelic et al. 1980; Love et al. 1996) and this would suggest that bacteria them- selves would inhibit subsequent invasion; however, reduced fluid flow might promote disease pathogen- esis by allowing increased diffusion of destructive or toxic bacterial products towards the pulp and/or peri- radicular tissues (Pashley 1996).

These studies demonstrate that tubule invasion and development of the intertubular bacterial flora follows that seen in colonization of tooth surfaces and the

formation of plaque biofilms; that is, initial attachment and colonization by primary streptococcal colonizers, which then allow colonization by late colonizers such as P. intermedia and P. gingivalis. It is highly likely that other bacterial interactions between host proteins and other bacteria influence tubule invasion.

The degree of dentin permeability influences bac- terial invasion and varies between different areas of a tooth, with the number of patent dentinal tubules present, and by tubule contents (Pashley 1990). The diameter of dentinal tubules is normally larger than that of bacteria and does not physically impede invasion; however, it determines the rate of solute diffusion (e.g., nutrients and waste products; Pashley 1992) and this influences the depth of bacterial invasion. The ongo- ing physiologic sclerosis of laying down intratubu- lar dentin results in a decrease in size, and ultimately obliteration, of the dentinal tubules, with about 40% decrease in the overall numbers between the ages of 20 and 80 years (Nalbandian et al. 1960; Tronstad 1973; Carrigan et al. 1984). Sclerotic or obliterated tubules will physically impede bacterial invasion and reduce the diffusion of bacterial and tissue products through dentin. The mean numbers of tubules at any given age within coronal, cervical, and mid-root dentin are simi- lar (Carrigan et al. 1984). However, significantly fewer dentinal tubules are found in apical dentin (Nalbandian

40 Endodontic Microbiology

(a)

(b)

Fig. 3.8 Regional variation in bacterial invasion of radicular dentine of an upper left canine with infection of the root canal system and resulting periapical inflammatory disease. Bacterial colonization of the root canal from a coronal to apical direction and more advanced dentinal tubule sclerosis in the apical radicular dentin results in low and superficial invasion of apical dentine (a) and heavy and deep invasion in mid-root and cervical radicular dentin (b).

et al. 1960; Carrigan et al. 1984), indicating that the formation of intratubular dentin occurs more rapidly in the apical region of the root and progresses towards the crown as a tooth matures (Nalbandian et al. 1960). The pattern of dentinal tubule sclerosis results in regional differences of invasion between different areas of a root. Invasion of cervical and mid-root radicular denti- nal tubules occurs readily while the extent and depth of invasion are significantly less in apical tubules (Love 1996a) (Figure 3.8).

Intact cementum is crucial to limiting bacterial inva- sion of radicular dentinal tubules from the pulpal surface of an infected root canal system. Bacterial penetration is enhanced when the overlying cemen- tum is resorbed (Valderhaug 1974; Haapasalo and Ørstavik 1987; Love 1996a), a common occurrence in the presence of inflammatory periapical disease (Fig- ure 3.9) and after traumatic injuries that damage the periradicular tissues. Thus, dentin infection will be heavier and deeper in areas where the cementum has been resorbed and diffusion of bacterial prod- ucts toward the periradicular tissues will be enhanced

resulting in an increased periradicular inflammatory response.

Failure to eradicate microbes from infected radicu- lar tubules may result in persistent root canal infec- tion (Haapasalo and Ørstavik 1987; Ørstavik and Haapasalo 1990). It is noteworthy that Enterococ- cus faecalis and other bacterial species, such as S. gordonii/sanguinis, Streptococcus mitis, P. micra, A. naeslaundi, and P. intermedia, which are identified in cases with persistent disease, possess the ability to invade dentinal tubules as mono-cultures in ex vivo experiments (Love 2001, 2004). For bacteria to be involved in the pathogenesis and maintenance of per- sistent apical periodontitis they must be able to sur- vive in the inhospitable environment of the filled root canal where the nutrient supply is limited. Investiga- tors have focused on E. faecalis in an attempt to elicit mechanisms involved in pathogenesis. Studies have shown that E. faecalis is able to withstand a high alka- line environment such as produced in radicular dentin by calcium hydroxide (Haapasalo and Ørstavik 1987), which appears to be related to a cell proton-pump

Microbiology of Dental Caries and Dentinal Tubule Infection 41

Fig. 3.9 Photomicrograph showing resorption of cementum and dentin and exposure of dentinal tubules to the periradicular tissues. As a consequence, the tubules are patent on both the pulpal (inner) and external dentin surfaces and this results in enhanced permeability of the dentin and deeper bacterial invasion from the pulpal to the external root surface (haematoxin and eosin stain, x 200 magnification).

that is necessary for its survival at high pH (Evans et al. 2002), and can form biofilms in calcium hydrox- ide medicated canals (Distel et al. 2001). In addition, under starved conditions it shows resistance to sodium hypochlorite (LaPlace et al. 1997), heat, hydrogen per- oxide, acid, and ethanol (Giard et al. 1996). E. fae- calis can also survive extended periods of starvation in water (Hartke et al. 1998) and within water-filled dentinal tubules (Ørstavik and Haapasalo 1990), and human serum (Love 2001), which likely reflects the nutritional supply within nonvital radicular dentinal tubules. The upregulation of stress-induced proteins has been shown to be important for cell survival in a stressed environment (Hartke et al. 1998). It is highly likely that bacterial cells within dentinal tubules would be in a state of starvation and some of these mecha- nisms may come into play. It is suggested that follow- ing root canal therapy this ability may allow residual bacterial cells in radicular dentinal tubules to recolo- nize the obturated root canal and participate in chronic failure of endodontically treated teeth.

Bacterial invasion of radicular dentin from the peri- odontal pocket of periodontally diseased teeth has been demonstrated by light microscopy (Kopczyk and Con- roy 1968; Langeland et al. 1974; Adriaens et al. 1987a) and by microbiological studies (Adriaens et al. 1987b; Giuliana et al. 1997). The majority of species recov- ered from radicular dentin are Gram-positive bacte- ria (P. micra, Streptococcus intermedius, and A. naes- lundii), with lower numbers of Gram-negative organ- isms (P. gingivalis, P. intermedia, Tannerella forsythia, F. nucleatum, and Veillonella parvula (Giuliana et al. 1997).

While it is clear that bacteria are able to invade radicular dentin from the periodontal pocket, it is not clear whether bacteria invade healthy cementum prior to dentin penetration or if bacteria gain assess to dentin only via breaches in the cementum layer. A number of studies have described invasion of the cementum of periodontally diseased teeth (Daly et al. 1982; Adri- aens et al. 1987a,b; Giuliana et al. 1997). However, it was not evident from any of these studies if the invaded cementum was intact, healthy, or diseased. Exposed cementum is a thin, often discontinuous layer (Moskow 1969), and commonly shows surface defects (e.g., at sites where Sharpey’s fibres attach to the cementum matrix; Adriaens et al. 1987a). Exposure of cementum to crevicular fluid, bacterial enzymes, or acidic metabolites may induce physicochemical and structural alterations, such as localized resorptive lacu- nae or demineralization (Daly et al. 1982; Eide et al. 1984; Adriaens et al. 1987a). It seems likely therefore that bacterial invasion of exposed cementum associ- ated with periodontal disease occurs after the cemen- tum has been altered by physiological, bacterial, or environmental factors. Similarly, removal of cemen- tum as a consequence of periodontal treatment (e.g., scaling) will enhance bacterial invasion of radicular dentin.

3.5 Clinical aspects of dental caries microbiota and dentinal tubule infection

Treating progressing and recurrent dental caries is a significant clinical challenge. The polymicrobial

42 Endodontic Microbiology

infections of dental caries, which may or may not be characterized by S. mutans, suggest that new strategies are needed in caries management. If microbial mon- itoring is indicated, then current data would suggest that relying on screening only for S. mutans is not suffi- cient. PCR assays have been developed for many of the caries-associated taxa, including mutans streptococci, several Lactobacillus species, and Gram-positive rods in the actinomyces/bifidobacterium families, including the development of multispecies microar- rays, suggesting the potential for improved microbial assessment to assist determining microbial end points of therapy, as has been available in periodontology. Other clinical considerations are treating dentinal tubule infection that has extended from deep carious lesions, secondarily from endodontic or periodontal infections.

3.5.1 Invasion of coronal dentin: influence on the progression and management of pulp disease

Bacterial invasion through coronal dentin is the pri- mary route of infection of the pulp and root canal system. Bacteria may enter the root canal system directly via carious lesions or via pulp exposure following trauma. However, many infections of the pulp occur as a result of supragingival or subgingi- val bacteria penetrating exposed dentin, enamel-dentin cracks, and around restorations (Pashley 1990; Love 1996a; Peters et al. 2001) and then invading denti- nal tubules. Microleakage is defined as the clinically undetectable passage of bacteria, fluids, molecules, or ions between a cavity wall and the restorative material applied to it (Kidd 1976). Studies have demonstrated that microleakage of oral bacteria around restora- tions allows bacterial invasion of exposed dentinal tubules at the base of the cavity (Brännström and Nyborg 1971; Vojinovic et al. 1973) resulting in pul- pal inflammation (Vojinovic et al. 1973) or periapical disease (Ray and Trope 1995). Likewise, microleak- age through enamel cracks and fractures as a result of trauma may lead to bacterial invasion of the pulp– dentin complex and act as a cause of endodontic dis- ease (Love 1996a). Hence, sealing of dentin from exogenous substances and bacteria in the oral cavity, in both vital and nonvital teeth, is a critical step in tooth restoration.

3.5.2 Invasion of radicular dentin: influence on the progression and management of periapical disease

The principles of complete debridement of vital and necrotic pulp tissue, removal of microorgan- isms and affected dentin, and disinfection of the root canal system and radicular dentin are cornerstones in successful management of periapical disease of endodontic origin. The ability to achieve these aims is partly dependent on the choice of root canal prepa- ration technique and disinfection regimen (for a more extensive discussion of endodontic disinfection pro- cedures see Chapter 13). Here, the basic principles of canal preparation and disinfection as they relate to radicular dentinal tubule infection are outlined.

A preparation technique that results in a flared root canal with minimal canal transportation is favored (Figure 3.10) as this results in the most effective debridement of the intraradicular space (Walton 1976) and infected radicular dentinal tubules, and produces a root canal shape that is conducive to filling and sealing the root canal (Ayar and Love 2004). In addi- tion, the regional differences in the pattern of tubule invasion in the root suggest that a technique that results in moderate apical preparation, because of mild and limited depth of dentin infection in this region, but that flares coronally debriding heavily infected mid-root and cervical dentin, should be employed (Love 1996b).

The complex anatomy of the root canal system cannot be cleaned by mechanical means alone. The concept of chemomechanical preparation, utilizing a chemical irrigant as an adjunct to mechanical instrumentation, maximizes soft tissue and microor- ganism removal. Traditionally, sodium hypochlorite (NaOCI) has been used to accomplish both functions (Byström and Sundqvist 1983) and is commonly used in solutions ranging from 0.5% to 6%. Its soft tis- sue dissolving and antimicrobial properties may be enhanced when the solution is warmed, similarly its antimicrobial effectiveness may also be improved in conjunction with ultrasonic energy.

Chemicals should also aid penetration of disinfec- tion agents into radicular tubules. Whenever dentin is cut or abraded a smear layer of debris packs into and occludes the dentinal tubules and cannot be readily removed (Love et al. 1996) (Figure 3.11). Dentinal smear layers decrease the area available for diffusion

Microbiology of Dental Caries and Dentinal Tubule Infection 43

Fig. 3.10 Radiograph of an endodontically treated upper right first molar with four root canals. Successful disinfection of the root canal and radicular dentin is dependent on chemomechanical instrumentation of all of the root canal system to produce a well-centred canal flaring from the apical terminus (A) to the canal orifice (B) at the pulp chamber. The root filling materials should three-dimensionally fill the root canal space and form an apical seal at the apical terminus and extend coronally to fill the root canal at or 1–2 mm short of the canal orifice; however, the root filling should not extend into the pulp chamber. A coronal seal (C) is formed over the coronal extent of the root filling using a restorative material such as a zinc oxide/eugenol or glass ionomer cement material and a well-sealed permanent restoration (D) that returns the tooth to function and form is placed.

and markedly reduce the permeability of dentin. This inhibits the ability of irrigants and intracanal medica- ments to diffuse into infected radicular dentinal tubules and eliminate microorganisms. To facilitate penetra- tion into radicular dentin the smear layer should be removed during chemomechanical preparation (Fig- ure 3.12). This is commonly accomplished using a

regime of ethylene-diamine-tetra-acetic acid (EDTA, 17% w/v), a chelation agent that dissolves the hard tissue components of the smear layer, alternating with sodium hypochlorite irrigation.

Chemomechanical preparation greatly reduces the number of bacteria in the root canal. However, to date no preparation technique or irrigation material can predictably render a root canal and radicular dentin sterile. As a consequence, bacteria can survive and multiply within the root canal system and dentinal tubules, necessitating the use of intracanal medicaments between endodontic appointments. Intracanal medicaments have been recommended for a number of different reasons; for example, to reduce periapical inflammation, induce healing of calcified tissue, eliminate apical exudate, and neutralize tis- sue debris. However, the main reason for their use is to eliminate residual bacteria within the root canal and dentinal tubules after chemomechanical prepara- tion. Clinicians should choose an intracanal medica- ment that is capable of disinfecting deeply infected dentin so as to effectively eradicate potentially heavy and deep infection of cervical and mid-root denti- nal tubules and deeply penetrating bacteria at sites associated with external resorption of the cementum. A number of intracanal medicaments are available; how- ever, calcium hydroxide is a popular medicament that has been shown to eliminate bacteria within dentinal tubules (Ørstavik and Haapasalo 1990) and effectively disinfect the prepared root canal system after 1 week (Sjögren et al. 1991).

However, after chemomechanical instrumentation and intraradicular dressing there may be residual viable bacteria within radicular dentinal tubules and under favorable growth conditions (e.g., nutritional supply from periradicular fluid) it takes only a few viable bacteria to subsequently reinfect the root canal system. As such, a root filling is placed within the root canal system (Figure 3.10) to control reinfection of the root canal from any residual bacteria. It does this primarily by producing an inhospitable environ- ment in the root canal and radicular dentin for bacte- rial growth; it denies bacteria nutrition by inhibiting microleakage into the root canal and tubules of tis- sue fluid that would sustain growth, it denies space for bacteria to multiply in (an essential part of colo- nization), and it alters the redox conditions within the root canal. There are numerous root filling techniques and materials and all conform to the same principles

44 Endodontic Microbiology

Fig. 3.11 A scanning electron micrograph of a dentin smear layer produced by instrumenting a root canal wall with an endodontic hand-file; note the presence of bacteria on the smear layer. The smear layer has occluded the underlying dentinal tubules and will inhibit the penetration of antimicrobial medicaments into dentinal tubules.

and attempt to produce a three-dimensional fill of the root canal that is closely associated with the radicular dentin so as to form a fluid-tight seal along the whole length of the root canal from the apical preparation (apical seal) to the canal orifice (coronal seal). The provision of a coronal seal (Figure 3.10) is mandatory to prevent reinfection from the oral cavity of the intraradicular space and radicular dentinal tubules both between endodontic appointments and after filling of the canal. A number of restorative materials can be used as a coronal seal.

3.5.3 Invasion of radicular dentin: influence on the progression and management of periodontal disease

The effect of periodontal disease and associated infected radicular dentin on the development of pulp

and periapical disease is not clear. The effects on the pulp are probably degenerative and inflammatory (Langeland et al. 1974); however, studies and clinical practice suggest that if pulpal pathosis does develop as a consequence of periodontal disease, it occurs as a result of exposure of a lateral/accessory canal (Rubach and Mitchell 1965) or develops late in the periodon- tal disease progression such as when it involves the apex of the tooth (Czarnecki and Schilder 1979). Sim- ilarly, it has been shown that the presence of periapical disease 4–7 years after root canal treatment was sig- nificantly associated with marginal bone loss >1/3 of the root length (Koch et al. 2014).

The effect of infected radicular dentin on pro- gression of periodontal disease may also be signifi- cant. It has been suggested that the dentinal tubule microflora associated with a periodontal pocket could act as a reservoir for recolonization of the pocket after

Fig. 3.12 A scanning electron micrograph of an instrumented root canal wall that was treated with an ethylene diamine tetraacetic acid/sodium hypochlorite regimen to remove the smear layer. The patent dentinal tubules allow maximum diffusion of antimicrobial medicaments into tubules to eradicate invading bacteria. Additionally, root canal filling materials can better form a seal with the root canal wall and occlude the tubules when the smear layer is removed.

Microbiology of Dental Caries and Dentinal Tubule Infection 45

debridement (Adriaens et al. 1987a,b; Giuliana et al. 1997). The presence of an infected root canal system and associated radicular dentinal tubules in treated periodontitis-prone patients was shown to result in increased pocket depth and radiographic evidence of loss of attachment (Jansson et al. 1993a,b), and impaired healing of pockets (Ehnevid et al. 1993). Sim- ilarly, the presence of a root canal infection in patients with periodontitis was associated with higher levels of attachment loss as assessed by probing depths and radiographs. It was suggested that a root canal infec- tion in periodontitis-involved teeth may potentiate pro- gression of the periodontal disease by spreading of endodontic pathogens through patent accessory canals and dentinal tubules (Jansson et al. 1995; Jansson and Ehnevid 1998). As a consequence, asymptomatic endodontic pathosis should be promptly treated in patients with periodontal disease, while the presence of an infected root canal system should be investigated in teeth where the response to periodontal treatment is less than expected or in teeth with isolated loss of peri- odontal attachment. Similarly, if advanced periodontal techniques are proposed (e.g., tissue regeneration) then the presence of root canal infection in associated teeth should be assessed and treated as necessary prior to the periodontal treatment.

3.6 Conclusions

Bacterial invasion of tooth tissue and the clinical con- sequences have been recognized for over a century. Updated microbiological methods incorporating 16S rRNA gene sequencing for bacterial identification have expanded our understanding of the microbiota of the oral cavity, including that of dental caries and denti- nal tubule invasion. There are several bacterial taxa that are associated with deep dentinal caries and pre- cursors to endodontic infection. Prevention of bacterial spread into the root canal from carious dentin or dentin infected from periodontal pockets will likely depend on the bacterial composition in these areas and the changes in environmental factors within the root canal system.

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Chapter 4 Culture-Based Analysis of Endodontic Infections Gunnar Dahlén

4.1 Introduction 4.2 Historical perspectives 4.3 Culture-based analysis in clinical

practice 4.3.1 Sampling 4.3.2 Transportation 4.3.3 Laboratory considerations 4.3.4 Sensitivity and specificity

4.4 Clinical interpretations 4.4.1 How to interpret the primary

infection 4.4.2 Interpretation of growth

during treatment 4.4.3 Use of specific antiseptics

and antibiotics 4.5 Route of infection in vital and necrotic

pulp 4.5.1 Root canals with vital pulps 4.5.2 Root canals with necrotic pulps

4.6 Apical periodontitis 4.6.1 Acute symptomatic infection 4.6.2 Bacteria and symptoms 4.6.3 Abscess and fistula formation 4.6.4 Chronic infection 4.6.5 The microflora of the root canal

versus the deep periodontal pocket

4.7 Treatment aspects 4.7.1 Why is it so difficult to eliminate

root canal microorganisms? 4.8 Persisting infections at root-filled teeth 4.9 Culture versus molecular biology

methods 4.10 Conclusions 4.11 References

4.1 Introduction

Much of our knowledge of the endodontic microflora is based on culture studies. This is simply because we had no real alternatives in the past. Microscopic stud- ies have been used; however, they have severe limita- tions when it comes to exact identification, to evaluate the composition, to characterize various microorgan- isms and to carry out further experimental studies on isolated species. Microscopy of smears from the root canal is limited to the main morphotypes. Microbial staining of histologic sections has the advantage of localizing the microbes in situ (Figure 4.1). Electron

microscopic imaging (transmission or scanning) has been valuable to distinguish main morphotypes in var- ious locations (Nair 1987). During the last decade, numerous studies using various types of molecular biology techniques have been used to characterize more closely the microbial composition of the root canal flora (see Chapter 5). These methods have def- initely showed that the root canal flora is much more complex than previously thought. This has made the clinical interpretations, diagnosis, and treatment strate- gies more difficult. Still, culture is a “gold standard” for dentists in the clinic to identify specific targets for treatment and to evaluate treatment strategies. This

52 Endodontic Microbiology

Fig. 4.1 Histologic section showing a stained (blue) microbial invasion into the root canal. Courtesy of Dr Dominico Ricucci.

chapter aims to describe the knowledge we have gained by culture studies for microbial composition in various phases of endodontic infection. Culture studies have the advantages of demonstrating the bacterial load, iso- lating bacterial strains for antibiotic susceptibility test- ing, and for examination of virulence factors. Culture is also used in experimental models, which have dis- closed the dynamics of the infection and the nature of the microorganisms. This is outlined, with the insight that we still do not have the complete picture of the microbiota and that new techniques will complement or even change our future opinions on the natural his- tory and complexity of the root canal flora.

4.2 Historical perspectives

In the late nineteenth century, Miller (1894) described how the open pulp chamber was filled with various bacteria. Endodontic problems were an early focus in the discussion on focal infections, and difficulties

in adequately diagnosing and treating endodontically involved teeth frequently led to extraction. Culture studies at this point recovered predominantly aerobic and facultatively anaerobic microorganisms. With the historical perspective in mind, we can today conclude that studies in the first half of the twentieth century were hampered by insufficient culture techniques, poor sampling methods, neglected antiseptic measures, and efforts to avoid contaminations (Figure 4.2). The microbial findings from infected root canals were pre- dominated by facultatives and by species and groups that today we regard as contaminants from the oral cavity or from the surroundings by careless handling in the dental office or laboratory. Anaerobes were sel- dom isolated from root canals during the first half of the twentieth century. Molecular methods (real time PCR, checker-board DNA-DNA hybridization, etc.) do not reveal the total microbiota unless a full sequencing of the recovered DNA is performed and are there- fore aimed at specific targets, for example, black- pigmented Gram-negative anaerobes (Gomes et al. 2005). It can be seen in Figure 4.2 that the low recov- ery or lack of streptococci/lactobacilli in some studies (Bergenholtz 1974; Sundqvist 1976) have used trau- matized teeth rather than teeth endodontically involved due to caries. Even if all these problems still exist, at least in part, a number of important milestone studies have been carried out, throwing light on the complexity of endodontic microbiology. The importance of ade- quate antiseptic measures, sampling procedures, trans- portation, and laboratory techniques (media, anaero- biosis, identification, and interpretation) were shown in the classic work by Möller (1966). He described how to avoid contaminations, how to take a relevant sample, the invention of the VMG III transport medium (today designated VMGA III after some later modifications; Dahlén et al. 1993), and the evaluation of various cul- ture media. His recommendations are still in use today.

Anaerobic techniques were introduced by several researchers in the 1960s and 1970s (Möller 1966; Bergenholtz 1974; Kantz and Henry 1974; Wittgow and Sabiston 1975; Sundquist 1976). The study by Sundquist (1976) illustrates well the complexity of root canal microbiology, as it utilized anaerobic techniques combined with a thorough phenotypical identification of the bacterial isolates. In this study, a number of bacterial species were discovered that were not previously detected in the root canal environment.

Kakehashi et al. (1965) is often mentioned as hav- ing performed the key study on germ-free animals

Culture-Based Analysis of Endodontic Infections 53

Som m

er and Crow ley 1940

M orse and Y

ates 1941

Brow n and Rudolf 1957

M cD

onald 1957

H obson 1959

W inkler and van A

m erongen 1959

Shovelton and Sidaw ay 1960

Engström and Frostell 1961

Craw ford and Shankle 1961

M öller 1966

Bergenholtz 1974

K antz 1974

W ittgow

1975

Sundquist 1976

G om

es et al. 2005*

Streptococci and Lactobacilli

Anaerobes

Other microorganisms

Uncultivated

Fig. 4.2 Microorganisms isolated by different investigators in initial samples from root canals with necrotic pulps. *Note that Gomes et al. (2005) only covers black-pigmented Porphyromonas and Prevotella species.

to prove that microbes were necessary for the devel- opment of apical periodontitis. Möller et al. (1981) showed in monkeys that infected teeth developed periapical lesions while noninfected teeth did not. The importance of anaerobes in the pathogenesis of root canal infection was further emphasized in experimen- tal studies (Figure 4.3) on monkeys by Fabricius et al. (1982a,b). Table 4.1 shows the microbiologic outcome in later studies on primary endodontic infections. The heterogeneity within and between the studies is quite substantial. However, the general trend is that 1–12

predominating species in each specimen are involved and the number of recovered cells varies from <102

to >108 (Sundqvist 1992b). Anaerobic streptococcal species, Gram-positive anaerobic rods, and species of Prevotella, Fusobacterium, and Campylobacter are usually present. Treponema species are also likely to be present; however, they are regularly missed in cul- ture analysis. The genus Treponema apparently holds a number of species previously not studied (see Chap- ter 5, Figure 5.6). Other bacteria primarily detected by molecular biology methods can be disclosed by culture

Fig. 4.3 Mean percentage of anaerobic and facultatives after different times of experimental infection. Source: Dahlén et al. (1982a). Reproduced with permission of John Wiley and Sons.

54 Endodontic Microbiology

Table 4.1 Frequency (percentage of total number of isolated strains) of microorganisms in root canal samples from teeth with necrotic pulps in some culture studies. Species >10% in bold

Wasfy et al. Sundqvist Le Goff et al. Lana et al. Peters et al. Gomes et al. Chu et al. Microorganisms (1992) (1992b) (1997) (2001) (2002) (2004) (2005)i

S. aureus – – – – – 0.6 – Other

Staphylococcus spp.

2 – 2 0.7 2 4 0.5

Streptococcus spp. (psp)a

6 4 1 4 – 11 2

Streptococcus spp. (non-psp)b

17 4 3 9 5 11 5

E. faecalis – 2 – 0.7 – 1 0.5 P. micrac 2 6 3 1 13 27 5 Other anae

streptococcid 0.4 9 – 7 2 – 16

Neisseria spp. – – – – – 0.6 5 Veillonella spp. 5 2 2 3 3 4 4 Bacillus spp. – – – – – – – Clostridium spp. – – – 8 – 2 1 Corynebacterium spp – – 6 – – – 2 Lactobacillus spp.e – 7 3 14 3 2 6 Propionibacterium

spp. 3 2 13 0.7 8 1 3

Actinomyces spp. 8 4 3 4 15 5 12 Eubacterium spp.f 20 17 7 2 7 4 4 Enteric rods – 0.3 – – – – – Capnocytophaga spp. – 2 7 0.7 7 0.6 5 Campylobacter spp.g – 5 12

C. gracilis – – 2

C. gracilis 8

Eikenella spp. – 0.3 – – – – 0.5 Porphyromonas spp. 2 9 2 – – 2 3 Prevotella

intermedia/ nigrescens

12 6 2 6 15 5 4

Other Prevotella spp.h 14 6 14 9 11 8 18 Fusobacterium spp. 6 14 7 8 8 11 6 Spirochetes – – – – – – – Candida spp. – – – 2 – – – Total number of

isolated strains 259 353 84 138 131 171 395

Number of teeth 85 65 26 31 58 41 88

aPolysaccharide producing (psp) streptococci including S. sanguis, S. salivarius, S. mutans, S. oralis, S. mitis. bNon-polysaccharide producing (non-psp) streptococci including S. anginosus, S. constellatus, S. intermedius, Gemella morbillorum. cParvimonas micra, formerly Micromonas micros, earlier Peptostreptococcus micros. dOther anaerobic streptococci including peptostreptococci, peptococci, Finegoldia spp. eLactocbacillus includes both anaerobic and facultative species, Olsenella uli and Bifidobacterium spp. fEubacterium also including Colinsella aerofaciens, Eggerthella lenta, Filifactor alocis. gCampylobacter includes formerly designated Wolinella spp. and Bacteroides gracilis. hOther Prevotella includes both pigmented and nonpigmented species and Bacteroides species such as B. capillosus, B. uniformis, and others. iChu et al. (2005) includes both samples from root canals “exposed” and “non-exposed” to the oral cavity.

Culture-Based Analysis of Endodontic Infections 55

Prewashing with H2O2 and taking away the surface of the sealing cement

Sterilization of the operation field

Inactivation of the iodine

Control of the sterility

Thiosulphate solution

Iodine tincture

Fig. 4.4 The main steps in the preparation and sterilization of the operation field before entering the root canal. Drawing by Mrs. Gunilla Hjort.

analysis once you know how to grow and identify them. Such bacterial species as the Gram-negative Dialis- ter spp., Selenomonas spp., and Catonella morbi, and the Gram-positive Filifactor alocis, Pseudoramibacter alactolyticus, Slackia exigua, Mogibacterium timidum, Granulicatella adiacens, and so on, are found in acute dental abscesses (Robertson and Smith 2009; Siqueira and Rôças 2009). It is clear that the microflora varies between studies because of a number of factors; for example, the diagnosis, type of teeth, sampling pro- cedures, and laboratory measures. Conclusively, the primary endodontic infection is a polymicrobial, pre- dominantly anaerobic, infection with little microbial specificity.

4.3 Culture-based analysis in clinical practice

4.3.1 Sampling

A critical measure for correct sampling and avoiding false positives is the sterilization and control of the operative field (Figure 4.4). After rubber dam application, prewashing with hydrogen peroxide (30%) should be carried out in order to make the following sterilization procedure more efficient. Leakage between the rubber dam and the tooth must be carefully controlled. Disinfection of the operative field is performed by flushing with 10%

56 Endodontic Microbiology

Remove the sealing cement

Add sampling solution (VMGA I) and execute pumping movements with a file

Repeat the procedure

Sampling with charcoaled points until all liquid is absorbed

All points are transferred to transportmedium VMGA III

Fig. 4.5 Microbiologic sampling from the root canal. Drawing by Mrs. Gunilla Hjort.

iodine tincture. The surface layer of the temporary filling can preferentially be removed and the procedure repeated to eliminate microorganisms at the borderline between the filling and the tooth. Likewise, if caries or defective restorations are present, these should be removed, and the sterilization protocol repeated. Möller (1966) recommended taking a control sample from the operative field in order to check sterility. This is especially recommended for the inexperienced dentist to learn how to avoid contaminations and to work aseptically. If a control sample is taken, an inactivation procedure of iodine with 5% thiosulfate solution should be carried out to avoid false negatives,

for example, viable but not cultivable bacteria that may be bacteriostatically affected by iodine.

The root canal sample is taken after removing the temporary filling (Figure 4.5). Interappointment dress- ings should be removed by irrigation using saline or sampling solution (VMG I; Möller 1966). Executing pumping movements with a file is recommended in order to obtain a suspension with bacteria from the root canal wall, dentine, and apex delta (Figure 4.6). Sam- pling is then performed using charcoaled paper points that are transferred to a transport medium; for exam- ple, VMGA III (Möller 1966; Dahlén et al. 1993). The sampling procedure is repeated with additional points

Culture-Based Analysis of Endodontic Infections 57

Fig. 4.6 A molar tooth indicating the location where special attention has to be taken for reaching bacteria at sampling. (a) Longitudinal section. (b) Horizontal or cross-sectional view of a lower molar tooth.

until all liquid is absorbed. It should be noted that the last point is the most important because it will absorb the liquid from the most distant areas of the apical region.

4.3.2 Transportation

The purpose of a transport medium VMGA III is to keep the viability of the microorganisms during trans- portation and being bacteriostatic in the sense that no multiplication takes place. The VMGA III medium has also a general inactivating ability, which inactivates medicals and antiseptic substances used in the root canal, which otherwise would prevent bacterial cells from growing in the laboratory media. Furthermore, the VMGA III medium contains reducing substances (cysteine) to keep the medium from being oxygenized and a redox indicator showing that this does not happen. VMGA III cannot be obtained commercially; however, a full description of composition and procedures has been published (Dahlén et al. 1993).

4.3.3 Laboratory considerations

The goals of the laboratory measures are as follows:

� To decide whether viable bacteria are present in the sample

� To identify microorganisms in the sample according to species or genus levels that are of clinical impor- tance

� To obtain a semiquantitative measure of bacterial load in the sample.

A fairly simple methodology that could be used by most laboratories is shown in Figure 4.7. This pro- vides the clinician with important information for the diagnosis of infection indicated by growth/no growth, and for treatment decision by the number and type of microorganisms present.

Liquid media are primarily recommended because they most often allow fastidious and/or dormant bacterial cells to grow. For practical purposes, media such as thioglycolate, trypticase broth, or brain heart infusion broth could be used. However, it is clear that more sophisticated media such as HCMG SuIa (Möller 1966) give a higher frequency of samples with growth. The liquid media also have the advantage that no extra equipment is necessary for the anaerobic incubation if the tubes are flushed with oxygen-free gas (e.g., nitrogen) when inoculated. If the tubes are prepared under anaerobic conditions, the sample is inoculated in the bottom layer of the tube, and the tubes are capped tightly (rubber stopper), the medium itself will ascertain anaerobic conditions even for the most oxygen-sensitive bacterial species. An important factor for the growth of a sample with few bacterial cells, sometimes in a “bad” condition, is to allow them sufficient incubation time. In the Laboratory of Oral Microbiology at University of Gothenburg, we give a preliminary reply to the dentist after 5–6 days on growth/no growth and then continue the incubation for 14 days. If growth appears after the preliminary reply has been delivered, the dentist is informed by a phone call.

Solid media (e.g., Brucella blood agar supplemented with hemolyzed blood and vitamins such as hemin and menadione), one for aerobic incubation and one for

58 Endodontic Microbiology

Fig. 4.7 The laboratory procedures of root canal samples in the Laboratory of Oral Microbiology at Göteborg University. Psp, polysaccharide producing.

incubation in anaerobic jars, are used to complement the liquid medium in order to disclose the diversity and presence of various microbial species and to give a semiquantification of each colony morphology type. Specification to genus or species level is based on Gram stain, selective media, and simple biochemical tests. In practice, a detailed specification to species level on all present bacteria is not necessary as this will have little impact on the diagnosis and the choice of treatment procedure. The final reply after 14 days also includes the finding recovered from the solid media including semiquantification.

4.3.4 Sensitivity and specificity

The sensitivity of cultural methods is fairly good and acceptable for practical purposes as long as it is car- ried out appropriately. Molecular biology methods have disclosed more microbial species. Many of these represent not-yet-cultured species but in other cases they may also represent nonviable (dead) cells or even remaining DNA. The persistence of dead-cell bacterial DNA has shown to last up to 2 years in root canals, suggesting preservation by binding to hydroxylapatite (Brundin et al. 2010, 2013). If so, this will increase the

false positive rate using molecular biology methods and decrease their sensitivity. The sensitivity calcula- tions for each method will be hampered by its limi- tations. For the more easily cultured species such as facultatives, theoretically only one viable cell is needed for growth in the liquid medium or to form a colony on the agar plate. In that sense, the sensitivity of the culture analysis should be regarded as high. However, it should be noted that in samples with a high num- ber of bacterial cells, such as in primary infected and untreated teeth, bacteria in low numbers will not be detected because of dilution or overgrowth of predom- inant species. An important factor for the sensitivity, irrespective of the detection method used, is the sam- pling method and how well the present microorgan- isms can be reached and sampled (Figure 4.6). The sensitivity of the complete procedure including both sampling and analyses is complex and cannot be fully evaluated.

A sample showing no growth (negative sample) indi- cates that the root canal is free from microorganisms and the treatment goal is achieved. To avoid false neg- atives, an adequate and representative sample must be taken, which might be somewhat demanding. During treatment, microorganisms are most easily eliminated

Culture-Based Analysis of Endodontic Infections 59

from the main root canal while it is much more difficult to eradicate them from dentinal tubules, lateral canals, and apical deltas (Figure 4.6). All possible precautions should be taken for transportation and culture to give viable and cultured bacteria the best chance to grow. Still, there is the problem with uncultivable or difficult to culture microbial cells that give an unknown num- ber of false negatives. Future studies should focus on whether remaining uncultivable microorganisms are of significance for failing outcomes of the endodontic treatment.

False negative samples are especially difficult to avoid when taking samples at revision of a previously root-filled tooth. Even if the gutta-percha or sealer is removed mechanically, the remaining bacteria can hide in peripheral parts of the root canal system and may be in a stressed situation, which does not allow them to grow instantly in the laboratory. This reason for false negative samples is rather likely when using dressings and antiseptic irrigations that do not kill the microorganisms but leave them in a dormant phase or in a biofilm (Figure 4.8). This can be avoided by the

Fig. 4.8 Hypothetical outline of a stress protein response in biofilm communities of root canal bacteria. The stress induces production of stress proteins, which are released into the biofilm matrix. These proteins will provide beneficial effects for the community. Source: Chavez de Paz (2004). Reproduced with permission of John Wiley and Sons.

60 Endodontic Microbiology

use of specific or unspecific inhibitors. Such inhibitors are sodium thiosulfate (5%) for halogen-containing antiseptics (iodine and chlorine), L-alpha lecithin in Tween 80 for chlorhexidine (Zamany and Spangberg 2002), or more unspecific inactivating agents such as charcoal or VMGA III. Another possibility is to leave the canal free from dressings between appointments. It was shown by Reit and Dahlén (1988) that a sec- ond sample (true sample) did show more culture posi- tives than a first sample (indicator sample), indicating a significant risk of false negatives if the sample is taken immediately after removing the Ca(OH)2 paste. A third possibility for a false negative sample is when the bacteria are retained on the external root surface, in the apical root cementum, or in resorptions around the apical orifice of the root canal (Figure 4.9). These bacteria cannot be reached by sampling through the root canal but only by surgical access to the root tip (see Chapter 6). This can only be performed in specific nonhealing cases, and is generally not a recommended procedure in the general dental practice.

Specificity in endodontic sampling is high because the number of false positives can be significantly reduced and controlled. A false positive test means that the samples show growth by contaminating microor- ganisms of various kinds. The most common reason is probably an inadequately sterilized operative field or leakage despite rubber dam application. It was rec- ommended by Möller (1966) to take a separate sam- ple from the operative field as a control of the anti- septic technique used. Bacteria present in the saliva and plaque may appear in the operative field sam- ples and if they occur concomitantly in the root canal sample, a contamination can be suspected. Facultative anaerobic species such as polysaccharide-producing streptococci (S. mutans, S. sanguis, S. oralis, and S. salivarius), Corynebacterium spp., Neisseria spp., and Haemophilus spp. are oral bacteria known not to establish themselves in the anaerobic and nonsaccha- rolytic environment in the root canal and are thereby strongly indicative of a leakage. If, by semiquantitative culture, the bacterial growth is still heavy, despite root

Fig. 4.9 Histologic section of the apical region of a root showing resorbtion where present bacteria will be difficult to sample. Source: Chavez de Paz (2004). Reproduced with permission of John Wiley and Sons.

Culture-Based Analysis of Endodontic Infections 61

canal treatment being initiated, a leakage to the oral cavity is likely because of remaining fillings, crown, and bridges, fractures, or inadequate temporary fill- ings. Presence of micrococci, coagulase-negative staphylococci, spore-forming bacteria (e.g., Bacillus spp.), and enteric rods are most likely contaminants by careless handling of the samples in the office or laboratory (Table 4.2). It should be noted, however, that enteric rods and Staphylococcus aureus, some- times although rarely, can in the root canal infection usually as monoinfections. Such infections are impor- tant to disclose because serious complications (e.g., osteomyelitis and other acute dentoalveolar infections) may follow and special treatment strategies have to be considered.

4.4 Clinical interpretations

4.4.1 How to interpret the primary infection

The reason for taking a sample from a primary infected tooth is not always rational. An untreated root canal with a necrotic pulp and an apical lesion, with or without symptoms, is always infected. We know that in most of these cases the microflora is polymicro- bial, predominantly anaerobic, and treatment proce- dure using mechanical debridement and antiseptic irrigation is the first choice. In an acute infection with general symptoms and risk of spreading, the administration of systemic antibiotics must be done instantly and based on the infection, usually anaero- bic, and sensitive for penicillins and/or metronidazole. There is generally little clinical benefit of a micro- biologic sample in primary endodontic infections. In refractory acute infections, a microbiologic diagno- sis can be performed in order to disclose infections with more virulent microorganisms such as S. aureus and enterics, which may need a different treatment strategy.

4.4.2 Interpretation of growth during treatment

A microbiologic sample taken during treatment as a control for bacterial growth may be important. Some studies have shown that if there is absence of viable bacteria prior to filling, the prognosis of root canal treatment (Sjögren et al. 1997) or retreatment (Sundqvist et al. 1998) is improved. If cultivable

bacteria persist in the canal, the long-term outcome may depend on the quality of the root canal filling (Möller et al. 2004; Fabricius et al. 2006) and the healing of lesions may be delayed (Waltimo et al. 2005). The use of intracanal medicaments between appointments improves the chances of bacterial elimination, but does not guarantee it (see Chapter 13). If the bacterial sample shows a low number of persisting bacteria, additional antiseptic procedures and interappointment dressings should be considered. If a high number of bacteria of polymicrobial and anaerobic nature still are present, a leakage (through rubber dam, fractures, remaining fillings, and crowns) should be suspected. In rare cases, more specific infections, for example, S. aureus, enteric rods, and Candida spp., could be disclosed, which need special considerations and treatment procedures.

4.4.3 Use of specific antiseptics and antibiotics

The choice between various antiseptics may, to a cer- tain degree, be based on the type of remaining bac- teria. Anaerobes are usually very sensitive for most antiseptics used for irrigation and as interappointment dressings, and no specific considerations are neces- sary. It should be remembered that irrigation is usually of short duration and the effect is limited compared to an interappointment dressing that can extend its effect over days. A very important basis for the antiseptic effect is that as much organic materials (necrotic pulp material and microorganisms) as possible should be removed. Otherwise, this will rapidly inactivate any antimicrobials. Calcium hydroxide paste has become useful as an interappointment dressing because of its ability to fill up the root canal lumen and prevent the remaining bacteria from growing. However, calcium hydroxide has a rather weak (bacteriostatic) antimi- crobial effect and many bacteria may survive, espe- cially Gram-positive facultative species. If such bacte- ria remain in the root canal, other antiseptics such as iodine or chlorhexidine should be considered.

There is no antibiotic that is efficient for all types of microorganisms occurring in the infected root canal and local antibiotics in the canal are therefore not recommended at this point. In primary acute infec- tions with pus formation, antibiotics may be used systemically in order to prevent spreading of the infection. As the main character of this infection is polymicrobial, predominantly anaerobic, penicillin or

62 Endodontic Microbiology

Table 4.2 Clinical significance of various microorganisms occurring in endodontic samples

Significance in endodontic samples

Resistance to Risk of Microorganisma Frequency Pathogend treatmente contamination Main appearance

Micrococci Staphylococcus aureus Staphylococcus epidermidis

+ + +

– +++

–

– +++

+++ ++ ++

Careless handling in clinic or in laboratory

Streptococci (polysaccharide- producing) S. mutans S. salivarius S. sanguis

+ + ++ +++ Primary sample, through leakage or saliva contamination

Streptococci (other) S. anginosus (S. milleri) S. oralis (S. mitior) S. mitis S. intermedius

+++ ++ ++ + Primary samples, persistent disease and root-filled teeth with apical periodontitis

Enterococci E. faecalis

++ + +++ + Persistent disease and root-filled teeth with apical periodontitis

Peptostreptococci (Parvimonas micra)b

++ ++ + – Primary samples and abscesses

Other anaerobic streptococci

++ ++ + –

Gram-negative cocci Neisseria spp. (+) – – +++ Saliva contamination Veillonella spp. + + – ++ Primary samples or

through leakage Sporeformers

Bacillus spp. (+) – ? +++ Careless handling in clinic or in laboratory

Gram-positive rods Corynebacterium spp. Actinomyces spp. Lactobacillus spp. Propionibacterium spp.

+ ++ ++ ++

– ++ + +

+ ++ ++ ++

+++ + + +

Primary samples, Persistence to treatment and at root-filled teeth with apical periodontitis

Other Gram-positive anerobic rods

++ + + + Primary samples. Saliva contamination

Eubacterium spp. ++ ++ – – Primary samples Enterobacteriaceae (enteric

rods) (+) ++ ++ +++ Careless handling in clinic

or in laboratory Gliders and corroding

rods + ++ – – Primary samples and

abscesses Capnocytophaga spp. Campylobacter spp. Eikenella spp.

Prevotella/Porphyromonas Porphyromonas gingivalis/P.

endodontalis

++ +++ – – Primary samples and abscesses

Culture-Based Analysis of Endodontic Infections 63

Table 4.2 (Continued)

Significance in endodontic samples

Resistance to Risk of Microorganisma Frequency Pathogen d treatment e contamination Main appearance

Prevotella intermediac ++ +++ – – Primary samples and Other Prevotella spp. ++ ++ + – abscesses Fusobacterium spp. ++ ++ + – Primary samples and

abscesses Spirochetes + ++ – – Microscopy

Seldom found in culture analysis

Fungi (yeasts) Candida spp + + + ++

Through leakage, persistence after treatment, and careless handling in clinic or in laboratory

aThe microorganisms are grouped as species, genus or other groups according to its clinical relevance. bParvimonas micra, formerly Micromonas micros, earlier Peptostreptococcus micros. cFormerly Bacteroides intermedius. dThe pathogenicity is based on test in animal experiments and their presence in clinical samples. eResistance against adequate mechanical and chemical treatment.

amoxicillin with or without metronidazole remain the drugs of choice (see Chapter 12).

4.5 Route of infection in vital and necrotic pulp

4.5.1 Root canals with vital pulps

The bacteria gain access to the pulp and root canal through various routes: (i) through the dentinal tubules, for example, the caries process or by deep mechanical preparations; (ii) through the exposed pulp by trauma or through fractures; and (iii) through the apical fora- men either through the periodontal ligament (deep pockets or trauma) or hematogenically (Bergenholtz 1977, 1981). Caries is probably the most common cause for bacterial access to the pulp. Bacteria invad- ing the dentine cause inflammation in the pulp tissue that in time leads to necrosis. If the pulp loses its vital- ity prior to becoming infected (by trauma and physical and chemical injuries), the bacteria are attracted to the necrotic tissue through the dentine tubules, fractures, and other routes and subsequently grow and infect the root canal.

As long as the pulp is vital, there is a host response mechanism in function that prevents the bacteria from

invading deeper into the pulp tissues. There is no infec- tion in the strict sense, but bacteria are present in the carious dentine adjacent to the exposed pulp (Bergen- holtz 1977, 1981). Bacteria that could be isolated here are predominantly species that are associated with the caries process (e.g., various Lactobacillus and Bifi- dobacterium species and species of Streptococcus, Propionibacterium, Actinomyces, Corynebacteria and Eubacterium) but very few Gram-negative anaerobic species (Edvardsson 1974; Hoshino et al. 1985). Con- clusively, vital pulps including those exposed to caries and those exposed by trauma may accidentally have bacteria on the surface, but usually in low numbers and without penetration into the vital pulp tissues.

4.5.2 Root canals with necrotic pulps

The pulp can easily become necrotic because of its limited and fragile blood supply at the apical fora- men. Younger teeth with open apices often withstand injuries better than older ones where the apical fora- men is narrow. Necrosis is the terminal end of the inflammatory process which, when it becomes large enough, causes the tissue to collapse as a result of the heavy bacterial load. Bacteria easily invade the necrotic pulp tissue because there is no host defense.

64 Endodontic Microbiology

(a)

(b)

Fig. 4.10 The predominant bacteria: (a) facultative anaerobic and (b) anaerobic bacteria in the main canal, dentine, and apical region of root canals of monkey teeth left open to the cavity for 7 days and then sealed for 6 months. Note the high number of enterics (G-facultative rods) typical for monkeys. Source: Dahlén et al. (1982a). Reproduced with permission of John Wiley and Sons.

The invading bacteria seem to go through some selec- tion mechanism by the route of infection and through the ecologic pressure in the root canal system. A ran- dom process occurs only if the pulp chamber is left open to the oral cavity. We should therefore not expect the numbers of participating species in the closed root canal to be as rich and diverse as those root canals that have been left open to the oral cavity.

The intensity of the infection process is related to the bacterial activity and presence of certain growth and virulence factors that favor some, but not all, bac- teria in their root canal environment (Figure 4.10). The bacterial growth is caused by an anaerobic, pro- teolytic bacterial metabolism in the root canal system and necrotic protein containing pulp. Lack of carbo- hydrates and especially sugars disfavor saccharolytic bacterial species and the low oxygen level does not give the facultatives any advantage over the strict anaer- obes. The local and systemic host defense systems in the root canal are destroyed, and cannot act until the infection front line reaches the vital tissues in the apical region. This is the environment in which the root canal flora of teeth with necrotic pulps develops (Table 4.1).

An inflammatory reaction is formed in the periapical tissues (apical periodontitis), which can be either acute or chronic (symptomatic or asymptomatic).

4.6 Apical periodontitis

4.6.1 Acute symptomatic infection

Acute infection is characterized by an increased bacterial metabolism and nonregulated multiplication. The body’s response is rapid and mainly unspecific and the neutrophilic granulocytes (polymorphonuclear leukocytes, PMNs) predominate in the periapical area. The battle between the growing bacteria and the phagocytic cells (PMN cells) can be very dramatic and the tissues destroyed (collapses). At this stage, the body has one main goal and that is to prevent the infection from spreading. A fibrotic capsule can be formed in order to build a barrier more difficult for the bacteria to penetrate; however, at the cost of a total destruction of the tissues within the barrier. This results in an abscess with pus. This is a common situation in the clinic because patients usually have

Culture-Based Analysis of Endodontic Infections 65

Table 4.3 Frequency (percentage of total number of isolated strains) of microorganisms in root canal samples from teeth with abscess (pus) in some culture studies

Brook Oguntebi Williams Lewis Brook Sakamoto Khemaleelakul et al. et al. et al. et al. et al. et al. et al.

Microorganisms (1981) (1982) (1983) (1986) (1991) (1998) (2002)

S. aureus – – – – 1 – – Other Staphylococcus

spp. – 4 5 – – 3 9

Streptococcus spp. (psp)

10 24 (S. mitis)

– 2 18 7 14

Streptococcus spp. (non-psp)

8 4 2 17 4 16 9

E. faecalis – 12 – – 4 – – P. micraa 20 4 12 ns 23 3 6 Other anaerobic

streptococci – 8 7 28 – 10 –

Neisseria spp. – – – – – – – Veillonella spp. 12 – – 2 2 6 2 Bacillus spp. – – – – – – – Clostridium spp. – – – – – – 3 Corynebacterium spp. – – – – – – 11 Lactobacillus spp. 6 – 5 2 – 1 3 Propionibacterium spp. – – – 1 1 1 3 Actinomyces spp. 6 12 5 1 – 1 4 Eubacterium spp. 2 – – – 2 – 4 Enteric rods – – – – – – – Capnocytophaga spp. – – – – – – – Campylobacter spp. – – – – – 3 – Eikenella spp. – – – – – – 1 Porphyromonas spp. 4 – – 8 10 4 2 Prevotella

intermedia/ nigrescens

2 8 – 3 3 4 5

Other Prevotella spp.b 29 – 48 23 17 21 19 Fusobacterium spp. 8 28 15 4 12 11 4 Spirochetes – – – – – – – Candida spp. – – – – – – – Total number of

isolated strains 59 25 40 168 78 112 118

Number of teeth 12 10 10 50 32 23 17

Species >10% in bold. psp, polysaccharide producing streptococci; non-psp, non-polysaccharide producing streptococci. aParvimonas micra formerly Micromonas micros earlier Peptostreptococcus micros. bIncluding isolates designated as Bacteroides spp.

symptoms, sometimes severe, and seek immediate treatment. Table 4.3 summarizes the microbial composition in acute symptomatic endodontic infec- tions with periapical abscess and presence of pus. Species of Peptostreptococcus (including Parvi- monas micra), Prevotella spp., and Fusobacterium spp. prevail together with various Gram-positive anaerobic/microaerophilic rods and cocci.

4.6.2 Bacteria and symptoms

Acute symptomatic infection is also a common reason to investigate the microbial composition in order to find out if there is some kind of specificity and if this has any treatment implications. Table 4.4 shows the results of studies that evaluated the association between spe- cific bacteria and symptoms such as pain, swelling,

66 Endodontic Microbiology

Table 4.4 Studies indicating microorganisms associated to symptoms

No. of Method of Microorganisms associated Frequency in teeth Study teeth detection with symptoms with symptoms (%)

Griffee et al. (1980) 12 Culture B. melaninogenicusa 92 Van Winkelhoff et al.

(1985) 17 Culture P. endodontalis 53

Haapasalo (1986) 35 Culture B. buccaeb 37 Haapasalo et al. (1986) 31 Culture Black-pigmented

Bacteroides B. intermediusb

B. gingivalisc

54 32 19

Yoshida et al. (1987) 11 Culture P. magnusd 55 Sundqvist et al. (1989) 72 Culture BPB

B. intermediusb 73

Hashioka et al. (1992) 25 Culture Eubacterium spp. Peptococcus spp. Peptostreptococcus spp. Porphyromonas spp.

15–35 7–24 11–18 9–24

Gomes et al. (1994) 30 Culture P. micrae

F. nucleatum BPB S. milleri groupf

17% (of isolates) 7% (of isolates) 13% (of isolates) <23% (of isolates)

Baumgartner et al. (1999)

40 PCR BPB P. nigrescens P. intermedia

55 50 36

Chavez de Paz (2002) 28 Culture F. nucleatum 3 Rôças et al. (2002) 20 PCR T. denticola

T. forsythia P. endodontalis P. gingivalis

50 40 40 30

Fouad et al. (2002) 24 PCR Streptococcus spp. F. nucleatum

Odds ratio 13 Odds ratio 3.2

Foschi et al. (2005) 62 PCR T. denticola 56

aNow divided into 6–9 different black-pigmented bacteria (BPB). bNow Prevotella spp. (P. buccae, P. intermedia, P. nigrescens). cNow Porphyromonas spp. (P. gingivalis, P. endodontalis). dNow Fingoldia magna. eParvimonas micra, formerly Micromonas micros, earlier Peptostreptococcus micros. fNow including S. anginosus, S. intermedia, S. constellatus.

tenderness, abscess formation, and sinus tract. Con- clusively, anaerobes, predominantly Gram-negatives rods, are most commonly present; however, the specificity is low. Correlations with some anaer- obes (Peptostreptococcus spp., Eubacterium spp., Pre- votella spp., and Fusobacterium spp.) have been claimed. Especially, the black-pigmented bacteria (BPB) have gained much attention. Notably, Por- phyromonas spp. (P. gingivalis and P. endodon- talis), which are considered more virulent than Prevotella species and associated with peri- odontitis, are less frequent in acute endodontic

infections and are outnumbered by black-pigmented Prevotella species, including P. intermedia (including P. nigrescens, P. pallens) in particular.

The importance of anaerobes in acute infection has been confirmed in numerous experimental ani- mal studies (for review see Dahlén 2002). In one study, eight bacterial species isolated from the same infected root canal in a monkey were inoculated into 12 experimentally devitalized teeth of monkeys in the same proportions (Fabricius et al. 1982b). Fig- ure 4.11 shows the proportion of those eight species in the 12 teeth after being followed for 6 months.

Culture-Based Analysis of Endodontic Infections 67

Fig. 4.11 Mean viable counts of eight strains in percentage of total counts in samples from 12 teeth after 6 months (experimental infection). The eight strains were originally isolated from a monkey tooth (original infection), pure cultured in the laboratory and inoculated in equal numbers experimentally in 12 monkey teeth. Source: Dahlén et al. (1982b). Reproduced with permission of John Wiley and Sons.

Note that the four anaerobic species, F. nucleatum, F. necrophorum (now both classified as Fusobacterium simiae), Peptotreptococcus anaerobius, and Prevotella oralis (formerly Bacteroides oralis), are predominat- ing both in the original infection as well as in the 12 experimentally infected teeth. The same study (Fabri- cius et al. 1982b) also showed that the streptococci and enterococci survived as pure cultures in the root canals but induced only weak periapical reactions, indicating that the virulence of the various bacterial combinations is mainly associated with the anaer- obic bacteria. The eight strains were also inoc- ulated into steel net wound chambers implanted in the back of rabbits and the dynamics were followed for >30 days (Dahlén et al. 1987). It was clear that the pus and abscess was formed when three of the anaerobic species started to grow and multiply (Figure 4.12). However, it was

necessary to include facultative anaerobes in the bacterial collection in order to let the anaerobes survive the initial phase of the infection, supposedly by reduc- ing the redox potential by consuming available oxy- gen. This appearance has been confirmed in numerous other experimental animal studies using subcutaneous injections (for review see Dahlén 2002). Sundquist et al. (1979) carried out a transmission study and found that a Bacteroides strain (later identified as P. endodontalis) was essential for the transfer of an infection between animals by bacterial combinations originating from infected root canals. No infections developed using this strain alone. It seems that the specificity in these anaerobic infections is low and numerous combinations of normally low virulent oral bacterial species have the capacity to induce an acute infection in the root canal and periapical tissues. The low virulence is compensated by the increase in

Fig. 4.12 Total viable counts (10th logarithm) at various time points of each strain of the eight strain collection in wound chambers in rabbits followed for 35 days. Source: Dahlén et al. (1987). Reproduced with permission of John Wiley and Sons.

68 Endodontic Microbiology

Table 4.5 Commonly isolated bacterial species in teeth with acute periapical lesions and some of their virulence factors

Bacterial species Virulence factors Toxins

P. anaerobius/P. micra Weak proteolytic activity Lipotechoic acid Metabolic acids

F. nucleatum/F. necrophorum Capsule polysaccharides (?) Proteolytic activity

Endotoxin Wide spectrum of metabolic acids Sulfur products Leukotoxin (F. necrophorum)

P. intermedia/P. nigrescens Thin polysaccharide capsule Proteolytic activity

Endotoxin Metabolic acids

P. endodontalis Capsule polysaccharides (?) Proteolytic enzymes (Gingipains)

Endotoxin Wide spectrum of metabolic acids

P. gingivalis Thick polysaccharide capsule Strong protelytic activity (Gingipain R

and K, and collagenase) Some strains highly invasive

Endotoxin Wide spectrum of metabolic acids

Treponema spp./T. denticola Strong proteolytic activity (Gingipains)

Endotoxins Wide spectrum of metabolic acids Sulfur products Ammonia

Source: Olsen and Dahlén (2004).

numbers by the growth and multiplication and by the polymicrobial nature of the primary endodontic infec- tion. The character of anaerobic infections, in gen- eral, is that they develop when the local and gen- eral defense is hampered (Finegold 1977). When the blood supply is inhibited or strangled, the decrease in oxygen level disfavors the PMNs’ oxygen-dependent killing mechanisms and the growth of anaerobic bac- teria is concomitantly favored. This is very much true in the root canal with a necrotic pulp, where the bac- teria can grow extensively without being reached by the defense system (PMNs, antibodies, complement factors) and by their production of toxic metabo- lites, proteolytic enzymes, and so on, which increase the challenge of the host defense system (Olsen and Dahlén 2004). The concomitant outgrowth of bacte- ria through apical foramen into the external periapical tissues cannot be prevented because the bacteria are in an active growing phase, sometimes even stimu- lated by host factors such as blood components and serum. The fate of the periapical acute infection or abscess is probably much dependent on the communi- cation through the apical foramen (Sundqvist 1992b). If that communication is wide, for example, as in younger teeth, this will probably favor the bacteria because of a better nutrient supply and the infection

route may be more dramatic. Even if the root canal infection is polymicrobial and unspecific, it does not mean that specific features do not exist. Some bacte- rial species are more common in these infections than others and some bacteria produce unique virulence fac- tors (capsule, leukotoxins, complement resistance, and immunoglobulin degrading enzymes), which make them more adapted to survive and grow in the lesion than others and to invade the tissues and actively partic- ipate in the pathologic destruction (Table 4.5) (see also Chapter 7).

4.6.3 Abscess and fistula formation

When bacteria grow in the necrotic root canal sys- tem, the periapical tissues are involved and the host defense system is activated. It is important to empha- size that this reaction is general for all acute infections of the body. If the bacteria maintain a high metabolic activity and growth, the body’s main defense goal is to prevent the infection from spreading. A fibrotic barrier may be formed and encapsulate the infection process into an abscess. This reaction is time dependent and it is sometimes too late to prevent bacteria and bacterial products from spreading through the tissues. Such bac- teria will be cleared by the lymphatic drainage and the

Culture-Based Analysis of Endodontic Infections 69

local lymph nodes which become swollen and painful. This is a stage when systemic antibiotic treatment is indicated with the purpose of inhibiting bacterial mul- tiplication and growth and spread of the infection. In the spreading periapical abscess (if no antibiotics are given), the bacteria may still grow, leading to an expan- sion of the abscess through the tissues. The nature of this expansion follows the route of the least resistance and in most cases ends up with drainage into the oral cavity through either the periodontal pocket or through the mucosal membrane. The latter condition is called a sinus tract and is frequently seen in the clinic. The microbiota in sinus tract is also mixed anaerobic (Haa- pasalo et al. 1987). Fortunately, less frequently the infection is spread to other compartments of the head and neck region, where serious complications can fol- low (see Chapter 10). The sinus tract is usually the termination of the acute phase of the infection, the symptoms decline, and the whole process becomes chronic. However, as long as the primary root canal infection is not subjected to intervention, the bacte- ria still remain in the tooth and maintain the process and the sinus tract can remain for a long period. Peri- apical infections are further considered in Chapters 6 and 10.

4.6.4 Chronic infection

Chronic infection is characterized by remaining or per- sisting bacteria and bacterial biofilms that are in a low metabolic stage with no or little growth (multiplica- tion). The host defense reaction is also changing into a chronic inflammation, predominated by lymphocytes and antibody-producing plasma cells. The tissue is reorganized into a granulomatous tissue whose main purpose is to keep the infection and bacteria local- ized and to prevent the bacteria from spreading. The formation of granulomatous tissue is favored by com- ponents of the immune system (e.g., antibodies). This process is usually quiescent, with no or few symptoms, and the risk of spreading is limited. This was shown in monkeys where immunization was performed with those species that later were experimentally introduced in the root canal (Dahlén et al. 1982a). The periapi- cal lesions in immunized monkeys that developed at infected teeth were clearly visible on radiographs as a sharp demarcation and sometimes even a sclerotic zone in the bone surrounding the lesion. Histologically, the sharp demarcation was confirmed and the inflamma- tory infiltration was only seen adjacent to the apical

Fig. 4.13 Histologic picture of the periapical area of a tooth in an immunized monkey. Note root resorption, cell infiltrate adjacent to the root and the thick fibrotic capsule. From Dahlén et al. 1982a.

foramen and surrounded by a thick fibrotic capsule (Figure 4.13). However, in the nonimmunized control monkeys this capsule was not formed, the inflamma- tory cells were spread deeper in the tissues includ- ing the bone (osteitis), and the radiographic lesions were more diffuse and sometimes not detectable. In patients, the same type of lesion as seen in the immu- nized animals is frequently seen among adults and especially the elderly. Long-term exposure for the anti- gens present in the tooth may stimulate the immune response and antibody formation. In monkeys, it was shown that antigens, for example, lipopolysaccharides (LPS) present in the root canal, can induce a specific antibody response (Dahlén et al. 1982b). Patients are usually not aware of this type of chronic lesions that are only detected on radiographs. These lesions should be treated because the infection will exacerbate sooner or later.

70 Endodontic Microbiology

183 cases with a first root canal sample submitted presenting growth

In 166 cases a second sample was submitted, 78 presented growth

In 69 cases a third sample was submitted, 11 presented growth

In 9 cases a fourth sample was submitted.

none with growth

2 9

+ + + + + +++–

– –

+ + +

First sample Second sample

Fourth sample Third sample

Fig. 4.14 Frequency of culture positive samples from 183 root canals undergoing treatment (first sample), 166 cases after the second appointment (second sample), 69 cases after the third appointment (third sample), and 9 cases after the fourth appointment (fourth sample). Courtesy of Dr. Chavez de Paz.

4.6.5 The microflora of the root canal versus the deep periodontal pocket

The bacterial flora of the untreated root canal (Table 4.1; Figure 4.14) is characterized predomi- nantly by anaerobic bacteria. They are mainly Gram- negative anaerobic rods and the whole flora resem- bles that of the deep periodontal pocket (Socransky et al. 1998; Haffajee and Socransky 2000; Marsh 2004). Thus, the main metabolic activities at both sites are anaerobic and proteolytic, and the access to oxy- gen and sugars/carbohydrates are limited. However, there are different selection mechanisms and prevail- ing ecologic pressures that lead to striking differences (Sundqvist and Figdor 2003). The access to nutri- ents, for example, blood and serum, is much higher in the periodontal pocket by the excessive exudate flow due to inflammation. This leads to higher bacterial metabolic activity and growth, leading to much higher

numbers of bacterial cells in the periodontal pocket compared to the root canal (Table 4.6). In addition, there is open communication between the periodontal pocket and the oral cavity, which results in higher num- ber of species and a more complex flora with hundreds of different microorganisms. While some are more vir- ulent and have etiologic association to periodontitis, others are innocent bystanders. The endodontic micro- biota show some distinct features. First, there are fewer cultivable species in endodontic infections because of limited communication with the oral cavity, unless the root canal has been left open. This is an important rea- son for not leaving the pulp chamber open to avoid a heavier invasion of bacteria. An open communication can only favor the infection and is of no benefit to the patient. Second, there is a selective ecologic mecha- nism that allows some species to be more common in the root canal flora as detected by culture studies than that of the periodontal pocket. Table 4.6 shows

Culture-Based Analysis of Endodontic Infections 71

Table 4.6 Comparison between the root canal flora and the flora in the deep periodontal pocket

Similarities and Deep periodontal pocket at differences Root canal flora at primary endodontic infection periodontitis

Similarities Polymicrobial predominantly anaerobic infection Polymicrobial predominantly anaerobic infection

Differences General characteristics

<12 predominant cultivable species 105–106 bacterial cells in a paper point sample Low access to nutrients

>Hundreds of species 107–108 bacterial cells in a paper

point sample High access to nutrients

Differences Specific species or groups

Highly frequent species or groups Prevotella spp. Fusobacterium spp. Prevotella intermedia Eubacterium spp. Propionibacterium spp. Actinomyces spp. Non-polysaccaharide producing streptococci P. micraa

Other anaerobic streptococci Treponema spp. (underscored in culture studies)

Frequent species or groups Polysaccharide producing streptococci Lactobacillus/Bifidobacterium Porphyromonas spp.

Highly frequent pathogens The red complex

P. gingivalis T. forsythia T. denticola A. actinomycetemcomitans

Frequent pathogens P. intermedia/nigrescens C. rectus P. micraa

P. tannerae F. alocis P. endodontalis

Frequent less pathogenic species Prevotella spp. F. nucleatum E. corrodens S. intermedia and more

aParvimonas micra, formerly Micromonas micros, earlier Peptostreptococcus micros.

microbiologic characteristics of endodontic and peri- odontal infections. Species such as Aggregatibacter actinomycetemcomitans, Haemophilus spp., Neisseria spp., some Streptococcus species (especially polysac- charide producing streptococci such as S. salivarius, S. sanguinis, S. gordonii, S. oralis, S. mutans) can reg- ularly be found in subgingival samples but seldom in root canals. These bacterial species are facultative and mainly saccharolytic. They may not be able to sur- vive in the strict anaerobic environment in the root canal with no access to sugars where there is also no advantage to their strong adherent capacity both to epithelial cells (mucosa) and enamel (salivary gly- coproteins), which makes them predominant on the oral mucosa and teeth. On the contrary, Lactobacillus spp., Bifidobacterium spp., Propionibacterium spp., and enterococci have low adhering capacity to the enamel surface, while they apparently are more easily established in the root canal system, especially under more hash conditions. Some species and genera, for example Tannerella forsythia (formerly Bacteroides

forsythus), Treponema species (spirochetes), Eubac- terium spp., Selenomonas spp., and Campylobacter spp., are underscored in culture studies because they are difficult to grow. Studies to disclose more specific but less frequent bacteria by culture have shown that these species may be detected in the root canal but in lower frequencies than in the deep periodontal pocket. By molecular biology methods a number of new bac- terial species, not yet cultured or difficult to culture, have been discovered both in the periodontal pocket and in the primary infected root canals. Thus, more quantitative and qualitative differences might be dis- closed between the two types of infections in the future.

The root canal represents a special environment in which selective pressures result in the establishment of a restricted number of microorganisms (Sundqvist 1992a). In the primary infection, the microbiota commonly consists of 10–12 predominantly anaer- obic cultivable bacteria. Bacterial interactions and access to nutrients are key factors in determining the

72 Endodontic Microbiology

outcome of the infection. Endodontic treatment should not only eliminate bacteria, but also disrupt the bal- ance within the microbial community established in the root canal. This balance is stabilized by the fact that persisting bacteria remain in biofilm communi- ties in various parts of the root canal system (Fig- ure 4.8). The root canal biofilm may not be as com- plex as the dental plaque biofilm; however, it gives the microorganisms a number of advantages and support (Box 4.1). Dental biofilms are considered difficult ther- apeutic targets (Socransky and Haffajee 2002) and similar aspects can be made for biofilms in the root canal system. The increased resistance for antimi- crobials should be specifically emphasized because it explains the difficulties in completely eliminating the remaining microorganisms from the root canal (Chavez de Paz et al. 2007).

Box 4.1 General properties of a biofilm

Protection from host defense Protection from dehydration Protection from antimicrobial agents (antiseptics

and antibiotics) � Surface-associated phenotype∗ � Slow growth rate � Poor penetration � Inactivation and neutralization

Novel gene expression and phenotype∗

Persistence in a flowing system Spatial and environmental heterogeneity Metabolic interaction and food web Elevated concentrations of nutrients

∗Increased resistance to antimicrobial agents may be due to altered gene expression. Source: Adapted from Marsh and Martin (1999).

4.7 Treatment aspects

Endodontic treatment has two major aims. First, infec- tions and microorganisms should be eliminated. A root canal free from microorganisms is a primary goal. Second, the root canal should be mechanically debrided and enlarged in a way that it can be ade- quately obturated with a permanent root filling. These two goals go well together because it is quite clear that

a well-prepared and filled root canal system also has the best chance to be free of microorganisms. These two goals are currently being pursued through a com- bination of mechanical debridement, irrigation, and interappointment dressings. These goals are not con- tradictory and a thorough debridement and enlarge- ment of the root canal lumen highly favors the chance to eliminate bacteria from the root canal. The use of irrigation and interappointment dressing is to further make it possible to kill, reduce, or eliminate bacteria from the root canal. There is an ongoing debate as to whether this is necessary or possible, and if this should or could be performed in one visit or if two steps or multiappointments and repeatable treatments are necessary, or at least gives a higher success rate (Molander et al. 2007). The success rate in practice is measured by the rate of future failures. However, what is a failure? Is it restricted to future exacerbations and new acute infections, remaining symptoms, persistent lesions observed radiographically, and/or those lesions above a certain size? All these questions are related to the attitudes of the dentist, the possibility to perform the procedures aseptically, whether the tooth could be restored, and the patient’s wish, together with practi- cal and economic factors. A series of publications by Chavez de Paz et al. (2003, 2004, 2005, 2007) illus- trate not only the possibility to render the root canal bacteria-free, but also the problem with the strategy to eliminate persistent bacteria (Figure 4.15) as analyzed by culture. Taken together, the root canal displayed a negative culture sample after 1–4 repeated treatments in 155 of 183 cases. The remaining 28 cases were

100 90 80 70 60 50 40 30 20 10 0

Untreated teeth

Teeth under treatment

Root-filled teeth

Gram pos cocci

Peptostreptococci Gram pos rods

Gram neg anae rods

Others No bacteria

Fig. 4.15 Microbial composition (%) between untreated teeth (Sundquist 1992a), teeth under treatment (Chavez de Paz et al. 2003), and root-filled teeth (Molander et al. 1998).

Culture-Based Analysis of Endodontic Infections 73

dropouts (Figure 4.14). A negative preobturation sam- ple will probably improve the prognosis of endodontic treatment; however, over many years only a few longi- tudinal studies have been performed with this aspect as a purpose (Sjögren et al. 1997; Sundqvist et al. 1998; Waltimo et al. 2005; Molander et al. 2007).

4.7.1 Why is it so difficult to eliminate root canal microorganisms?

There are several reasons why it is difficult in practice to eliminate microorganisms from the root canal. Some even believe that it is impossible or doubt its importance because bacteria may be entombed and die in the filled root canal. Even if it is difficult to eliminate bacteria, this argument cannot be accepted as a reason for not trying hard enough, for doing a number of shortcuts, or even for neglecting the importance of the antimicrobial efforts. First, the importance of careful antiseptic measures in all treatment steps cannot be overestimated. It seems obvious that a rubber dam should isolate the tooth and a careful disinfection of the operative field is a must (as discussed previously) to avoid reinfection. Second, the anatomic condition and variations can be extremely problematic (Figure 4.6), especially in molars with 3–4 canals, and with apical resorptions, isthmi, and accessory canals. Bacteria may penetrate, especially in long-standing infections, into the dentine tubules which makes it difficult to reach them by mechanical or chemical procedures. Cross-sectionally, it is obvious that the canals are very seldom circular and especially the isthmus area in roots with two canals (lower molars) is difficult to reach mechanically (Figure 4.6). It is even more diffi- cult in the teeth of older people with much hard tissue formation (calcification and secondary dentine) on the root canal walls. In the apical region, this hard tissue may give rise to a very complicated apex delta that will not be possible to reach mechanically. Bacteria could also penetrate through the apex and be present exter- nally on the tooth, in particular after an acute infection when abscess has occurred. Bacteria may thus be present on the periapical root surface which may be difficult to reach by intracanal treatment procedures. This problem is aggravated in root surface resorptions (Figure 4.9). Third, mechanically unreachable bacteria remain in niches of the root canal system forming biofilms (Nair et al. 2005). Biofilms are to the benefit of bacteria and offer them a number of advantages (Figure 4.8). Notably, the increase of resistance against

all types of antimicrobial agents should be considered. Finally, a number of species are true “persisters” by the fact that they have a natural ability to survive more harsh environments and stressed conditions (Chavez de Paz 2007). Gram-positive bacteria generally survive better than Gram-negative bacteria when it comes to dehydration, lack of nutrients, changed ion strength/osmotic pressure, and presence of antiseptics. This has been appreciated specifically for enterococci and the use of Ca(OH)2 paste as an interappointment dressing (Chavez de Paz et al. 2007). However, this is also true for other Gram-positive organisms such as streptococci, lactobacilli, Actinomyces spp., propionibacteria, and yeasts (Waltimo et al. 1997; Chavez et al. 2003, 2004, 2005). Conclusively, there is a striking difference between primary infected teeth and teeth undergoing treatment (Figure 4.16). Particularly, the increase of Gram-positive facultative cocci and the reduction of Gram-negative rods should be noted.

4.8 Persisting infections at root-filled teeth

Apical periodontitis associated with root-filled teeth is very common among endodontic patients. The fre- quency of periapical destructions as evaluated on radiographs varies between 14% and 72%, depending on the number of teeth included in the study, type of teeth, age of the patient, and in which country it is per- formed (Frisk and Hakeberg 2005). In a later Swedish follow-up study (Frisk et al. 2008) was reported an improving technical quality of root fillings over time without a concomitant improvement of the periapical status in root-filled teeth. This may be interpreted as that the remaining infections are still as frequent and that technical quality cannot generally save the root filling to become successful. Table 4.7 gives the suc- cess rate in some studies after 2 years or more for teeth that had been bacteria-free (as could be determined by a root canal sample for culture analysis) at the time of the permanent root filling. Remaining bacte- ria in the root canal may prevent the periapical lesion from healing and radiographic signs may persist for years. The patient is not aware of the process because these are chronic infections with few or no symptoms. The remaining microorganisms have a low metabolic activity, and the apical lesion formed is characteristi- cally a granulomatous tissue with a predominance of

74 Endodontic Microbiology

Fig. 4.16 Pie charts showing the proportions of organisms isolated in studies of untreated necrotic pulps, cases undergoing treatment, and root-filled teeth with apical periodontitis. Courtesy of Dr. Chavez de Paz.

lymphocytes and plasma cells. Apical periodontitis lesions are usually well recognized on radiographs because of the concomitant loss of bone. The size can differ greatly from small and hardly discernable lesions to lesions ≥10 mm in diameter. Their progression is usually slow and makes the dentist uncertain whether to carry out revision or retreatment or not. Lesions are often just followed and therapy is postponed until 2–4

years after initial therapy, when a definitive decision is usually made.

There is an ongoing discussion as to if and when these lesions should be retreated. A number of culture studies have been conducted to see to what extent these lesions show presence of microorganisms and of what kind. This is not easy because it is very likely that the present microorganisms are unreachable for sampling

Table 4.7 Studies showing the success rate of the root canal treatment/filling with or without bacteria present as determined by culture

Follow-up Success rate in Success rate in Number and type period case of negative case of positive

Study of teeth (years) culture sample (%) culture sample (%)

Sjögren et al. (1997) 55 single rooted human teeth 5 94 68 Fabricius et al. (2006) 175 mixed monkey teeth 2 80 44 Molander et al. (2007) 101 mixed human teeth 2 72 21

Culture-Based Analysis of Endodontic Infections 75

or they are eliminated when the root filling material is removed. Therefore the risk of false negative samples is high when culture is used. The frequency of positive samples by culture or molecular methods has increased in recent studies, when the difficulties have been more carefully considered (Table 4.8). Only 1–3 species are generally isolated, and in low numbers.

They are predominantly Gram-positive cocci and rods, for example, Enterococcus faecalis, streptococci, lactobacilli, Actinomyces spp., peptostreptococci, and yeasts (Figure 4.16). In Figure 4.16, the change in microbiologic character from the primary untreated tooth, cases in treatment, and the root-filled tooth with apical periodontitis is illustrated. Molander et al.

Table 4.8 Microflora (percentage of isolates) in root-filled teeth with apical periodontitis as indicated in some recent studies. Species frequency >10% in bold

Sirén Molander Sundqvist Pinheiro Gomes Adib et al. et al. et al. et al. et al. et al.

Microorganisms (1997) (1998) (1998) (2003) (2004) (2004)

S. aureus – – – – – 1 Other Staphylococcus

spp. 7 6 5 2 3 21

Streptococcus spp. (psp) 26 6 6 9 14 13 Streptococcus spp.

(non-psp) Nsb 6 12 6 11 19

E. faecalis 16 27 29 25 17 11 P. micraa 5 1 6 5 8 – Other anae streptococci – – – 7 8 7 Neisseria spp. 2 – – – – – Veillonella spp. – 1 – 4 – – Bacillus spp. – 1 – – – – Clostridium spp. – – – 1 – – Corynebacterium spp. – – – – – – Lactobacillus spp. 1 12 3 4 3 2 Propionibacterium spp. – 3 10 – 6 4 Actinomyces spp. 2 2 6 4 6 9 Eubacterium spp. – 1 6 – 4 6 Enteric rods 7 13 – – – 9 Capnocytophaga spp. – – – 1 – 2 Campylobacter spp. 1 1 10

C.gracilis – – –

Eikenella spp. – – – – – – Porphyromonas spp. 2 – – – 3 – Prevotella

intermedia/nigrescens 5 – – 3 3 –

Other Prevotella spp. – 4 – 6 11 1 Fusobacterium spp. 12 4 3 3 – 2 Spirochetes – – – – – – Candida spp. 3 3 6 2 – 3

Total number of isolated strains

147 117 31 108 36 90

Number of teeth 40 100 40 60 19 8 Teeth with detected

microorganisms 100% (selected cases)

68% 44% (root canals)

85% No data 100% (selected cases)

aParvimonas micra, formerly Micromonas micros, earlier Peptostreptococcus micros. bns, not specified.

76 Endodontic Microbiology

(1998) reached a frequency of 68% positive samples and it was suggested that the prevalence is, in fact, 100%. Later studies have confirmed an even higher prevalence of positive bacterial cultures from root- filled teeth than the figures reported in the Molander et al. (1998) study (Table 4.8). With the microbial pattern of the persisting flora during treatment in mind, it is likely that the bacteria observed in root-filled teeth persist from the initial treatment (Figure 4.16). A secondary invasion by coronal leakage or other routes is possible, but unlikely in the adequately treated and restored case. This has been experimentally proven in monkeys (Möller et al. 2004; Fabricius et al. 2006), where 175 teeth were infected using a collection of 4–5 strains previously isolated from an infected root canal of a monkey (Fabricius et al. 1982a). After estab- lishing an infection in all teeth, they were subjected to treatment with mechanical debridement and irrigation with NaOCl but no interappointment dressing. After two appointments all teeth were permanently root filled and followed for 2 years. Eighty teeth were radiographically and histologically healed while 95 did not heal (Table 4.7). Only 19 (21%) of the teeth that contained bacteria healed, while 61 (72%) teeth with a negative sample at the root-filling occasion healed. It was also found that no other bacteria occurred than those primarily inoculated into the root canals, indicating that coronal leakage during the experimental period was negligible. Furthermore, the root-filling quality was of less importance in the sense that in root-filled teeth without bacteria the lesions healed irrespective of whether the root filling was extended beyond or short of apex. However, lesions remained at many teeth with bacteria even if the root filling was adequate. Conclusively, remaining bacteria at permanent root filling is a stronger risk factor for a nonhealed periapical lesion than the technical quality of the root filling. These studies clearly show that the root canal should be bacteria-free according to culture before root filling and that careful antiseptic measures at the appointments are necessary.

4.9 Culture versus molecular biology methods

It is clear that endodontic microbiology has developed tremendously through the years by using culture anal- ysis. Much current knowledge is based on samples and experiments where culture analysis has been used.

Culture analysis can also easily be quantitative, which indicates the bacterial load in the root canal. We have also learned about the characteristics of the isolated bacteria and under what conditions they participate in various stages of the root canal infection. We could also carry out experimental infections to prove their pathogenic nature. We must also admit that culture analysis has only disclosed part of the true microbial condition of the infected root canal. The problems of avoiding false negative samples because of sampling problems are highlighted as well as the ability for dor- mant (viable but uncultivable/difficult to grow) bacte- ria to grow simply because we do not have the right medium or conditions.

By molecular biology techniques, a significant num- ber of microorganisms, designated as “not yet cul- tured” have been detected (see Chapter 5). It is not clear whether all these new findings, in fact, corre- spond to viable infectious cells or to what degree they are remains of dead cells. A limitation in using some molecular biology methods such as specific PCR is that the targets have to be preselected by primers and other microorganisms are not detected. In fact, it seems that culture-dependent and culture-independent methods recover different parts of the microbiota and the ben- efit of combined methods for detection of the micro- bial diversity has been recently emphasized (Anderson et al. 2012).

Conclusively, for a number of years culture will be the gold standard for most clinicians while new techniques are being further developed, disclosing new knowledge and complementing current knowledge in endodontic microbiology.

4.10 Conclusions

Endodontic infections are major problems in the dental clinic and treatment of the root canal constitutes a seri- ous challenge for the dentist. The root canal constitutes an excellent environment for microorganisms because it offers a protected compartment with no or weak host defense systems and little chance for the host to eliminate them. The common condition involves necrosis of the pulp, which leaves a nutrient source for the bacteria to grow and multiply. The infection reaches the root apex, penetrates through the api- cal foramen, and forms a periapical lesion that may develop into an acute infection with symptoms or into a chronic infection where there are few symptoms. It

Culture-Based Analysis of Endodontic Infections 77

is important to emphasize that the distinction between acute and chronic periapical infections is the metabolic activity of the bacteria, their growth, and the bacte- rial load rather than the composition of the flora. Cul- ture studies have an advantage over molecular-based studies in quantitatively disclosing the predominant viable and cultivable flora, which then could be fur- ther subjected for antibiotic susceptibility testing and experimental studies. In both acute and chronic root canal infections, the microbiota is polymicrobial and predominantly anaerobic, harboring up to 12 species according to culture-based analysis. Species of anaer- obic streptococci, Eubacterium, Campylobacter, Pre- votella, and Fusobacterium usually prevail and the flora thus resembles the flora of the periodontal pocket.

Treatment includes mechanical debridement, irri- gation, and interappointment dressings performed under strict aseptic measures. The combination of the high risk of reinfection during treatment and the difficulties in reaching all the bacteria in the root canal system leads to an unacceptable situation, with root canals often permanently filled with remaining microorganisms. Culture analysis of samples from root canals undergoing treatment shows that the persisting microorganisms are mainly Gram-positive and facul- tative such as streptococci, enterococci, lactobacilli, propionibacteria, and Actinomyces. These microorgan- isms are resistant against most antimicrobial agents and form biofilms in locations in the root canal system, particularly where it is especially difficult to eliminate them. They can survive for years after the permanent root filling is placed and are the predominating flora in teeth that are subjected to revision of treatment. Sev- eral experimental and clinical follow-up studies show that healing of periapical lesions occurs significantly more often if the bacteria are eliminated as determined by culture-based analysis. Reduction or elimination of remaining bacteria, providing a technically optimal root filling and an adequate permanent restoration are the most important steps for a successful outcome of endodontic treatment.

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Chapter 5 Molecular Analysis of Endodontic Infections José F. Siqueira, Jr, and Isabela N. Rôças

5.1 Introduction 5.2 Limitations of culture methods

5.2.1 Difficulties in culturing: the as-yet-uncultivated majority

5.2.2 Difficulties in identification: species with uncommon phenotypes

5.3 Molecular biology techniques 5.3.1 Impact in medical microbiology

5.4 Gene targets for microbial identification 5.5 PCR and its derivatives

5.5.1 Species-specific PCR 5.5.2 Multiplex PCR 5.5.3 Nested PCR 5.5.4 Reverse transcriptase PCR 5.5.5 Quantitative PCR 5.5.6 PCR-based microbial typing 5.5.7 Broad-range PCR and clone

library analysis 5.5.8 Phylogenetic tree

5.6 Denaturing gradient gel electrophoresis 5.7 Terminal restriction fragment length

polymorphism 5.8 DNA–DNA hybridization assays

5.8.1 Checkerboard DNA–DNA hybridization

5.8.2 DNA microarrays 5.9 Fluorescence in situ hybridization

5.10 Next-generation DNA sequencing technologies

5.11 Metagenomics 5.12 Advantages and limitations of molecular

methods

5.12.1 The too-high sensitivity issue 5.12.2 The dead-cell issue

5.13 Unraveling the endodontic microbiome with molecular biology methods 5.13.1 The five generations of

endodontic microbiology studies

5.13.2 Impact of molecular methods in endodontic microbiology

5.14 Microbial diversity in endodontic infections 5.14.1 Primary intraradicular infections 5.14.2 Uncultivated bacteria 5.14.3 Newly cultivated and

characterized species 5.14.4 Geographic influence

5.15 Persistent and secondary intraradicular infections 5.15.1 Bacteria at the root canal-filling

stage 5.15.2 Microbiome in root

canal-treated teeth 5.16 Extraradicular infections 5.17 Other microorganisms in endodontic

infections 5.17.1 Archaea 5.17.2 Fungi 5.17.3 Viral infections

5.18 Next-generation DNA sequencing analyses of the endodontic microbiome

5.19 Conclusions 5.20 References

82 Endodontic Microbiology

5.1 Introduction

Research in endodontic microbiology is moving at a rapid pace and, consequently, our understanding of the etiology and pathogenesis of endodontic diseases has intensified and continues to evolve. The last two decades have witnessed an overwhelming volume of new information about diverse aspects of endodontic infections. Much of the substantial progress in this area has been a result of improvement in laboratory techniques, particularly the introduction and further widespread use of culture-independent molecular biol- ogy techniques. This chapter reviews the molecular biology techniques that have been used in endodontic microbiology research, their advantages and limita- tions, as well as the contribution they have made to the field of endodontic microbiology.

5.2 Limitations of culture methods

Traditionally, microbiological culture has been the pre- ferred means for examination of the endodontic micro- biota (for review see Chapter 4). Culture is the process of propagating microorganisms in the laboratory by providing them with proper environmental conditions. Ingredients necessary for microbial pathogens can be supplied by living systems (e.g., growth in an animal host or in cell culture) or artificial systems (by gather- ing the required nutrients and conditions for growth). Artificial systems have been widely used for microbi- ological diagnosis of most bacterial and fungal infec- tions that affect humans. In order for microorganisms to multiply on or in artificial media, they must have available the required nutrients and proper physico- chemical conditions, including temperature, moisture, atmosphere, salt concentration, and pH (Slots 1986).

Essentially, culture analyses involve the follow- ing steps: sample collection and transport, dispersion, dilution, cultivation, isolation, and identification. Oral samples are collected and transported to the labora- tory in a viability-preserving, nonsupportive, anaero- bic medium. They are then dispersed by sonication or by vortex mixing, diluted, distributed on to vari- ous types of agar media, and cultivated under aero- bic or anaerobic conditions. After a suitable period of incubation, individual colonies are subcultivated and identified on the basis of multiple phenotype-based aspects, including colony and cellular morphology,

Gram-staining pattern, oxygen tolerance, comprehen- sive biochemical characterization, and metabolic end- product analysis by gas-liquid chromatography. The outer cellular membrane protein profile as examined by gel electrophoresis, fluorescence under ultraviolet light, and susceptibility tests to selected antibiotics can be needed for identification of some species (Engelkirk et al. 1992). Marketed packaged kits that test for pre- formed enzymes have also been used for rapid identi- fication of several species. Recently, matrix-assisted laser desorption ionization-time of flight (MALDI- TOF) mass spectrometry (MS), and 16S rRNA gene sequencing have become powerful and accurate tools for identification of bacteria isolated by culture (Seng et al. 2010, 2013; Fournier et al. 2013; Pfleiderer et al. 2013).

Culture analyses of endodontic infections have pro- vided a substantial body of information about the etiology of apical periodontitis, composition of the endodontic microbiota in different clinical conditions, effects of treatment procedures in microbial elimina- tion, susceptibilities of endodontic microorganisms to antibiotics, and so on. Advantages and limitations of the culture method are listed in Table 5.1. As one can tell, some important limitations of culture meth- ods make a comprehensive analysis of the endodontic microbiota difficult to achieve. The difficulties in cul- turing or in identifying many microbial species are of special relevance and deserve more discussion.

5.2.1 Difficulties in culturing: the as-yet-uncultivated majority

Microorganisms survive and reproduce in their natu- ral environments, where their nutritional and physi- ologic needs are met. Successful cultivation of these microorganisms relies on our ability to determine and reproduce their growth requirements in the labora- tory. Unfortunately, not all microorganisms can be cultivated under artificial conditions and this is sim- ply because the nutritional and physiologic needs of most microorganisms are still unknown. There are several instances of microbial ecosystems that were thought to be well-characterized by culture-dependent approaches, but which proved to be far different when assessed by culture-independent techniques (Hugen- holtz and Pace 1996).

Investigations of many aquatic and terrestrial envi- ronments using culture-independent methods have

Molecular Analysis of Endodontic Infections 83

Table 5.1 Advantages and limitations of culture methods

Culture techniques

Advantages Limitations

1. Broad-range nature, identification of unexpected species

2. Allow quantification of all major viable microorganisms in the samples

3. Allow determination of antimicrobial susceptibilities of the isolates

4. Physiologic studies are possible 5. Pathogenicity studies are possible 6. Widely available

1. Impossibility of culturing a large number of extant microbial species 2. Not all viable microorganisms can be recovered 3. Once isolated, microorganisms require identification using a number

of techniques 4. Misidentification of strains with ambiguous phenotypic behavior 5. Low sensitivity 6. Strict dependence on the mode of sample transport 7. Samples require immediate processing 8. Costly, time-consuming and laborious 9. Specificity is dependent on the composition of media and experience

of the microbiologist 10. Extensive expertise and specialized equipment is needed to isolate

strict anaerobes 11. It takes several days to weeks to identify most anaerobic bacteria 12. Bacteria grown artificially may not present the same phenotypes as

those grown in unique biologic systems or niches

revealed that the cultivable members of these systems represent less than 1% of the total extant population (Ward et al. 1990; Amann et al. 1995). These fig- ures have been calculated by comparing the number and types of bacteria directly observed by microscopy with the number and types of bacteria that are culti- vated from the same sample. The discrepancy between the directly observed and the cultivable bacteria has been referred to as the “great plate count anomaly” (Handelsman 2004). This problem assumes promi- nence when one considers that there is a strong bias towards the cultivable minority—it is estimated that more than 99.9% of the microbiologists work on the 1% of cultivable microorganisms (Lewis 2007).

Culture-independent molecular biology methods that involve amplification of the 16S rRNA gene fol- lowed by cloning and sequencing (discussed later) have been used to determine the bacterial diversity in diverse environments. Not surprisingly, the number of recognized bacterial phyla has exploded from the orig- inal estimate of 11 in 1987 to near 61, of which 31 still have no cultivable representatives (Rappe and Giovan- noni 2003; Keller and Zengler 2004; Vartoukian et al. 2010). Of the phyla containing cultivable members, the great majority are still characterized by a large propor- tion of not-yet-cultured representatives (Hugenholtz 2002; Riesenfeld et al. 2004).

Bacteria that are difficult or impossible to cultivate have been classified into two nonexclusive categories: the as-yet-uncultivated phylotypes and the viable but not cultivable (VBNC) bacteria (Puspita et al. 2012). The former category consists of bacterial species with no cultivated representatives, which are known only by a 16S rRNA gene sequence and for which ade- quate conditions for culture have still to be determined. The latter category is represented by bacteria that have already been cultivated and phenotypically character- ized, but whose cells may enter a dormant nondividing state when exposed to certain conditions, particularly when the bacterial cell is under stress. These cells, while still alive, do not grow in artificial media (Oliver 2010).

Several terms, such as uncultivable, as-yet- uncultivated, noncultured, nonculturable, not-yet- cultured, and so forth, have been used to refer to bac- teria that are known only through culture-independent approaches. It has been suggested that terms like not-yet-cultured or as-yet-uncultivated bacteria be used instead of nonculturable or uncultivable bac- teria, because conceptually all bacteria are able to grow under the proper nutritional and physicochem- ical conditions (Clarridge 2004). In this chapter, we refer to these bacteria as as-yet-uncultivated. Also, the term phylotype is used for those as-yet-uncultivated

84 Endodontic Microbiology

species that are known only by a 16S rRNA gene sequence.

As-yet-uncultivated bacteria can be either bacteria that are truly culture-resistant or that are relatively easy to cultivate on ordinary media but for some rea- son have not yet been cultivated (Siqueira and Rôças 2013a). Many bacteria may have not been cultivated and phenotypically characterized merely by chance. A given species occurring in low abundance in the environment can pass unnoticed as more dominant species are detected. Moreover, slow-growing species may be overcome by fast-growing ones and remain undetected. Some species may be difficult to identify by phenotype-based approaches and may have been identified in previous studies only to the genus level or even misclassified.

Even though many species thought to be uncul- tivable were in fact overlooked by culture meth- ods by chance, the problem of “uncultivability” is real and has been focus of intense research. Bacte- ria live in their environments usually organized in biofilm communities with a multitude of interactions among the community members and between them and the environment. In the natural environment, opti- mal conditions for growth are met, including nutrients, growth factors, signaling molecules, oxygen tension, and other physicochemical conditions. Successful cul- tivation of these bacteria in the laboratory depends upon our ability to reproduce these conditions in vitro.

There are several possible reasons why many bacte- rial species remain to be grown in vitro and phenotyp- ically characterized:

1. Lack of essential nutrients, growth factors, and/ or signaling molecules in the artificial culture medium;

2. Overfeeding conditions, so slow-growing species are overcome by faster-growing species;

3. Toxicity of the culture medium itself, which can inhibit the growth of some species;

4. Metabolic dependence on other species for growth; and

5. Disruption of bacterial quorum-sensing and other signaling systems induced by separation of bacteria from biofilm communities on solid culture media (Koch 1997; Connon and Giovannoni 2002; Wade 2002; Siqueira and Rôças 2005a; Vartoukian et al. 2010).

It is worth pointing out that the fact that a given species has not been cultivated does not imply that this species will remain indefinitely impossible to cultivate. For instance, a myriad of obligate anaerobic bacteria were unable to be cultivated 100 years ago, but further developments in anaerobic culturing techniques have to a large extent helped to solve this problem. It must be assumed that no single method or culture medium is suitable for isolating the vast diversity of microorgan- isms present in most environments (Green and Keller 2006). There is a growing trend to develop specific approaches and culture media that allow cultivation of previously uncultivated bacteria, many of which can be clinically important.

Strategies to cultivate the so-called uncultivated bac- teria may rely on application of conditions that are as close as possible to the natural environment from which samples were taken. Recent efforts to accom- plish this have met with some success. Examples of strategies to culture the uncultivated portion of envi- ronmental communities include the following:

1. Use of culture media with little or no added nutri- ents (Vartoukian et al. 2010; Sizova et al. 2012);

2. Long-term cultivation (Hugenholtz 2002; Song et al. 2009; Vartoukian et al. 2010; Puspita et al. 2012);

3. Serial dilution to extinction, which reduces the inoculum size and decreases the chances of compe- tition by faster-growing species present in the com- munity (Puspita et al. 2012; Sizova et al. 2012);

4. Addition of specific growth factors in the culture media (Gibbons and Macdonald 1960; Wyss 1989); and

5. In vivo incubation (Kaeberlein et al. 2002; Gavrish et al. 2008; Sizova et al. 2012).

5.2.2 Difficulties in identification: species with uncommon phenotypes

Successful cultivation of a given microorganism does not necessarily mean that this microorganism can be successfully identified. Culture-dependent identifica- tion is based on phenotypic traits observed in reference strains, with predictable biochemical and physical properties under optimal growth conditions. However, many phenotype-related factors can lead to difficulties in identification and even to misidentification:

Molecular Analysis of Endodontic Infections 85

1. Not all strains within a given species may exhibit a common phenotype, with some strains showing a divergent behavior (Beighton et al. 1991; Tanner et al. 1992);

2. Strains of different species may show a similar phe- notype characterizing a convergent behavior (Tan- ner et al. 1992; Siqueira and Rôças 2005a);

3. The phenotype is not static and can change under some conditions, such as stress (Ochman et al. 2005; Petti et al. 2005);

4. The same strain may show different results after repeated tests (Tardif et al. 1989);

5. Databases do not usually include newly named species and, obviously, as-yet-uncharacterized species;

6. Test results are sensitive to even small alterations in the assay, with consequent false results (Bosshard et al. 2004); and

7. Test results rely on individual interpretation and expertise (Bosshard et al. 2004).

As a consequence of these factors, phenotype-based identification does not always allow an unequivocal identification.

The 16S rRNA gene sequencing approach has become the reference method for bacterial identifi- cation and taxonomy (Patel 2001; Clarridge 2004). In addition to being widely used to identify both cul- tivable and as-yet-uncultivated bacteria without the need for cultivation, the 16S rRNA gene sequencing approach can also be used for identification of bacteria isolated by culture. By this method, an isolate can be identified after obtaining its 16S rRNA gene sequence and comparing it with sequences deposited in well- annotated and adequately maintained public databases, like the Human Oral Microbiome Database (HOMD) or Greengenes (see later). This molecular technique can provide a more precise and reliable identification of bacteria that are difficult to identify or that cannot be precisely identified by available phenotypic tests (Tang et al. 1998; Drancourt et al. 2000; Bosshard et al. 2003; Song et al. 2003; Petti et al. 2005; Siqueira and Rôças 2005a). Also, the 16S rRNA gene sequencing approach has the advantage of being able to accurately identify rare isolates, poorly described bacteria, as-yet- uncultivated and uncharacterized bacteria, and newly named species.

Some of the as-yet-uncultivated bacteria revealed by molecular studies are actually cultivable but as- yet-uncharacterized species that can grow in ordinary

culture media and have not been previously isolated by chance. Studies using culture followed by identifica- tion of the isolates by 16S rRNA gene sequencing have revealed many species-level taxa that were previously identified only by culture-independent approaches and regarded as uncultivated phylotypes (Munson et al. 2002, 2004; Siqueira et al. 2007; Tanner et al. 2011). Siqueira et al. (2007) used ordinary anaerobic culture coupled with 16S rRNA identification for analysis of endodontic infections and found isolates from the gen- era Prevotella, Fusobacterium, and Actinomyces that were previously deemed as uncultivated phylotypes. In a comprehensive study of the bacteria isolated from severe early childhood caries, Tanner et al. (2011) iden- tified more than 5000 isolates using 16S rRNA gene sequencing and found 45 previously uncultivated taxa; most of the previously uncultivated taxa belonged to the genera Streptococcus, Selenomonas, Actinomyces, and Capnocytophaga.

5.3 Molecular biology techniques

The recognition that the as-yet-uncultivated microbial world far outsizes the cultivable world has caused a great revolution in microbiology. Fortunately, tools and procedures have become available and substan- tially improved to achieve a more realistic description of this unseen world.

There are a plethora of molecular biology methods for the study of microorganisms and the choice of a particular approach depends on the questions being addressed. This chapter restricts discussion to the most commonly used approaches applied to the research of the endodontic microbiome and some with potential to be used with this intent.

5.3.1 Impact in medical microbiology

A significant contribution of molecular biology meth- ods to medical microbiology relates to the iden- tification of previously unknown human pathogens (Fredricks and Relman 1999; Relman 1999). Further- more, molecular studies have revealed a previously unanticipated breadth of bacterial diversity associated with the human body. It has been demonstrated that 20–80% of the species-level bacteria identified by 16S rRNA gene sequencing, depending on the human body site, still remain to be cultivated in vitro (Dethlefsen et al. 2007). Surveys of the human gut microbiome

86 Endodontic Microbiology

have shown that each individual may harbor 500– 3000 bacterial species in the gut, 80% of which are composed of taxa that have not yet been cultivated and characterized (Suau et al. 1999; Eckburg et al. 2005; Dethlefsen et al. 2008). As for other body sites, uncultivated phylotypes have been shown to represent about 40–50% of the species-level taxa detected in the esophagus (Pei et al. 2004), stomach (Bik et al. 2006), vagina (Verhelst et al. 2004; Fredricks et al. 2005), and skin (Dekio et al. 2005). Studies of the human oral microbiome have demonstrated that about 40–60% of the approximately 1000 bacterial species inhabiting the oral cavity still remain to be grown in the labo- ratory (Siqueira and Rôças 2013a). The HOMD lists over 200 oral taxa as still unnamed and uncultivated (Dewhirst et al. 2010). It is not difficult to realize that a number of as-yet-uncharacterized pathogens occur in this uncultivated proportion of the human micro- biome. Actually, many as-yet-uncultivated phylotypes have been associated with oral and extraoral diseases (Siqueira and Rôças 2013a).

5.4 Gene targets for microbial identification

Each living organism carries sequences within cer- tain genes that are uniquely and specifically present only in its own species. Indeed, each particular indi- vidual within a species also has its signature DNA sequences. These unique sequences bring important genomic information that makes it possible to iden- tify each species, and even each individual within a species, by using molecular biology methods.

Molecular approaches for microbial identification rely on certain genes that contain revealing information about the microbial identity. Ideally, a gene to be used as a target for microbial identification should contain regions that are unique to each species. Genes encod- ing housekeeping functions are preferable to infer phy- logenetic classification because they are usually ubiq- uitous and tend to exhibit functional constancy, evolv- ing slowly with time (Woese 2000; Wade 2004).

Several genes have been chosen as targets for bacte- rial identification. Some of these genes are shared by a vast majority, if not all, bacterial species. Genes pro- posed for bacterial identification include the 16S rRNA and 23S rRNA genes, the 16S-23S rRNA gene inter-

nal transcribed sequences (ITS), the rpoB gene encod- ing the β-subunit of RNA polymerase, the groEL gene encoding the heat-shock protein, the gyrB gene encod- ing the β-subunit of DNA gyrase, the tuf gene, and homologous recombination-encoding recA (Ke et al. 1999; Drancourt and Raoult 2005). Of these, the gene encoding the 16S rRNA has been widely accepted and used for bacterial identification.

Following the pioneer studies by Woese (1987), the genes encoding rRNA molecules, which are present in all cellular forms of life, namely, the domains Bac- teria, Archaea, and Eucarya, have been extensively used for comprehensive identification of virtually all living organisms and inference of their natural rela- tionships. The rRNA is the central component of the highly complex translation apparatus of the cell, and because fidelity and maintenance of this translation function are critical, some regions of the rRNA are so highly conserved that they can be used to align genes from different organisms (Woese 2000). Other regions less critical to translation of the code are under less selective pressure and show enough variation so that each species has a unique sequence. The advantages of using the small subunit rRNA genes for microbial identification is that it is found in all organisms, is long enough to be highly informative and short enough to be easily sequenced (particularly with the advent of automated DNA sequencers), and affords reliability for inferring phylogenetic relationships (Woese 1987). Thus, the 16S rRNA gene (or 16S rDNA) of Bacteria and Archaea and the 18S rRNA gene (or 18S rDNA) of fungi and other eukaryotes have been extensively examined and used for identification and phylogenetic studies. The conserved regions of these genes are vir- tually identical in all representatives of each domain, while the variable regions contain unique signatures of the genus and species of the organism.

Data from small subunit rRNA gene sequences can be used for accurate and rapid identification of known and unknown bacterial species, using techniques that do not require cultivation. For instance, the 16S RNA gene of virtually all bacterial species in an envi- ronment, including as-yet-uncultivated and unchar- acterized bacteria, can be amplified by polymerase chain reaction (PCR) using broad-range (or universal) primers that are complementary to conserved regions of this gene. Sequencing of the variable regions flanked by the broad-range primers will provide information for accurate bacterial identification. Primers or probes

Molecular Analysis of Endodontic Infections 87

Fig. 5.1 The 16S rRNA gene (rDNA). Areas in orange correspond to variable regions, which contain information about the genus and the species. Primers designed on these regions are used in species-specific assays. Red areas correspond to conserved regions of the gene. Primers designed on these areas are used in broad-range assays.

that are complementary to variable regions can also be designed to detect specific target species or groups of species directly in clinical samples (Figure 5.1). The analytical sensitivity of most species- or group- specific PCR assays is usually higher than broad-range PCR assays (Maiwald 2004).

5.5 PCR and its derivatives

The PCR process was conceived by Kary Mullis in 1983 and ever since has revolutionized the field of molecular biology by enabling the amplification of as few as one copy of a gene into millions to billions of copies of that gene in just a matter of minutes to a few hours (Mullis et al. 1994). The impact of PCR on biological and medical research has been remarkable. Nowadays, it is possible to isolate essentially any gene from any organism using PCR, which makes this tech- nique a cornerstone of genome sequencing projects (Lee and Tirnady 2003). Since its introduction, PCR has spawned an increasing number of associated tech- nologies for diverse applications. Perhaps the most widespread advance in clinical diagnostic technology has come from the application of PCR for detection of microbial pathogens (Whelen and Persing 1996; Tang et al. 1997; Louie et al. 2000).

The PCR method is based on the in vitro replication of DNA through repetitive cycles of DNA melting,

primer annealing, and extension steps. Briefly, the method consists of three steps that are repeated in several cycles of amplification:

1. The target DNA serving as template is melted (con- verted from double-strand helix into single strands) at temperatures high enough to break the hydrogen bonds holding the strands together, thus liberating single strands of DNA.

2. Two short oligonucleotides (primers) anneal to complementary sequences on opposite strands of the target DNA. Primers define the two ends of the amplified stretch of DNA.

3. A complementary second strand of new DNA is synthesized through the extension of each annealed primer by a thermostable DNA polymerase in the presence of excess deoxyribonucleoside triphos- phates. All previously synthesized products act as templates for new primer-extension reactions in each ensuing cycle. The result is the exponential amplification of new products.

PCR has unrivaled sensitivity. While it can detect as few as 10 bacterial cells in a sample, other meth- ods of identification show much higher detection lim- its. For instance, culture using nonselective media can detect 104–105 cultivable cells in a sample (Zambon and Haraszthy 1995). It is easy to understand this low sensitivity if we do some calculations. After 10-fold serial dilutions of the sample for cultivation, bacterial

88 Endodontic Microbiology

counts are usually performed on plates containing 30– 300 colonies. As a consequence, a single colony would represent 0.3–3% of the total cultivable population. A negative result for a target species usually means that it was absent or at least present at lower amounts (i.e., <0.3% of the population). Based on the calculation that we can detect one colony of a given species among 300 colonies grown on the surface of a blood agar plate (0.3% of the population), then in a root canal sample containing a total number of 108 cells, roughly 3 × 105 cells of this species must be present so that it can be detected by culture. When selective media are used, the sensitivity of culture method can increase to 103 cells (Zambon and Haraszthy 1995). Immunologic methods have a detection limit ranging from 103 to 104 cells. DNA–DNA hybridization assays can detect 103–104

cells in a sample. Thus, PCR methodology is at least 10- to 100-fold more sensitive than the other more sensitive identification method (Siqueira and Rôças 2003d).

There are several methods to check if the intended PCR product was generated. The most commonly used method for detecting PCR products is electrophore- sis in an agarose gel. Aliquots of the PCR reac- tion are loaded into the gel and an electrical gradi- ent is applied through a buffer solution. The products migrate through the gel according to size, with larger products running a shorter distance in the gel because they experience more resistance in the gel matrix. DNA ladder digests represent DNA fragments of known size and are run in the same gel to serve as molecular size standard. This allows the size of the PCR products to be estimated. The gel is stained (with ethidium bromide, SYBR stain, or GelRed) and viewed under ultraviolet transillumination. Designed primers are expected to generate a PCR product of a given size and observation of a band of the predicted size in the electrophoretic gel is consistent with a positive PCR result. Identity of PCR products should be confirmed preferentially by sequencing of the PCR product.

Numerous derivatives in PCR technology have been developed since its inception. The most used PCR- derived assays are described in the following sections.

5.5.1 Species-specific PCR

One of the simplest approaches to detect a target species in a sample is to use a species-specific PCR assay. By this method, primers designed to anneal to signature genomic DNA sequences of a given species

are used to detect this species directly in clinical sam- ples even in the presence of a background of non- targeted species and without the need for cultivation. Most assays use the variable regions of the 16S rRNA gene to design primers specific for bacterial species. Using public databases that contain the 16S rRNA gene sequences from a vast number of oral bacteria, primers can be designed to specifically detect virtually every cultivable and as-yet-uncultivated oral species. The presence of a species-specific PCR product of predicted size is usually determined by agarose gel electrophoresis and represents a positive result for the occurrence of the target species in the sample. The best way to check the specificity of the assay is by sequencing the PCR product. This approach can be used not only in single PCR assays, but also in nested PCR and multiplex PCR, furnishing qualitative results (presence or absence) about one (in single and nested PCR techniques) or more (the multiplex tech- nique) target species. Species-specific detection can also be performed using a quantitative real-time PCR assay, which detects and monitors the appearance of the amplification product throughout the reaction.

Specificity of the PCR assay can be increased by using a touchdown procedure. By this approach, the annealing temperature in the initial PCR cycle is set several degrees above the calculated melting temper- ature (Tm) of the primers. In subsequent cycles, the annealing temperature is decreased in steps of 0.5–2◦C per cycle until a temperature is reached that is equal to, or 2–5◦C below, the Tm of the primers. Touchdown techniques have been considered useful to avoid the amplification of spurious DNA fragments (non-rRNA gene fragments and/or fragments with improper sizes) (Don et al. 1991).

5.5.2 Multiplex PCR

Most PCR assays have concentrated on the detection of a single species by means of individual reactions. Mul- tiplex PCR is a process where multiple primer pairs are used to simultaneously amplify several sequences in a single reaction (Chamberlain et al. 1988). As more than one unique target sequence in a clinical specimen can be amplified at the same time, mul- tiplex PCR assays permit the concomitant detection of different species. Multiplex PCR assays have been used to minimize the time and expenditure needed for detection approaches. Primers used in multiplex assays must be designed carefully to have similar annealing

Molecular Analysis of Endodontic Infections 89

temperatures and avoid complementarity among them (Dieffenbach and Dveksler 1995; Hayden 2004).

5.5.3 Nested PCR

Nested PCR (nPCR) is a conventional PCR method that amplifies a target region of DNA with an outer primer pair in an initial reaction, followed by a sec- ond amplification using an internal primer pair (Haqqi et al. 1988). The first PCR products are used as tem- plate in the second round of amplification with a sep- arate primer set, which anneals internally to the first products and generates a shorter amplified fragment. This approach shows increased sensitivity when com- pared to single PCR. Increased sensitivity is due to the large total number of cycles. In addition, target DNA is amplified in the first round of amplification, with sub- sequent reduction of nontargeted DNA and inhibitors present in the sample. The set of primers used in the second round of PCR results in additional speci- ficity. The second reaction is performed with reduced background of eukaryotic DNA and other regions of the bacterial DNA (Siqueira and Rôças 2003d). Even if nonspecific DNA amplification occurs in the first round of amplification, the nonspecific PCR product does not serve as template in the second reaction, because it is highly unlikely to possess regions of DNA complementary to the second set of specific primers (McPherson and Moller 2000).

5.5.4 Reverse transcriptase PCR

Reverse transcriptase PCR (RT-PCR) was developed to amplify RNA targets and exploits the use of the enzyme reverse transcriptase, which can synthesize a strand of complementary DNA (cDNA) from an RNA template. Most RT-PCR assays employ a two-step approach. In the first step, reverse transcriptase con- verts RNA into single-stranded cDNA. In the second step, PCR primers, DNA polymerase, and nucleotides are added to create the second strand of cDNA. Once the double-stranded DNA is formed, it can be used as template for amplification as in conventional PCR (Sambrook and Russell 2001). The RT-PCR process may be modified into a one-step approach by using it directly with RNA as the template. In this approach, an enzyme with both reverse transcriptase and DNA polymerase activities is used, such as that from the bacteria Thermus thermophilus (Tth).

5.5.5 Quantitative PCR

Conventional PCR assays are qualitative or can be adjusted to be semiquantitative. Of the PCR technolo- gies that provide quantitative results, the real-time PCR method has been the most widely used. This is a high- throughput technique that is more accurate and precise than the other qPCR assays, and requires no post-PCR manipulation steps, reducing the risks of contamina- tion (Raoult et al. 2004; Sharma et al. 2007). Real- time PCR assays allow the quantification of individ- ual target species as well as total bacteria in clinical samples.

Basically, by adding a fluorescent dye to the assay and monitoring the appearance of fluorescence dur- ing the reaction, the amount of synthesis of new DNA can be measured. The fluorescent signal is propor- tional to the amount of DNA being synthesized and is measured in a closed tube format by a fluorimeter combined to the PCR thermocycler. There are several different real-time PCR approaches. The most used real-time PCR chemistries are SYBR-Green (Higuchi et al. 1992) and TaqMan (Heid et al. 1996). SYBR- Green is the simplest and most affordable method, and consists of a fluorescent dye that binds to double- stranded DNA. During extension, increasing amounts of dye bind to the increasing amount of newly formed double-stranded DNA. Fluorescence is measured at the end of the extension step of every PCR cycle to moni- tor the increasing amount of amplified DNA. Dye that remains unbound exhibits little fluorescence in solu- tion. The SYBR-Green assay is very sensitive but has diminished specificity, as the dye binds to all double- stranded DNA present, and primer dimers may result in a false reading (Bustin 2000). However, a study found no significant difference between the TaqMan and SYBR-Green chemistry with regard to specificity, quantitativity, and sensitivity for detection of oral bac- teria (Maeda et al. 2003). The advantage of the SYBR- Green assay over the TaqMan assay is that the proto- col is usually simpler and less expensive (Maeda et al. 2003).

The TaqMan method can be more specific than the SYBR-Green assay. Increased specificity of the TaqMan assay results from the utilization of a spe- cific labeled oligonucleotide probe along with the primers (Holland et al. 1991; Heid et al. 1996). The TaqMan probe is a 20- to 30-base-long oligonu- cleotide sequence that specifically anneals to a sequence flanked by the two primers. The TaqMan

90 Endodontic Microbiology

probe contains a reporter fluorescent dye at the 5′

end and a quencher dye at the 3′ end that quenches the emission spectrum of the reporter dye. As long as the probe remains unbound, it is intact and no signal is generated. During the extension step of real-time PCR, the Thermus aquaticus (Taq) DNA polymerase enzyme cleaves the TaqMan probe, resulting in sepa- ration of the reporter from the quencher. This results in increased fluorescence emission.

5.5.6 PCR-based microbial typing

PCR technology can also be used for clonal anal- ysis of microbial isolates. An example of the PCR techniques used for this purpose includes the arbitrar- ily primed PCR (AP-PCR), also referred to as ran- dom amplified polymorphic DNA (RAPD) (Welsh and McClelland 1990; Power 1996). AP-PCR is a relatively rapid tool to determine whether two iso- lates of the same species are related. This method is based on the use of a single 10- to 20-base-long random sequence primer that anneals to unspecified DNA target sites under conditions that allow for mis- matched base-pairing. The use of a random-sequence primer at low stringency allows for priming at sites with imperfect matches. Amplification will only occur when two primers anneal close enough to one another, in the proper “forward” and “reverse” directions nec- essary for the product to be formed. Genetic variations between two DNA templates result in discriminative DNA fingerprints because of the differences in the priming sites. The amplicons generated form a strain specific pattern of about 5–15 bands per species in the electrophoretic gel (Spiegelman et al. 2005). The advantage of AP-PCR is its ability to furnish highly specific DNA profiles with no prerequisite for know- ing the DNA sequences. Primers may also be designed to target known genetic elements, such as enterobacte- rial repetitive intergenic consensus sequences (ERIC- PCR) (de Bruijn 1992; Arora et al. 1996) and repetitive extragenic palindromic sequences (REP-PCR) (Hig- gins et al. 1982). Clonal analysis may help elucidate whether certain strains of a given species are more associated with signs or symptoms of a given disease. Clonal analysis also may help to track the origin of microorganisms infecting a given site. For instance, by comparing bacterial strains isolated from the root canal and the gingival sulcus or other oral sites, one can have information as to where bacteria present in the root canal system came from. Clonal analysis can also

track the origin of the microorganisms present in a sus- pected focal disease by comparing the isolates found in the secondarily infected site with others present in the suspected original focus of infection.

5.5.7 Broad-range PCR and clone library analysis

Broad-range PCR has been extensively used to inves- tigate the whole microbial diversity in diverse envi- ronments. In broad-range PCR, primers are designed to be complementary to conserved regions of a par- ticular gene that is shared by a group of microorgan- isms. For instance, primers that are complementary to conserved regions of the 16S rRNA gene have been used with the intention of exploiting the variable inter- nal regions of the amplified sequence for sequenc- ing and further bacterial identification (Göbel 1995). The strength of broad-range PCR lies in the relative absence of selectivity, so that (in principle) any kind of bacteria present in a sample can be detected and iden- tified. This aspect is in analogy to cultivation using nonselective nutrient media and in contrast to species- specific molecular approaches (Maiwald 2004). Thus, broad-range PCR can detect the unexpected and in this regard it is far more effective and accurate than culture. Broad-range PCR has allowed the identification of sev- eral novel fastidious or as-yet-uncultivated bacterial pathogens directly from diverse human sites (Relman 1997; Pitt and Saunders 2000; Wade 2002). The most commonly used protocol is as follows. Initially, bulk DNA is extracted directly from samples. Afterwards, the 16S rRNA gene is isolated from the bulk DNA via PCR with primers specific for conserved regions of the gene (broad-range or universal primers). Ampli- fication of long fragments generally results in less sensitivity, but provides more variable sequence infor- mation for accurate identification and may reduce the risks of amplifying DNA from contaminants in PCR reagents, which appear to be fragmented into smaller sizes (Maiwald 2004). Because broad-range primers are used, a mixture of the 16S rRNA gene amplified from nearly all bacteria in the sample is formed. In mixed infections, direct sequencing of the PCR prod- ucts should not be performed because there are mixed products from the different species composing the con- sortium. PCR products are then cloned into a plas- mid vector, which is used to transform Escherichia coli cells, establishing a library of 16S rRNA genes from the sample. The cloning procedure is used to

Molecular Analysis of Endodontic Infections 91

separate the sequences so that they can be character- ized individually by sequencing.

DNA sequencing is usually performed by the Sanger approach, which comprises the following steps: DNA purification; DNA synthesis and labeling using the chain termination method with dye-labelled dideoxynucleotides (ddNTPs); capillary electrophore- sis; and fluorescence detection. After the cloned genes are individually sequenced, the obtained sequences are submitted for identification to databases, usually via the World Wide Web, such as the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/), the Ribosomal Database Project (RDP) (http:// rdp.cme.msu.edu/), SILVA (www.arb-silva.de), and Greengenes (http://greengenes.lbl.gov) databases. Currently, there are also phylogenetically curated databases specific for the oral microbiome, includ- ing HOMD (www.homd.org) (Dewhirst et al. 2010) and CORE (http://microbiome.osu.edu) (Griffen et al. 2011). Preliminary identification can be performed by using similarity searches in a public database. A 98.5– 99% identity in 16S rRNA gene sequence has been the most accepted criterion used to identify a bacterium to the species level (Drancourt et al. 2000, 2004; Paster et al. 2006).

If a sequence exhibits low similarity scores (<98.5– 99%) to the other sequences from defined species or phylotypes in public databases, it potentially repre- sents a new species (Drancourt et al. 2000, 2004; Paster et al. 2006). These new species are usually considered as uncultivated and hitherto unknown bac- terial taxa and an unofficial name is assigned. As- yet-uncultivated phylotypes cannot be given a formal species name, because naming a species requires culti- vation for phenotypic characterization. There is no rule for naming phylotypes, so there may be a huge redun- dancy when evaluating separate studies, with different authors giving different names for the same phylo- types. One of the purposes of the HOMD is to organize information for oral bacteria, creating a provisional naming system (Dewhirst et al. 2010).

As broad-range primers are used, there is a high risk for amplification of contaminant DNA from sources other than the site sampled (Millar et al. 2002). Con- taminating DNA can be introduced by inadvertent tube-to-tube contamination or by the use of contam- inated reagents. In PCR experiments, in general, and especially in broad-range reactions, a number of pre- cautions are necessary to avoid contamination: sep- arate room for pre- and post-PCR work; ultraviolet

decontamination of surface areas; use of high-quality reagents, including negative controls with no tem- plate in every reaction; and adequate sampling tech- niques and vials for clinical specimens (Dragon 1993; McPherson and Moller 2000; Millar et al. 2002; Mai- wald 2004).

5.5.8 Phylogenetic tree

In addition to the similarity search in public databases, phylogenetic analysis should be accomplished because it provides a much more accurate assessment (Lepp and Relman 2004; Maiwald 2004). Phylogeny can be regarded as the evolutionary history of a group of organisms. Because evolution involves inherited nucleotide sequence changes, analyses of differences in DNA sequences among microorganisms allows for reconstruction of their phylogenetic history. Phylo- genetic analysis is based on homology of nucleotide sequences, which are related by common ancestry. In the phylogenetic analysis of a group of bacteria, the 16S rRNA gene sequences from different bacteria are aligned based on conserved regions and the resulting mismatches, insertions, and deletions provide infor- mation on how the sequences have diverged over the years from a common ancestral sequence. Computa- tions based on the number of dissimilarities present in variable regions are used to construct a phylogenetic tree (Leys et al. 2006). The relationships among vari- ous bacterial phylotypes (or taxa) are shown in dendro- grams, or phylogenetic trees, such as that depicted in Figure 5.2. The 16S rRNA gene can be used to estab- lish phylogenetic relationships among bacteria even when sequences are derived from previously uncul- tivated and uncharacterized bacteria. In this partic- ular case, phylogenetic trees are important adjuncts for identification of sequences belonging to as-yet- uncharacterized phylotypes, permitting assignment of phylogenetic classification to some level.

The phylogenetic tree depicts relationships between different species and is composed of nodes and branches. The internal nodes represent points in the evolutionary history where an ancestor diverged and gave rise to two new taxa. These two taxa then start to accumulate differences over their independent evolution. The branch length comprises the number of nucleotide changes that have occurred over the evolutionary history of the taxon. Some researchers also include an outgroup to root the tree, which is usually a bacterial species less related to the other

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92 Endodontic Microbiology

Fig. 5.2 Phylogenetic tree based on 16S rRNA gene comparisons showing several candidate endodontic pathogens, their respective phyla, and the clinical conditions they have been associated with. Scale bar shows number of nucleotide substitutions per site.

Molecular Analysis of Endodontic Infections 93

species included in the analysis. A horizontal scale is usually provided to give an approximate measure of genetic distance. The vertical distance in the tree is of no importance.

5.6 Denaturing gradient gel electrophoresis

Techniques for genetic fingerprinting of microbial communities can be used to determine the diversity of different microorganisms living in diverse ecosys- tems and to monitor microbial community behavior over time. A commonly used strategy for genetic fin- gerprinting of complex microbial communities encom- passes DNA extraction, amplification of the 16S rRNA genes using broad-range primers, and then the analy- sis of PCR products by denaturing gradient gel elec- trophoresis (DGGE) (Siqueira et al. 2010).

In DGGE, DNA fragments of the same length but with different sequences can be separated (Myers et al. 1985; Muyzer et al. 1993). The DGGE technique is based on electrophoresis of PCR-amplified 16S rRNA gene (or other genes) fragments in polyacrylamide gels containing a linearly increasing gradient of DNA denaturants (a mixture of urea and formamide). As the PCR product migrates in the gel, it encounters increas- ing concentrations of denaturants and, at some position in the gel, it will become partially or fully denatured. Partial denaturation causes a significant decrease in the electrophoretic mobility of the DNA molecule. Molecules with different sequences may have a differ- ent melting behavior and will therefore stop migrating at different positions in the gel. The position in the gel at which the DNA melts is determined by its nucleotide sequence and composition (Gasser 1998). A GC-rich sequence (GC-clamp) is added to the 5′ end of one of the primers used in the PCR reaction and makes the DNA unable to denature completely in the gel (Muyzer and Smalla 1998). DNA bands in DGGE can be visu- alized using ethidium bromide, SYBR-Green, or silver staining.

In DGGE, multiple samples can be analyzed con- currently, making it possible to compare the structure of the microbial community of different samples and to follow changes in microbial populations over time, including after antimicrobial treatment (Siqueira et al. 2005d). If identification is desired, specific bands can

be excised from the gels, re-amplified and sequenced (Machado de Oliveira et al. 2007a).

5.7 Terminal restriction fragment length polymorphism

The terminal restriction fragment length polymor- phism (T-RFLP) approach can be used to explore the microbial diversity in the environment and pro- vide insight into the structure of microbial commu- nities over time and after treatment (Marsh 1999). T- RFLP analysis measures the size polymorphism of terminal restriction fragments from a PCR amplified marker. When T-RFLP is used to analyze bacterial communities, PCR is first carried out to amplify the 16S rRNA gene from different species in the sample using one of the primers labeled with a fluorescent dye (Clement et al. 1998). PCR amplicons are then digested with restriction enzymes, generating fluores- cently labeled fragments of different lengths (the ter- minal fragments). These fragments are separated on high resolution sequencing gels in an automated DNA sequencer, which is used to read both the size and the intensity of terminally labeled restriction fragments (T- RF), creating a typical profile. In such a profile, size is represented on the horizontal axis and intensity (rela- tive to the abundance of a given fragment size) is repre- sented on the vertical axis (Spiegelman et al. 2005). In theory, each T-RF represents a single species. Exten- sive databases exist for 16S rRNA gene sequences and can be used to identify all T-RFs predicted from known sequences, considering a given set of primers and restriction enzymes (Matsumoto et al. 2005). T- RF lengths are predicted by finding the restriction site closest to the site where the labeled primer will anneal and counting the number of nucleotides in between. Multiple restriction enzymes (four or five) are usually necessary to provide reliable identification because distinct species may generate the same T-RF when only one enzyme is used (Sakamoto et al. 2005; Siqueira et al. 2010).

Through application of automated DNA sequencer technology, T-RFLP has considerably greater resolu- tion than gel-based community fingerprinting tech- niques, such as DGGE (Clement et al. 1998; Marsh 1999). The digital output of the method also elimi- nates an element of human error from the analysis process (Spiegelman et al. 2005).

94 Endodontic Microbiology

5.8 DNA–DNA hybridization assays

DNA–DNA hybridization methodology is the process of annealing the complementary bases of two single- stranded DNA molecules. It employs labeled single- stranded DNA probes that can locate and bind to a tar- get sequence, forming a new duplex molecule, which can then be detected (Li and Hanna 2004). Probes can be constructed from either whole genomic DNA or oligonucleotides. Whole genomic probes are more likely to cross-react with nontargeted microorganisms because of the presence of homologous sequences between different species. Oligonucleotide probes based on signature sequences of specific genes (such as the 16S rRNA gene) usually displays higher speci- ficity, because the known probe sequence allows calcu- lation of stringent hybridization temperatures and mis- matches are not tolerated because of the considerable reduction of bond strength between the short probe and the target (Theron and Cloete 2000; Juretschko et al. 2004). Also, oligonucleotide probes can differentiate between closely related species or even subspecies and can be designed to detect as-yet-uncultivated bacteria.

Some DNA–DNA hybridization techniques are suit- able for large-scale clinical studies. They include the checkerboard DNA–DNA hybridization and DNA microarray techniques.

5.8.1 Checkerboard DNA–DNA hybridization

This technique was introduced by Socransky et al. (1994) for hybridizing large numbers of DNA samples against large numbers of digoxigenin-labeled whole genomic DNA on a single support membrane. Briefly, denatured DNA from clinical samples is loaded in lanes on a nylon membrane using a Minislot appara- tus. After fixation of the samples to the membrane, the membrane is placed in a Miniblotter 45 apparatus with the lanes of samples at 90◦ to the lanes of the device. Digoxigenin-labeled whole genomic DNA probes are then loaded in individual lanes of the Miniblotter. After hybridization, the membranes are washed at high strin- gency and the DNA probes detected using antibody to digoxigenin conjugated with alkaline phosphatase and chemifluorescence or chemiluminescence detec- tion. The presence of a spot on the membrane in the crossing lanes means that hybridization of one probe to one sample occurred. The intensity of the spot is proportional to the amount of DNA from the target

species in the sample. The checkerboard method per- mits the simultaneous determination of the presence of a multitude of bacterial species in single or multiple clinical samples.

A modification of the checkerboard method was proposed by Paster et al. (1998) and consists of a PCR-based, reverse-capture checkerboard hybridiza- tion methodology. In this assay, the probe rather than the sample is fixed first to the membrane – thus the name “reverse-capture checkerboard.” The procedure circumvents the need for in vitro bacterial culture, nec- essary for preparation of whole genomic probes in the original checkerboard approach. Up to 30 reverse- capture oligonucleotide probes that target regions of the 16S rRNA gene are deposited on a nylon mem- brane in separate horizontal lanes using a Minislot apparatus. Probes are synthesized with a polythymi- dine tail, which are cross-linked to the membrane via ultraviolet irradiation or heat, leaving the probe available for hybridization. The 16S rRNA gene from clinical samples is PCR amplified using one of the primers labeled with digoxigenin. Hybridizations are performed in vertical channels in a Miniblotter appara- tus with digoxigenin-labeled PCR amplicons from up to 45 samples. Hybridization signals are detected using chemifluorescence or chemiluminescence procedures. The reverse-capture checkerboard has the potential to be more specific than the original method, mostly because of the use of oligonucleotide probes. Further- more, oligonucleotide probes can be designed to detect both cultivable and as-yet-uncultivated bacteria, while in the original method using whole genomic probes only cultivable species are targeted.

5.8.2 DNA microarrays

In essence, DNA microarrays can be regarded as a miniaturization of the checkerboard approach (Leys et al. 2006). DNA microarrays were first described in 1995 (Schena et al. 1995) and consist of a high-density matrix of DNA probes which are printed or synthesized on a glass or silicon slide (Mothershed and Whitney 2006). Targets incorporate either a fluorescent label or some other moiety that permits subsequent detection with a secondary label. Targets are applied to the array and those that hybridize to complementary probes are detected using some type of reporter molecule. Fol- lowing hybridization, arrays are imaged using a high- resolution laser scanner and analyzed by sophisticated computer software programs.

Molecular Analysis of Endodontic Infections 95

DNA microarrays can be used to enhance PCR product detection and identification. When broad- range PCR is used to amplify DNA from samples of mixed infections, microarrays can then be used to identify the PCR products by hybridization to an array that is composed of hundreds to thousands of species-specific probes (Palmer et al. 2006). A DNA microarray approach to detect oral species has been developed—the Human Oral Microbe Identification Microarray (HOMIM) (Boches et al. 2006). In this assay, 16S rRNA gene-based oligonucleotide probes targeting about 300 oral species/phylotypes are printed on aldehyde-coated glass slides. Broad-range PCR is used to amplify the 16S rRNA genes from clinical sam- ples. PCR products are labeled with a fluorescent dye (Cy3-dCTP) in a second nested PCR. When labeled bacterial DNA hybridizes to a species-specific probe on the slide, it provides a fluorescent signal that can be read with a microarray laser scanner. By using this method, a single hybridization approach can provide results as to the presence and levels of 300 target species/phylotypes at a time (Colombo et al. 2012; Lourenco et al. 2014).

5.9 Fluorescence in situ hybridization

This method uses fluorescently labeled rRNA-directed probes and fluorescence microscopy to detect intact microbial cells directly in clinical specimens, in situ (Moter and Gobel 2000). One of the greatest advantages of using fluorescence in situ hybridiza- tion (FISH) is that this technique allows identifica- tion while providing information about morphology, number, community architecture, and spatial relation- ships of microorganisms (Amann et al. 2001). Because oligonucleotide probes can be designed for use, FISH not only allows the detection of cultivable species, but also of as-yet-uncultivated phylotypes (Moter et al. 1998a,b). In FISH, microbial cells are fixed and then hybridized with rRNA-directed probes on a glass slide. Probes are short (15–25 base-long), labeled covalently at one end with a fluorescent dye. After stringent wash- ing, cells are visualized by using a conventional epi- fluorescence microscope or a confocal laser scanning microscope. Multiple species-specific probes, each labeled with different colored fluorescent dyes, can be used at the same time.

5.10 Next-generation DNA sequencing technologies

Recently, new technologies have become available that permit massive DNA sequencing with a much higher throughput than the conventional Sanger sequenc- ing approach. The first commercially available Next- Generation DNA Sequencing (NGS) platform was introduced in 2005 and as of then NGS methods have continuously evolved to revolutionize the field of genomic analysis (Voelkerding et al. 2009). The most used NGS technologies include the 454 pyrose- quencing (Roche Applied Science), HiSeq or MiSeq (Illumina), and SOLiD (Thermo Fisher). These plat- forms share a common technologic characteristic of performing massively parallel sequencing of clonally PCR amplified products based on physical separa- tion in a flow cell surface. This feature diverges from the classic Sanger technique, which is based on elec- trophoretic separation of chain-termination products.

The 454 pyrosequencing technology has been the most used NGS platform to study the human oral microbiome (Siqueira et al. 2012). This technology is a sequencing-by-synthesis method that involves a combination of emulsion PCR and pyrosequencing. In the pyrosequencing approach, DNA is isolated, fragmented, ligated to special adapters, and sepa- rated into single strands. Emulsion PCR is then car- ried out for clonal amplification. In this approach, an oil–water emulsion is formed in which the aqueous phase contains the PCR reagents and the DNA tem- plate to be sequenced. Capture beads containing one of the oligonucleotide primers attached to them are also included. The other PCR primer is placed in the solu- tion. Beads are included in excess of DNA template molecules, but not of the expected number of droplets to be formed in the emulsion. After controlled and vigorous agitation of the oil–water system, emulsifi- cation takes place and millions of aqueous droplets are formed, within which PCR amplification takes place. Optimization of the concentration of DNA template, beads and water droplets guarantees that only one tem- plate and one bead are included in each droplet. Even- tually, million copies of a unique DNA template are generated on each bead in a clonal PCR amplification (Higuchi et al. 2011).

Next in the process, the emulsion is broken, the DNA is denatured, and beads carrying single-stranded DNA are transferred to the wells of a picotiter plate in such

96 Endodontic Microbiology

a way that permits a single bead to be present in each of the several hundred thousand wells. Because each bead has a fixed location in the plate, each sequenc- ing reaction can be monitored. Beads containing the enzymes used in the pyrosequencing reaction steps are then deposited into each well (Mardis 2008; Rothberg and Leamon 2008).

The pyrosequencing reaction takes place using a mixture of the single-stranded DNA template, the sequencing primer, the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, and the substrates adenosine 5′ phosphosulfate (APS) and luciferin (Figure 5.3). The first one of the four dNTPs is added to the sequencing reaction and the DNA polymerase catalyzes its incorporation into the DNA strand. During each incorporation event, a phospho- diester bond between the dNTPs is formed, releas- ing pyrophosphate (PPi) in a quantity equivalent to the amount of incorporated nucleotide. In sequence, the enzyme ATP sulfurylase converts PPi to ATP in the presence of APS. ATP is used in the con- version of luciferin to oxyluciferin mediated by the enzyme luciferase. This gives rise to light in inten- sity that is proportional to the amount of ATP used. Light is detected by a charge coupled device camera and detected as a peak in a pyrogram. The height of each peak is proportional to the number of nucleotides incorporated. The system is regenerated with the enzyme apyrase, which degrades ATP and unincor- porated dNTPs. Then, the next dNTP is added. Addi- tion of dNTPs is performed one at a time. Generation of a signal indicates which nucleotide is the next one occurring in the sequence. As the process goes on, the complementary DNA strand grows and the nucleotide sequence is determined according to the signal peaks in the pyrogram. Roche discontinued the 454 platform in 2016.

One of the greatest advantages of the NGS approaches over the Sanger sequencing method is that hundreds of thousands of sequence reads can be obtained in a single run, generating sequence informa- tion data that are orders of magnitude larger (Engel- brektson et al. 2010). In NGS methods, sequences from different samples can be identified in the same run using the barcoding multiplex approach, in which unique sequences are incorporated into the primers and barcoded amplicons are generated (Tringe and Hugen- holtz 2008).

The assignment of taxonomy to sequences, anno- tations, and functional analysis can be done using a

Fig. 5.3 The 454 pyrosequencing approach. ADP, adenosine diphosphate; AMP, adenosine monophosphate; APS, adenosine 5′ phosphosulfate; ATP, adenosine triphosphate; dNDP, deoxynucleoside diphosphate; dNMP, deoxynucleoside monophosphate; dNTP, deoxynucleoside triphosphate; PPi, pyrophosphate. Source: Siqueira et al. http://www.journaloforalmicrobiology.net/index.php/jom/ article/view/10743

variety of online tools, mostly available in the public databases, such as GenBank, RDP, Greengenes, HOMD, and CORE. Another metric that is used for sequence analysis is UniFrac (Lozupone and Knight 2005; Costello et al. 2009), which is a valuable tool for comparing the structure of different bacterial communities.

The high throughput of the NGS methods has a great impact on both sampling depth (number of sequences per sample) and breadth (number of samples or indi- viduals analyzed) (Siqueira et al. 2012). A greater

http://www.journaloforalmicrobiology.net/index.php/jom/article/view/10743

Molecular Analysis of Endodontic Infections 97

sampling depth permits one to have a better coverage of each individual sample, increasing the chances of detecting low-abundance species. A greater sampling breadth permits one to examine more samples or sub- jects, providing results that are more robust for com- parisons. Depending on the goal of the study and the type of samples analyzed, the researcher should decide upon which is better suited to his/her purposes. When comparing microbial communities from different sites, it has been argued that NGS platforms can be most wisely utilized to study more samples (breadth) rather than more sequences per sample (depth) (Kuczynski et al. 2010). However, if the samples to be compared come from the same or closely related sites, deeper sequencing may be necessary to reveal minor differ- ences in the community composition (Lemos et al. 2011).

5.11 Metagenomics

The two fundamental questions in microbial ecol- ogy are “who is there?” and “what are they doing?” Molecular biology methods have provided a great deal of information about the species composition in diverse environments. Now, the important question to be answered refers to the role of different species in the consortium (i.e., what are they doing there?).

Data from 16S rRNA gene clone libraries have opened a window into a world of bacterial diversity that is astonishing in its breadth. The challenge now is to develop methods to move beyond cataloging 16S rRNA gene sequences toward an understanding of the physiology and functional roles of bacteria in different environments. The 16S rRNA gene repre- sents only about 0.05% on the average of the genome of a bacterial cell and has very little value to pre- dict the physiology and functional roles of such cell. For instance, assigning physiologies and functions to as-yet-uncultivated bacteria based on 16S rRNA gene sequences is complicated in many cases by the lack of characterized close relatives or even by the diver- sity of phenotypes among close relatives (Janssen 2006). There are many examples of strains that are related by more than 98% similarity at the 16S rRNA gene level but behave very differently physiologically and ecologically (Rodriguez-Valera 2002). While the 16S rRNA gene often provides accurate identifica- tion, the other 99.95% of the genome provides the blueprint for the vast array of metabolic, structural, and

virulence abilities of each bacterium. Because as-yet- uncultivated bacteria make up a large proportion of most environments, studies of the physiologic and functional roles of the community members should also rely on culture-independent approaches.

Physiology and function in a bacterial commu- nity can be inferred by the substances produced and released by the community members. Methods such as metatranscriptomics (RNA), metaproteomics (pro- teins), and metabolomics (metabolites) can provide valuable information in this regard. The genetic poten- tial of the community can be determined by metage- nomics. Metagenomics is the culture-independent analysis of the collective microbial genomes (termed the metagenome) in an environmental community, using an approach based either on expression or on sequencing (Handelsman 2004). Metagenomics treats the genomes of all microorganisms present in a specific habitat as an entity. Metagenomics is different from community analysis, as the latter focuses on microbial diversity as determined by a single target gene whereas in the former theoretically all genes in the commu- nity are analyzed. This not only allows identification of community members without the biases of PCR amplification, but also provides information as to the functional potential of the community. Of the molec- ular methods discussed in this chapter, metagenomics is the only one that remains to be used in endodontic microbiology research.

5.12 Advantages and limitations of molecular methods

Most of the advantages of molecular methods have already been pointed out in the previous sections. Like other methods, molecular biology techniques also have limitations. Advantages and limitations are depicted in Table 5.2. The issues related to the ability of PCR to detect either an extremely low number of cells or dead cells are of special interest when one interprets the results of PCR identification procedures in endodontic microbiology research. Therefore, these issues deserve a separate discussion.

5.12.1 The too-high sensitivity issue

The high detection rate of PCR may be a reason of concern, particularly when nonquantitative assays are

98 Endodontic Microbiology

Table 5.2 Advantages and limitations of molecular biology methods

Molecular biology techniques

Advantages Limitations

1. Detect both cultivable and as-yet-uncultivated species and/or strains

2. High specificity and accurate identification of strains with ambiguous phenotypic behavior

3. Detect species directly in clinical samples 4. High sensitivity 5. Rapid – most assays take no more than minutes to

a few hours to identify a microbial species 6. Do not require carefully controlled anaerobic

conditions during sampling, transportation, and handling

7. Can be used during antimicrobial treatment 8. Samples can be stored frozen for later analysis 9. DNA can be transported easily between

laboratories 10. Detect dead microorganisms

1. Most assays are qualitative or semiquantitative (exceptions: real-time PCR, DNA microarrays)

2. Most assays only detect one species or a few different species at a time (exceptions: broad-range PCR, DGGE, T-RFLP, checkerboard, DNA microarrays, NGS methods)

3. Most assays detect only the target species and fail to detect unexpected species (exceptions: broad-range PCR, DGGE, T-RFLP, NGS methods)

4. Some assays can be laborious and costly (e.g., broad-range PCR, T-RFLP, metagenomics)

5. Biases in broad-range PCR introduced by homogenization procedures, preferential DNA amplification, and differential DNA extraction

6. Hybridization assays using whole genome probes detect only cultivable species

7. Detect dead microorganisms 8. Sequencing errors may inflate richness estimates

(i.e., the number of taxa; NGS technologies)

DGGE, denaturing gradient gel electrophoresis; NGS, Next-Generation DNA Sequencing; PCR, polymerase chain reaction; T-RFLP, terminal restriction fragment length polymorphism.

employed. It has been claimed that because PCR can detect a very low number of cells of a given tar- get species, the results obtained by this method may have no significance with regard to disease causation. Nonetheless, the method’s high sensitivity can repre- sent a great advantage for microbiologic diagnosis in endodontics.

When taking samples from endodontic infections, difficulties posed by the physical constraints of the root canal system and by the limitations of the conventional sampling technique using paper points can make it difficult to obtain a good representative sample from the main canal (Siqueira and Rôças 2005a). If cells of a given species are sampled at a level below the detection limit of the diagnostic test, species prevalence will be underestimated.

It is also important to take into consideration the analytical sensitivity needed for the specific clinical sample. For example, a sensitivity of no more than 104

microbial cells per mL is required for urine, while a sensitivity of one cell may be of extreme relevance for samples from blood or cerebrospinal fluid (Boissinot and Bergeron 2002). There is no clear evidence as to the microbial load necessary for apical periodontitis

to be induced. Endodontic infections are characterized by a mixed community and individual species can have different roles in the consortium or dominate various stages of the infection. At least theoretically, all bacte- rial species established in the infected root canal have the potential to be considered endodontic pathogens (Sundqvist and Figdor 2003). Also, from an ecologic point of view, one cannot dismiss the ecologic role of the less abundant species in the consortium, exclu- sively on the basis of numbers, particularly if we con- sider that nothing is known about their functional role in the ecosystem. In other words, even less abundant species may exert a big difference in the ecosystem (Siqueira and Rôças 2009a). For instance, at 5% of the population, chemolithoautotrophs or secondary- metabolite producers do make a big difference to an ecosystem (Strous 2007). Also, it remains unknown whether or not the abundance of these species changes over time and what are the consequences of such changes. Based on this, it would be glaringly prudent to use the method with the highest sensitivity to detect all species colonizing the root canal.

PCR detection of very low numbers of cells in clin- ical samples may not be as common as anticipated.

Molecular Analysis of Endodontic Infections 99

Numerous factors influence PCR reactions, sometimes dramatically reducing sensitivity for direct microbial detection in clinical samples. Therefore, the analytical sensitivity of the method does not always correspond to its “clinical” sensitivity. It is well known that the effects of inhibitors are magnified in samples with low number of target DNA and therefore can significantly decrease the sensitivity of the method (Hayden 2004). Another impediment refers to the aliquots of the whole sample used in PCR reactions. Detection of 1–10 cells of a species in a sample would be possible only if the entire sample were used in the PCR assay. And this is not the case. In fact, aliquots that usually represent 1– 5% of the entire sample are used in each amplification reaction. Thus, for the PCR assay to detect 1–10 cells in the aliquot used in each PCR tube, 20–100 (detec- tion limit of 1 cell) to 200–1000 (detection limit of 10 cells) cells have to be present in the original sample, not considering the effects of inhibitors (Siqueira and Rôças 2003d). These numbers are still lower than the detection limits of other methods, but may represent more significance in terms of pathogenicity.

Therefore the use of highly sensitive techniques is welcome in the study of endodontic infections, decreasing the risks for potentially important species to pass unnoticed during sample analysis. Although qual- itative results do not lack significance, the use of quan- titative molecular assays, like real-time PCR, can allow inference of the role of a given species in the infec- tious process while maintaining high sensitivity and the ability to detect fastidious or as-yet-uncultivated bacteria.

5.12.2 The dead-cell issue

Detection of dead cells by a given identification method can be at the same time an advantage and a limitation. On the one hand, this allows for detection of hitherto uncultivated or fastidious bacteria that can die during sampling, transportation, or isolation pro- cedures (Wang et al. 1996; Rantakokko-Jalava et al. 2000; Siqueira and Rôças 2005a). On the other hand, if the bacteria were already dead in the infected site, they may also be detected and this might give rise to a false assumption of their role in the infectious process (Josephson et al. 1993; Keer and Birch 2003).

Several studies show that bacterial DNA is rapidly cleared from the host sites after bacterial death and that DNA from different species may differ as to the elimination kinetics at different body sites (Malawista

et al. 1994; Post et al. 1996; Aul et al. 1998; Wicher et al. 1998). It remains to be clarified how long bacterial DNA from dead cells can remain detectable in the infected root canal system. There is no in vivo study on the subject.

It is true that detection of microbial DNA sequences in clinical samples does not indicate viability of the microorganism. However, the fact that some microor- ganisms die during the course of an infectious process does not necessarily implicate that in a determined moment these microorganisms have not participated in the pathogenesis of the disease. It is important to point out that for DNA from dead cells to be detected by the molecular method, it has to be present in levels above the method’s detection threshold. Considering the clinical sensitivity of the method, even detection of DNA from dead bacteria may suggest that in the past they were important in disease causation. Viable bacte- ria detected in a cross-sectional study may be involved with maintenance of the disease but not necessarily with causation.

In addition, the fate of DNA from microorganisms that have entered and not survived in root canals is unknown. DNA from dead cells might be adsorbed to dentine because of affinity of hydroxyapatite to this molecule (Bernardi 1965). However, it remains to be shown if DNA from dead microbial cells can really be adsorbed on dentinal walls and, if even, it can be retrieved during sampling with paper points. In fact, it is highly unlikely that free microbial DNA can remain intact in an environment colonized by living microorganisms. The half-life of the DNA released in the environment is considered to be very short owing to the presence of DNases in a complex microbial background like that occurring in the infected root canal. DNases released by some living species as well as at cell death can degrade free DNA in the envi- ronment. It has been reported the presence of DNase activity on whole bacterial cells and vesicles thereof that can degrade DNA (Leduc et al. 1995). Bacte- ria displaying DNase activity include common puta- tive endodontic pathogens, such as Porphyromonas endodontalis, Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium species, Prevotella inter- media, and Prevotella nigrescens. DNase activity in P. intermedia culture resulted in degradation of extracted DNA to a nondetectable level by only 3 hours (Brundin et al. 2010). Thus, the free DNA molecule faces an onslaught of bacteria that can degrade macro- molecules (Paabo et al. 2004). Indeed, DNases are

100 Endodontic Microbiology

of concern during sample storage, because they can be carried along with the sample and cause DNA degrada- tion, with consequent false negative results after PCR amplification.

Moreover, in the clinical situation, tissue fluids and exudate can seep into the canal and comprise the main sustainable source of nutrients to intraradicular bac- teria. It has been demonstrated that serum, which can also contain DNases, can rapidly decompose free DNA to levels below the detection limit of molecular meth- ods (Brundin et al. 2010).

Under rare circumstances, such as when the tis- sue becomes rapidly desiccated after host death or the DNA becomes adsorbed to a mineral matrix, like bone or teeth, DNA may escape enzymatic and microbial degradation. Even so, slower but still relentless chem- ical processes start affecting the DNA. Many of these processes are similar to those that affect the DNA in the living cell, with the difference that, after cell death, these processes are not counterbalanced by cellular repair processes. Thus, damage accumulates progres- sively until the DNA loses its integrity and decom- poses, with an irreversible loss of nucleotide sequence information (Paabo et al. 2004).

DNA is not a stable molecule, and chemical pro- cesses, like oxidation and hydrolysis, induce dam- age over time. As a result, the DNA becomes frag- mented and difficult or even impossible to be detected and/or analyzed. In palaeomicrobiology, certain strate- gies have to be developed for successful detection of ancient DNA. One of the most important strategies consists of using primers that will amplify a small DNA target size, preferably below 200 bp. Even so, the sample has to be well-preserved, usually frozen or mummified. It has been stated that it is not the age of the DNA but the environmental conditions that are crit- ical in preservation (Donoghue et al. 2004). Thus, any comparisons between the use of molecular methods in palaeomicrobiology and endodontics can be consid- ered inappropriate at best.

Based on the discussion earlier, although there is a possibility of detecting DNA from dead cells in endodontic infections, this possibility is conceivably low. In the event DNA from dead cells is detected, the results by no means lack significance with regard to participation in disease causation. Nonetheless, the ability to detect DNA from dead cells poses a major problem when one is investigating the immediate effectiveness of antimicrobial intracanal treatment, as DNA released from cells that have recently died can

be detected. To circumvent or at least minimize this problem, one can use some adjustments in the PCR assay or take advantage of PCR technology deriva- tives, such as RT-PCR. Because smaller fragments of DNA may persist for a longer time after cell death than larger sequences, designing primers to gener- ate large amplicons may reduce the risks of positive results due to DNA from dead cells (McCarty and Atlas 1993). Moreover, assays directed towards the detection of RNA through RT-PCR can be more reli- able for detection of living cells. As RNAs are more labile and have a shorter half-life than DNA, they can be rapidly degraded after cell death (Keer and Birch 2003).

Another approach has been proposed to distinguish between the DNA from viable and dead cells in a sample. A viable/dead stain [propidium monoazide (PMA) or ethidium monoazide (EMA)] is used in combination with real-time PCR to inhibit amplifi- cation of DNA from dead cells that have taken up PMA or EMA (Nocker and Camper 2006; Nocker et al. 2006). These stains are selective in penetrat- ing only into dead bacterial cells with compromised membrane/cell wall integrity but not into live cells with intact membranes/cell walls. The stain is added to the test sample, penetrates the dead cells (if present) and binds to the DNA. Exposure to bright light for 1 minute leads to covalent binding and inactivation of the free stain. This process renders the DNA insoluble and results in its loss during subsequent DNA extraction. DNA from viable cells is unstained, while the DNA from the dead cells covalently bound to the stain is selectively removed and not amplified by PCR. Thus, only DNA from viable cells is available for detec- tion (Nocker and Camper 2006; Nocker et al. 2006). PMA shows superiority over EMA, because the latter can also penetrate live cells of some bacterial species (Nocker et al. 2006). The approach of treating micro- biologic samples with a viability dye prior to DNA extraction to selectively detect intact live cells has been called viability-PCR (Nkuipou-Kenfack et al. 2013).

5.13 Unraveling the endodontic microbiome with molecular biology methods

The limitations imposed by the ability of a microorgan- ism to be cultivated can be sidestepped by the use of

Molecular Analysis of Endodontic Infections 101

molecular identification methods. Several molecular methods have been or have the potential to be applied to the study of endodontic infections. The choice of a particular approach depends on the questions to be answered and a variety of techniques have been used to offer a better picture of the endodontic bacterial com- munities. If the purpose is to investigate the breadth of bacterial diversity in the endodontic environment, the broad-range PCR followed by 16S rRNA gene clone library analysis or next generation sequencing (NGS) can be the methods of choice. Analysis of bacterial community structures and identification of the community members can be performed via finger- printing techniques, such as DGGE and T-RFLP. NGS can also be used to compare communities and in this regard it has a much higher resolution than DGGE and T-RFLP. FISH can identify, measure abundance and provide information on spatial distribution of particu- lar species in tissues. If the purpose is to detect selected target species, checkerboard assays, DNA microar- rays, single PCR, nPCR, multiplex PCR, and real-time PCR can be used to survey a large number of clin- ical samples, each one with its own advantages and limitations. Figure 5.4 exemplifies the use of different molecular biology methods for the study of endodontic infections.

Traditionally, endodontic infections have been stud- ied by means of culture-dependent approaches (see Chapter 4). Such studies have resulted in the estab- lishment of a set of species thought to have an impor- tant role in the pathogenesis of apical periodonti- tis. Over the last 15 years, not only have findings from culture-based methods been confirmed, but they have also been significantly supplemented with those from molecular diagnostic techniques (Siqueira and Rôças 2009b). Molecular methods have confirmed and strengthened the association of many cultivable bacterial species with apical periodontitis and have revealed new suspected endodontic pathogens. Detec- tion of cultivable named species in higher preva- lence by molecular methods can be explained on the basis of the higher sensitivity of the methods when compared to culture, the fastidious nature of the microorganisms, or even the fact that not all strains within a species can be cultivated or accurately identified. Moreover, the list of candidate pathogens has expanded to include fastidious cultivable species or even as-yet-uncultivated bacteria that have never been previously found in endodontic infections by culturing procedures. Consequently, the endodontic

microbiota has been refined and redefined by molecu- lar methods.

5.13.1 The five generations of endodontic microbiology studies

Microbiologic studies for identification of the species participating in endodontic infections can be chrono- logically divided into five generations on the basis of the different strategic approaches used (Siqueira and Rôças 2009b, 2013b):

1. First generation: involves studies of the endodon- tic microbiota using open-ended (or broad-range) culture methods, which disclosed many cultivable species in association with apical periodontitis.

2. Second generation: consists of studies employing closed-ended molecular detection methods, such as species-specific PCR and its derivatives as well as the original checkerboard hybridization assay, to target cultivable bacteria. These methods allowed the inclusion of some difficult-to-grow species in the set of candidate endodontic pathogens.

3. Third generation: involves studies adopting open- ended molecular methods, such as broad-range PCR followed by cloning and Sanger sequencing or T-RFLP, which allowed an even more compre- hensive investigation of the bacterial diversity in endodontic infections. By these approaches, not only cultivable species, but also as-yet-uncultivated and uncharacterized bacteria have been identified. Technical hurdles make it difficult to analyze a large number of samples by cloning and sequencing, but cataloguing bacterial species in the oral cavity by this approach provided 16S rRNA gene sequence information that could be used to design primers and oligonucleotide probes to target both cultivable and as-yet-uncultivated bacteria.

4. Fourth generation: comprises studies involving closed-ended molecular analyses with PCR and DNA hybridization assays (e.g., reverse-capture checkerboard) in large-scale clinical studies to investigate prevalence and association of cultivable and as-yet-uncultivated bacteria with endodontic infecions.

5. Fifth generation: contemporary fifth generation of studies use the NGS technologies for a deep- coverage open-ended analysis of endodontic sam- ples, which has substantially expanded the knowl- edge of the diversity of the endodontic microbiome.

102 Endodontic Microbiology

Fig. 5.4 Molecular biology methods used, or with potential to be used, in the study of endodontic infections. The choice for a particular technique will depend on the type of analysis to be performed. DGGE, denaturing gradient gel electrophoresis; PCR, polymerase chain reaction; T-RFLP, terminal restriction fragment length polymorphism.

Molecular Analysis of Endodontic Infections 103

5.13.2 Impact of molecular methods in endodontic microbiology

Culture studies (first generation) identified a set of species thought to have an important role in the pathogenesis of apical periodontitis (Seltzer and Far- ber 1994; Sundqvist 1994). Further, not only have findings from culture-based methods been confirmed, but they have also been significantly supplemented with those from culture-independent molecular biol- ogy techniques, which comprise the other four gener- ations of endodontic microbiology studies (Siqueira and Rôças 2009b). Molecular methods have con- firmed and strengthened the association of many cultivable bacterial species with apical periodonti- tis and have also revealed new suspected endodon- tic pathogens (Siqueira and Rôças 2005b). The list of candidate pathogens has expanded to include culture- difficult species or as-yet-uncultivated bacteria that had never been found in endodontic infections by cul- turing approaches. The impact of molecular studies in endodontics is highly significant in terms of deci- phering the microbial diversity in endodontic infec- tions. Approximately 500 different microbial species (mostly bacteria) have been detected in different types of endodontic infections (Siqueira and Rôças 2009b). Of these, about 45% were exclusively reported by molecular biology studies, compared to 32% detected by culture studies alone (Siqueira and Rôças 2009b). The percentage of species detected by both approaches is 33%. As a consequence, it becomes evident that the endodontic microbiota has been refined and rede- fined by molecular methods (Siqueira and Rôças 2005c).

Another significant contribution of molecular meth- ods to the knowledge of endodontic infections comes from bacterial community-profiling analyses. By using methods that provide an insight into the community structure (richness and abundance), such as DGGE, T-RFLP, and NGS technologies, these studies have brought a new perspective related to the etiology and pathogenesis of apical periodontitis, with the focus moving from the “single-pathogen” concept to the “community-as-pathogen” concept. According to the latter, the community is the unit of pathogenicity and several factors influence its virulence, with special emphasis to the presence of certain pathogenic species, their multiple interactions in a multispecies consor- tium, and the accumulation of virulence factors in the biofilm (Siqueira and Rôças 2009a).

It has been shown that the bacterial community profiles seem to follow some patterns related to the different clinical presentations of apical periodonti- tis (Siqueira and Rôças 2009a). The most interest- ing results of community-profiling analyses of the endodontic microbiome are summarized as follows:

1. The different types of endodontic infections were confirmed as being composed of multispecies bac- terial communities (Siqueira et al. 2004b; Machado de Oliveira et al. 2007b; Chugal et al. 2011; Santos et al. 2011). This also applies to persis- tent/secondary intraradicular infections associated with treated teeth and posttreatment disease (Rôças et al. 2004a, 2008; Siqueira and Rôças 2004a; Blome et al. 2008; Sakamoto et al. 2008; Li et al. 2010; Chugal et al. 2011; Hong et al. 2013).

2. There is a great interindividual variability in the diversity of endodontic bacterial communities asso- ciated with the same clinical disease (Rôças et al. 2004a; Siqueira et al. 2004b, 2011; Machado de Oliveira et al. 2007; Li et al. 2010; Chu- gal et al. 2011). The fact that the composition of the endodontic microbiome differs consistently between individuals with the same disease indicates a heterogeneous etiology for apical periodontitis, where multiple species combinations can lead to similar disease outcomes.

3. In spite of the interindividual heterogeneity, bac- terial communities seem to follow a specific pat- tern according to the clinical condition (Rôças et al. 2004a; Siqueira et al. 2004b; Sakamoto et al. 2006; Santos et al. 2011). For instance, endodon- tic bacterial communities associated with symp- tomatic lesions are more similar to each other when compared with communities present in teeth with asymptomatic lesions (Siqueira et al. 2004b; Santos et al. 2011). Therefore, disease severity (intensity of signs and symptoms) or response to treatment may be related to the bacterial community composition.

4. There seems to be a geography-related pattern in community profiles. Interindividual variability is lower among individuals residing in the same geo- graphic location when compared with that observed for individuals living in distant countries (Rôças et al. 2004a; Siqueira et al. 2004b, 2008; Machado de Oliveira et al. 2007).

5. The apical portion of the root canal harbors a microbiome that is significantly different in com- position than that occurring in the more coronal

104 Endodontic Microbiology

aspects of the root canal (Alves et al. 2009; Rôças et al. 2010; Ozok et al. 2012). The apical bacterial communities are as diverse as those occurring at the middle/coronal thirds (Alves et al. 2009; Ozok et al. 2012). A high variability is observed for both interindividual (samples from the same root canal region but from different patients) and intraindivid- ual (samples from different root canal regions of the same tooth) comparisons (Alves et al. 2009).

5.14 Microbial diversity in endodontic infections

5.14.1 Primary intraradicular infections

Molecular methods have revealed that primary intraradicular infections are characterized by a mixed consortium composed of 10–30 species per canal (Siqueira and Rôças 2005b), but these figures may be even higher according to NGS-based studies (San- tos et al. 2011; Hong et al. 2013) (see later). The number of bacterial cells in an infected canal varies from 103 to 108 (Vianna et al. 2006b; Sakamoto et al. 2007). Primary infections display a large interindivid- ual variability (i.e., each individual shows a unique endodontic microbiome in terms of composition and species dominance as revealed by DGGE, T-RFLP, and NGS approaches; Siqueira et al. 2004b, 2008; Sakamoto et al. 2006; Machado de Oliveira et al. 2007; Santos et al. 2011; Hong et al. 2013). Com- parison of the bacterial community profiles between symptomatic (abscesses) and asymptomatic primary infections by community-profiling techniques have found marked differences between these conditions (Siqueira et al. 2004b; Sakamoto et al. 2006; San- tos et al. 2011). This means that there is a significant difference in the species composition and abundance associated with symptomatic and asymptomatic infec- tions. Symptomatic infections also harbor a signifi- cantly higher number of bacterial species (Siqueira et al. 2004b; Sakamoto et al. 2006; Santos et al. 2011).

Bacterial species/phylotypes detected in primary infections fall into 9 of the 13 phyla that have oral representatives: Firmicutes, Bacteroidetes, Fusobacte- ria, Actinobacteria, Proteobacteria, Spirochaetes, Syn- ergistetes, TM7 (also known as Candidatus Saccharib- acteria), and SR1 (Munson et al. 2002; Siqueira and Rôças 2005c; Saito et al. 2006; Sakamoto et al. 2006, 2007; Rôças and Siqueira 2008). However, there may

be representatives of many other phyla in endodontic infecions as revealed by NGS-based studies (Li et al. 2010; Santos et al. 2011; Siqueira et al. 2011; Ozok et al. 2012; Hong et al. 2013). It is highly likely that these species belonging to uncommon phyla are low- abundance members of the endodontic community.

Members of the phyla Spirochaetes, Synergistetes, TM7, and SR1 are usually difficult or impossible to culture and have been found in endodontic infec- tions only after the introduction of molecular methods. Culture-independent molecular studies have also dis- closed several as-yet-uncultivated phylotypes from the five other phyla that have cultivable representatives in the endodontic microbiome.

An important periodontal pathogen, Tannerella forsythia (formerly Bacteroides forsythus), a Gram- negative obligate anaerobe that had never been reported to occur in infected root canals by culture, was for the first time detected in primary endodon- tic infections in a study using species-specific sin- gle PCR (Conrads et al. 1997). Subsequent studies using different PCR assays, the checkerboard approach and DNA microarray, have confirmed that T. forsythia is a common member of the microbiome associated with different forms of apical periodontitis, includ- ing abscesses (Conrads et al. 1997; Jung et al. 2000; Siqueira et al. 2000b; Rôças et al. 2001; Siqueira and Rôças 2003a; Foschi et al. 2005; Vianna et al. 2005; Gomes et al. 2006; Rôças and Siqueira 2008; Sas- sone et al. 2008; Lacevic et al. 2009; Saito et al. 2009; Ribeiro et al. 2011; Montagner et al. 2012). Depending on the molecular identification approach, prevalence values for T. forsythia have ranged from 5% to 66% of the cases investigated (Figure 5.5).

Spirochetes are highly motile, spiral-shaped bacteria that have been frequently observed in samples taken from endodontic infections by microscopy, but had never been identified to the species level. The appli- cation of molecular diagnostic methods to the iden- tification of spirochetes has demonstrated that their occurrence in infections of endodontic origin has been overlooked by technical hurdles of culture techniques. Thus far, 10 oral Treponema species have been culti- vated and validly named. They can be classified into two groups according to the fermentation of carbo- hydrates: the saccharolytic species include T. pecti- novorum, T. socranskii, T. amylovorum, T. lecithi- nolyticum, T. maltophilum, and T. parvum, and the asaccharolytic species include T. denticola, T. medium, T. putidum, and T. vincentii. Using species-specific

Molecular Analysis of Endodontic Infections 105

Rôças et al., 2011 (rc- checkerboard)

Rôças et al., 2008 (rc- checkerboard)

Rôças et al., 2001 (PCR)

Siqueira & Rôças, 2003 (nPCR)

Siqueira et al., 2002 (PCR)

Siqueira et al., 2000 (checkerboard)

Conrads et al., 1997 (PCR)

0 20 40

60 80

Jung et al., 2000 (PCR&dot-blot)

Saito et al., 2009 (qPCR)

Foschi et al., 2005 (PCR)

Fouad et al., 2002 (PCR)

Vianna et al., 2005 (microarray)

Lacevic et al., 2009 (nPCR)

Gomes et al., 2006 (nPCR)

Fig. 5.5 Prevalence of Tannerella forsythia in primary endodontic infections as revealed by molecular studies using different methodologies.

PCR, we detected T. denticola, a recognized periodon- tal pathogen, for the first time in infected root canals (Siqueira et al. 2000a). Several other molecular stud- ies confirmed that this treponeme and all the other cultivable species can take part in the microbiome of primary endodontic infections, including abscesses (Siqueira et al. 2000b, 2001a; Jung et al. 2001; Fouad et al. 2002; Baumgartner et al. 2003; Rôças et al. 2003; Siqueira and Rôças 2003c, 2004b, 2009c; Foschi et al. 2005; Rôças and Siqueira 2005a, 2008; Vianna et al. 2005; Gomes et al. 2006) (Figures 5.6 and 5.7). The most frequent treponemes in endodontic infec- tions are T. denticola and T. socranskii (Siqueira et al. 2000a; Baumgartner et al. 2003; Rôças et al. 2003; Siqueira and Rôças 2004b). The species T. parvum, T. maltophilum, and T. lecithinolyticum have been

moderately prevalent (Jung et al. 2001; Baumgart- ner et al. 2003; Siqueira and Rôças 2003c, 2004b; Rôças and Siqueira 2005a). Sakamoto et al. (2009) investigated the diversity of spirochetes in primary endodontic infections by clone library analysis and revealed that all detected spirochetes belonged to the genus Treponema. Overall, 28 different taxa were identified: 9 cultivable and validly named species, 1 cultivable as-yet-uncharaterized strain and 18 as-yet- uncultivated phylotypes. However, the large major- ity of clones (94%) were from cultivable named species.

Dialister species are asaccharolytic, obligately anaerobic, Gram-negative coccobacilli that repre- sent another example of bacteria that have been consistently detected in endodontic infections only by

106 Endodontic Microbiology

Rôças et al., 2011 (rc-checkerboard)

Rôças et al., 2008 (rc-checkerboard)

Rôças et al., 2003 (nPCR)

Baumgartner et al., 2003 (nPCR)

Fouad et al., 2002 (PCR)

Jung et al., 2001 (PCR&dot-blot)

Siqueira et al., 2001 (single PCR)

Siqueira et al., 2000 (checkerboard)

0 20 40

60 80

Gomes et al., 2006 (nPCR)

Foschi et al., 2005 (PCR)

Vianna et al., 2005 (microarray)

Fig. 5.6 Prevalence of Treponema denticola in primary endodontic infections as revealed by molecular studies using different methodologies.

Fig. 5.7 Prevalence of named Treponema species in primary endodontic infections associated with different forms of apical periodontitis. Findings from the authors’ laboratory using group- and species-specific nested PCR.

Molecular Analysis of Endodontic Infections 107

molecular biology techniques. Dialister pneumosintes and Dialister invisus have been frequently present in the microbiome associated with asymptomatic and symptomatic primary endodontic infections (Mun- son et al. 2002; Rôças and Siqueira 2002; Siqueira and Rôças 2002, 2003e, 2005a,c; Saito et al. 2006; Sakamoto et al. 2006).

Black-pigmented Gram-negative anaerobic rods of the genera Prevotella and Porphyromonas have been commonly found in endodontic infections by culture (Haapasalo et al. 1986; Baumgartner et al. 1999), with some species suggested to be associated with symp- toms (van Winkelhoff et al. 1985; Sundqvist et al. 1989). Molecular studies have shown even higher prevalence of black-pigmented bacteria in primary

infections (Siqueira et al. 2001c; Baumgartner et al. 2004; Gomes et al. 2005; Seol et al. 2006; Rôças and Siqueira 2008), but have failed to disclose asso- ciation with symptoms (Jung et al. 2000; Siqueira et al. 2001b; Fouad et al. 2002). The most preva- lent species include P. endodontalis, P. gingivalis, P. intermedia, and P. nigrescens (Figure 5.8). Other Pre- votella species, such as Prevotella multissacharivorax and Prevotella baroniae, and Alloprevotella tannerae, have been detected in endodontic infections only after the advent of molecular methods (Xia et al. 2000; Baumgartner et al. 2004; Sakamoto et al. 2006; Brito et al. 2007; Rôças and Siqueira 2009). The subgingival plaque can be a source of black-pigmented bacteria for endodontic infections, as suggested by a study using

100

50 %

Porphyromonas endodontalis

Porphyromonas gingivalis

Prevotella intermedia

Prevotella nigrescens

Rôças et al., 2011 (rc-checkerboard)

Siqueira & Rôças, unpublished (nPCR)

Siqueira et al., 2001 (PCR)

Siqueira et al., 2001 (checkerboard)

Jung et al., 2000 (PCR&dot-blot)

Baum gartner et al., 1999 (culture)

Sundqvist 1992 (culture)

Haapasalo et al., 1986 (culture)

Rôças et al., 2008 (rc-checkerboard)

Seol et al., 2006 (m ultiPCR)

Gom es et al., 2005 (nPCR)

Baum gartner et al., 2004 (PCR)

Fouad et al., 2002 (PCR)

Fig. 5.8 Prevalence of four black-pigmented bacterial species in primary endodontic infections as revealed by culture and molecular studies.

108 Endodontic Microbiology

AP-PCR to compare periodontal and endodontic iso- lates of two black-pigmented species (Goncalves et al. 1999).

Fusobacterium nucleatum is one of the most com- monly encountered Gram-negative species in primary endodontic infections by culturing studies (Sundqvist 1992; Debelian et al. 1995). Molecular studies have confirmed these findings and have shown even higher prevalence values for this species in primarily infected root canals, including cases of abscesses (Jung et al. 2000; Fouad et al. 2002; Baumgartner et al. 2004; Sas- sone et al. 2008, 2012; Vianna et al. 2008; Siqueira and Rôças 2009c; Rôças et al. 2010, 2011). Different clonal types of F. nucleatum can be found in the same infected canal as revealed by PCR-based bacterial typing techniques (ERIC-PCR and AP-PCR) (Moraes et al. 2002).

Culture-dependent studies have shown that anaer- obic Gram-negative bacteria are the most common microorganisms in primary endodontic infections, but some Gram-positive bacteria may also be frequent members of the endodontic microbial consortium. Culture-independent analyses of primary endodontic infections not only have supported these findings, but also have shown higher prevalence for some species and included new Gram-positive species in the set of candidate pathogens. Gram-positive species found in similar or higher prevalence by molecular tech- niques when compared to culture include strepto- cocci (Siqueira et al. 2002a,b; Fouad et al. 2003), Pseudoramibacter alactolyticus (Siqueira and Rôças 2003f; Siqueira et al. 2004a, 2009; Ribeiro et al. 2011), Propionibacterium propionicum (Siqueira and Rôças 2003b), and Parvimonas micra (Siqueira et al. 2003; Vianna et al. 2008; Siqueira and Rôças 2009c; Rôças et al. 2011). Species isolated only sporadically or never found by culture that have been disclosed by molecular methods in relatively high frequencies include Filifactor alocis (Siqueira and Rôças 2003c; Gomes et al. 2006), Slackia exigua (Hashimura et al. 2001), Eubacterium infirmum (Fouad et al. 2003), and Olsenella species (Fouad et al. 2002; Munson et al. 2002; Rôças and Siqueira 2005c, 2008; Rôças et al. 2010).

Figure 5.9 shows data from the authors’ laboratory as to the most frequently detected species/phylotypes in primary endodontic infections associated with symptomatic (abscesses) and asymptomatic apical periodontitis, as revealed by a species-specific nested PCR approach.

5.14.2 Uncultivated bacteria

Molecular evaluations of the bacterial diversity in pri- mary endodontic infections using broad-range PCR and 16S rRNA gene clone library analysis have demonstrated the as-yet-uncultivated bacteria com- prise approximately 40–60% of the species-level taxa (Munson et al. 2002; Sakamoto et al. 2006, 2007; Vickerman et al. 2007; Ribeiro et al. 2011). Sakamoto et al. (2006) reported that uncultivated phy- lotypes accounted for approximately 55% of the taxa found in root canals of teeth with apical periodon- titis and in terms of abundance represented more than 38% of the clones sequenced. In pus aspirates from acute apical abscesses, as-yet-uncultivated phy- lotypes encompassed approximately 24–46% of the taxa found (Sakamoto et al. 2006; Flynn et al. 2012), and 6% to more than 30% of the clones sequenced (Sakamoto et al. 2006; Riggio et al. 2007). Uncul- tivated phylotypes from several genera have been identified, including Dialister, Treponema, Prevotella, Solobacterium, Olsenella, Fusobacterium, Eubac- terium, Megasphaera, Veillonella, and Selenomonas as well as phylotypes related to the family Lach- nospiraceae or the Synergistetes and TM7 phyla (Rolph et al. 2001; Munson et al. 2002; Rôças and Siqueira 2005b, 2006; Siqueira and Rôças 2005c; Siqueira et al. 2005c; Saito et al. 2006; Sakamoto et al. 2006, 2009). One of the most prevalent as-yet- uncultivated phylotypes found in endodontic infec- tions is Bacteroidaceae sp. HOT-272 (synonym, Bac- teroidetes oral clone X083) (Rôças and Siqueira 2008, 2009).

5.14.3 Newly cultivated and characterized species

Several species detected in endodontic infections that were considered as uncultivated have been success- fully cultivated, phenotypically characterized, and for- mally named. These include bacteria that are relatively easy to cultivate on ordinary media but have, for some reason, only recently been cultivated for the first time (e.g., Prevotella baroniae, Peptostreptococcus stoma- tis, Dialister invisus, Anaeroglobus geminatus) or bac- teria that are truly resistant to culture (e.g., Fretibac- terium fastidiosum) and require special strategies to be cultivated (Siqueira and Rôças 2013a). Table 5.3 shows examples of some of these species and their previous phylotype names.

Molecular Analysis of Endodontic Infections 109

Symptomatic

Treponema denticola

Dialister invisus

Porphyromonas endodontalis

Tannerella forsythia

Pseudoramibacter alactolyticus

Dialister pneumosintes

Filifactor alocis

Porphyromonas gingivalis

Prevotella baroniae

Prevotella multisaccharivorax

Propionibacterium propionicum

Treponema socranskii

Parvimonas micra

Catonella morbi

Treponema parvum

Treponema maltophilum

Veillonella parvula

Olsenella uli

Fusobacterium nucleatum

Campylobacter rectus

Campylobacter gracilis

Pyramidobacter piscolens

Eikenella corrodens

Enterococcus faecalis

Prevotella nigrescens

Synergistes clone BH017

Treponema lecithinolyticum

Prevotella intermedia

Centipeda periodontii

Treponema pectinovorum

Granulicatella adiacens

Fretibacterium fastidiosum

0 25 50

75 100

Streptococcus species

Bacteroidetes clone X083

Asymptomatic

Fig. 5.9 Most prevalent bacterial species/phylotypes in asymptomatic and symptomatic primary endodontic infections. Findings from the authors’ laboratory using species-specific nested PCR.

110 Endodontic Microbiology

Table 5.3 Examples of phylotypes found in endodontic infections that were recently characterized and named as a new species or were reclassified into established species

Formal species name Previous phylotype identification

Dialister invisus Dialister E1 strains E2.20, E3.07, E9.48, P2.65, E7.25, and clones GBA27, IS013B24, BS095 and 9N-1

Eubacterium nodatum Eubacterium oral clone BP1-89 F. nucleatum ss animalis Fusobacterium genomospecies C1 and C2 F. nucleatum ss polymorphum Fusobacterium oral clone BS019 F. nucleatum ss vincentii Fusobacterium oral clones CZ006 and R002 Mogibacterium timidum Mogibacterium oral clone BP1-36 Parvimonas micra Peptostreptococcus oral clones BS044 and FG014 Porphyromonas endodontalis Porphyromonas oral clone BB134 Prevotella oris Prevotella oral clone F045 Solobacterium moorei Solobacterium oral clone K010 Treponema denticola Treponema sp. II:10:D12 Veillonella parvula Veillonella oral clones AA050, X042 and BU083

5.14.4 Geographic influence

Data from epidemiologic microbiology studies carried out in some geographic regions have been intuitively considered as applicable to other distinct locations, with the clear idea that “everything is everywhere and playing the same role.” Nevertheless, studies have fostered the assumption that oral microbial commu- nities can differ significantly according to the geo- graphic location (Ide et al. 2000; Haffajee et al. 2004, 2005).

Findings from laboratories in different countries are often quite different regarding the prevalence of the species involved in endodontic infections. Although these differences may be attributed to variations in the identification methodologies, a geographic influence in the composition of the root canal microbiome has been suspected. Molecular approaches are the most appropriate methods to compare microbiologic find- ings from distinct geographic locations. Because sam- ples from different countries should ideally be ana- lyzed in the same laboratory, the time elapsed from collection to delivery to a distant laboratory may make samples improper for culturing analysis. Molecular methods detect DNA, which can remain relatively unaltered and thereby detectable for a long time when stored under proper conditions. Thus, samples can be submitted to a distant laboratory all at once and resist long time transportation.

Data from molecular studies that have directly com- pared the endodontic microbiome of patients resid- ing in different geographic locations suggest that

significant differences in the prevalence of some important species can actually exist. Comparisons of acute apical abscess samples taken from US and Brazilian patients have shown that the prevalence of P. intermedia, P. nigrescens, P. tannerae, F. nucleatum, P. gingivalis, T. denticola, and T. forsythia markedly differed regarding the two locations (Baumgartner et al. 2004; Rôças et al. 2006) (Figure 5.10). Analy- sis of samples from primary infections from Brazilian and South Korean patients revealed that the frequen- cies of P. endodontalis, D. pneumosintes, F. alocis, T. denticola, and T. forsythia were significantly dif- ferent between the two geographic locations (Siqueira et al. 2005a) (Figure 5.11). A study using the DGGE approach compared the bacterial community pro- files of the microbiome associated with acute apical abscesses from Brazilian and US patients (Machado de Oliveira et al. 2007b). Results displayed a great vari- ability among samples. This indicates that bacterial communities of abscesses are unique for each individ- ual in terms of diversity. The composition of the micro- biome in many samples showed a geography-related pattern. Several species were exclusive for each loca- tion and others shared by the two locations showed great differences in prevalence. Another study also using DGGE compared the endodontic bacterial com- munity profiles of teeth with chronic apical periodon- titis from Brazilian and Norwegian patients (Siqueira et al. 2008). Once again, DGGE fingerprints demon- strated a great interindividual variability in the bacte- rial community profiles, irrespective of the geographic

Molecular Analysis of Endodontic Infections 111

Fig. 5.10 Comparison of the prevalence of different bacterial species/phylotypes in acute apical abscess samples taken from two geographic locations.

Fig. 5.11 Comparison of the prevalence of different bacterial species in primary endodontic infections of patients from two geographic locations.

112 Endodontic Microbiology

location of the patient. Nonetheless, a geography- related pattern was also identified.

The factors that can lead to differences in the compo- sition of the endodontic microbiome and the impact of these differences on therapy, particularly in abscessed cases requiring systemic antibiotic therapy, remain to be illuminated.

5.15 Persistent and secondary intraradicular infections

Persistent or secondary intraradicular infections are the major causes of endodontic treatment failures. This statement is supported by two strong evidence-based arguments. First, most (if not all) root canal-treated teeth showing persistent apical periodontitis have been demonstrated to harbor an intraradicular infection (Lin et al. 1991, 1992; Sundqvist et al. 1998; Pinheiro et al. 2003; Rôças et al. 2004b; Siqueira and Rôças 2004a; Rôças and Siqueira 2012; Hong et al. 2013). Second, it has been demonstrated that there is an increased risk of adverse treatment outcome when microorganisms are present in the canal at the time of filling (Sjogren et al. 1997; Waltimo et al. 2005; Fabricius et al. 2006).

Molecular methods have been recently applied to the study of the microorganisms found at the root canal- filling stage, which have the potential to put the treat- ment outcome at risk, or in root canal-treated teeth with apical periodontitis, which can be participating in the already established treatment failure.

5.15.1 Bacteria at the root canal-filling stage

The impact of bacterial persistence on treatment out- come has been demonstrated and studies have intended to identity species resisting root canal procedures. Most culturing studies have revealed an overall higher occurrence of Gram-positive bacteria in both postin- strumentation and postmedication samples (Sjogren et al. 1997; Chavez de Paz 2005; Chu et al. 2006). With the recent findings showing as-yet-uncultivated bacteria as constituents of a significant proportion of the endodontic microbiome, studies on the effects of intracanal antimicrobial procedures should also focus on these bacteria. A study using broad-range PCR and 16S rRNA gene clone library investigated the bacteria persisting after chemomechanical prepa- ration using 2.5% NaOCl as irrigant and intracanal

medication with a calcium hydroxide paste (Sakamoto et al. 2007). Fifty-six percent of the taxa found in ini- tial samples consisted of as-yet-uncultivated bacteria. A mean of 11 taxa were detected in initial (S1) sam- ples, 4 taxa in post-instrumentation (S2) samples and 5 taxa in post-medication (S3) samples. Streptococ- cus species were detected in all posttreatment samples and were the most dominant taxa in these samples, except for a S2 sample in which Solobacterium clone K010 corresponded to 56% of the clones sequenced. Forty-two percent of the taxa found in posttreatment samples were as-yet-uncultivated bacteria. Studies by Paiva et al. (2013a,b) also using clone library analy- sis confirmed that as-yet-uncultivated phylotypes may persist after treatment procedures. These findings sug- gest that previously uncharacterized bacteria may also participate in persistent endodontic infections.

Several clinical studies have used quantitative real- time PCR assays with broad-range primers to ana- lyze the reduction in bacterial numbers after endodon- tic treatment procedures. All these studies revealed that chemomechanical procedures are highly effec- tive in reducing the bacterial counts in infected canals (Vianna et al. 2006b; Sakamoto et al. 2007; Neves et al. 2014; Paiva et al. 2013a,b Rôças et al. 2013). One study compared the bacterial reduction in infected canals after chemomechanical preparation using either 2.5% NaOCl or 2% chlorhexidine (CHX) as irrigant (Vianna et al. 2006b) and found that bacterial reduction in the NaOCl group was significantly greater than in the CHX group. Sakamoto et al. (2007) evaluated bacterial elim- ination after preparation with 2.5% NaOCl as irrigant (S2 samples) and intracanal medication with a calcium hydroxide paste (S3 samples) and found that although S3 samples showed an overall percentage decrease of 56% in the number of bacteria when compared with S2, there was no significant difference in bacterial counts between S2 and S3. Rôças et al. (2013) compared the intracanal bacterial reduction promoted by two instru- mentation techniques, one using hand nickel-titanium instruments and the other using rotary NiTi instru- ments, and found no significant difference between them. However, rotary instrumentation resulted in significantly fewer qPCR positive cases (60%) than hand NiTi instrumentation (95%). Neves et al. (2014) evaluated the antibacterial effectiveness of the self- adjusting file (SAF) and reported that this system was both quantitatively and qualitatively more effec- tive than hand instrumentation. Checkerboard analysis revealed that streptococci, some anaerobic and even

Molecular Analysis of Endodontic Infections 113

as-yet-uncultivated bacteria may resist the effects of chemomechanical procedures.

Studies have used broad-range PCR and reverse- capture checkerboard to evaluate the antibacterial effects of treatment procedures and identify bacteria persisting in the canal. Rôças and Siqueira (2011a) compared the antimicrobial effects of 2.5% NaOCl and 0.12% CHX when used as irrigants and found no significant differences between them in all tested parameters: incidence of negative PCR results in S2, reduction in the number of taxa per canal, and reduc- tion in the bacterial levels (semiquantitative analy- sis). In another study, the same authors (Rôças and Siqueira 2011b) found PCR-negative results in 46% of the samples taken after chemomechanical prepara- tion and 62.5% after intracanal medication with cal- cium hydroxide pastes. In a study using RT-PCR and the checkerboard approach, Rôças and Siqueira (2010) reported that detectable levels of bacterial ribosomal RNA, used as an indicator of viability, were observed in 60% of the cases after chemomechanical prepara- tion and 53% after intracanal medication. In all these studies using checkerboard, different bacterial species were identified following treatment, including Strep- tococcus species, Olsenella uli, Pyramidobacter pis- colens, Bacteroidetes clone X083, F. nucleatum, and Propionibacterium acnes (Rôças and Siqueira 2010, 2011a,b).

Paiva et al. (2013a) used several molecular meth- ods to evaluate the antibacterial effects of root canal treatment procedures involving instrumentation with 2.5% NaOCl irrigation (S2), a final rinse with 2% CHX (S3) and then 1-week interappointment medica- tion with calcium hydroxide/2% CHX paste (S4). They used broad-range PCR to evaluate the incidence of positive results for bacteria; DGGE to profile bacterial community structures; real-time PCR to quantify bac- terial reduction; and clone library analysis to identify persisters. Treatment procedures promoted a decrease in microbial diversity and significantly reduced the incidence of positive results and the bacterial counts. In general, each subsequent treatment step improved disinfection. No specific taxon or community pat- tern was associated with posttreatment samples. The supplementary steps consisting of a final rinse with CHX followed by calcium hydroxide/CHX interap- pointment medication promoted further decrease of the bacterial bioburden to levels significantly below those achieved by the chemomechanical procedures alone.

In another study, Paiva et al. (2013b) used the same molecular methods to evaluate the effects of passive ultrasonic irrigation (PUI) as a supplementary disin- fecting step after root canal preparation. Once again, it was observed that treatment procedures were sig- nificantly effective in reducing the incidence of posi- tive results for bacteria, the infectious bioburden, and bacterial diversity. However, the supplementary PUI approach did not succeed in significantly enhancing disinfection beyond that achieved by chemomechani- cal preparation.

Because molecular methods are more sensitive and specific than culture and can detect as-yet-uncultivated bacteria, they can provide a more realistic and detailed insight into the effects of antimicrobial treatment pro- tocols. However, as discussed early on in this chap- ter, molecular technologies have some limitations that may affect this kind of analysis. Of particular inter- est, the ability to detect DNA from dead cells poses a major problem when one is investigating the imme- diate effectiveness of antibacterial treatment, because DNA from cells that have recently died can still be detected. In an in vitro study using root canals exper- imentally contaminated with Enterococcus faecalis, Alves et al. (2012) demonstrated that whereas qPCR counts in initial (S1) samples were significantly higher than culture counts, no significant differences were observed for S2 samples. Clinical studies using qPCR have also shown a highly significant reduction in bacte- rial counts after treatment procedures, especially when NaOCl is used as the irrigant (Vianna et al. 2006b; Sakamoto et al. 2007). Dead bacteria may have been washed away from the canal along with their DNA and/or NaOCl may have degraded DNA released from dead cells and made it undetectable (Alves et al. 2012). NaOCl is known to kill bacteria and degrade DNA, with resultant fragments being undetectable by PCR (McCarty and Atlas 1993; Fouad and Barry 2005). A study showed that 1% NaOCl eliminated amplifi- able DNA within 60 seconds of exposure (Young et al. 2007). Hydroxyl ions from calcium hydroxide also exert oxidative damaging effects on DNA (Siqueira and Lopes 1999), and may contribute to degradation of free DNA from dead cells.

5.15.2 Microbiome in root canal-treated teeth

Culture studies have demonstrated that the microbiota of root canal-treated teeth with apical periodontitis

114 Endodontic Microbiology

usually include one to two species, which are pre- dominantly Gram-positive bacteria, with E. faecalis as the most prevalent one (Engström 1964; Möller 1966; Molander et al. 1998; Sundqvist et al. 1998; Peciuliene et al. 2000; Hancock et al. 2001; Pinheiro et al. 2003). Poorly filled root canals have been shown to contain a greater number of species than canals apparently well-treated (Sundqvist et al. 1998; Pin- heiro et al. 2003). A study using species-specific PCR revealed that the mean number of species in ade- quately treated cases was 3 (range 1–5), while cases poorly treated yielded a mean of 5 species (range 2– 11) (Siqueira and Rôças 2004a). This difference was statistically significant. Gram-positive bacteria were present in all cases, and at least one of the following species was detected—E. faecalis, P. alactolyticus, and P. propionicum—which occurred isolatedly, in pairs or in threes. DGGE analysis of the bacterial communities in root canal-treated teeth revealed an average of about

six species per canal (range 1–26) (Rôças et al. 2004a). The structure of the bacterial communities varied from individual to individual, suggesting that distinct bacte- rial combinations can have a role in treatment failure.

Several molecular studies have confirmed E. fae- calis as frequent species in root canal-treated teeth showing apical periodontitis, with prevalence values reaching up to 90% of the cases (Rôças et al. 2004b,c; Siqueira and Rôças 2004a; Foschi et al. 2005; Fouad et al. 2005; Sedgley et al. 2006; Williams et al. 2006; Gomes et al. 2008; Rôças and Siqueira 2012) (Fig- ure 5.12). Using quantitative real-time PCR analysis, Sedgley et al. (2006) reported that E. faecalis con- stituted a median of almost 1% (range 0.14–100%) of the overall bacterial load in root canal-treated teeth. Also using real-time PCR, Rôças and Siqueira (2012) observed that this species comprised 0.3–91% of the total bacterial counts (median 1.1%). Root canal-treated teeth are about nine times more likely

Fig. 5.12 Prevalence of Enterococcus faecalis in samples from root canal-treated teeth with apical periodontitis. Data from culture and molecular studies.

Molecular Analysis of Endodontic Infections 115

to harbor E. faecalis than cases of primary infections (Rôças et al. 2004c). This suggests that this species can be inhibited by other members of a mixed bacterial consortium commonly present in primary infections and that the bleak environmental conditions within filled root canals do not prevent its survival.

The fact that E. faecalis is the most commonly encountered species in treated teeth and the attributes of this species that make it to survive in treated canals have prompted many authors to nominate E. faecalis as the main pathogen involved in treatment failures. The consequence of this was an avalanche of in vitro studies focusing on E. faecalis (Spangberg 2006). However, findings from recent molecular studies carried out in independent laboratories have somewhat questioned the role of E. faecalis as the main causative agent of endodontic failures. Some studies have not succeeded in detecting enterococci in root-canal-treated teeth with lesions (Rolph et al. 2001) or have demonstrated that E. faecalis is not the dominant species in most retreatment cases (Rôças et al. 2004a, 2008; Sakamoto et al. 2008; Rôças and Siqueira 2012). Reports in the literature have demonstrated that E. faecalis can also be found in root canal-treated teeth with no lesions. A study detected enterococci in 6% of the root canal- treated teeth with apical periodontitis and in 23% of the treated teeth with no lesions (Kaufman et al. 2005). In another study (Zoletti et al. 2006), E. faecalis was found in 81.5% of the root canal-treated teeth with no apical periodontitis and in 78% of the treated teeth with disease. These findings put into question the sta- tus of E. faecalis as the main species causing treatment failure.

In addition to finding E. faecalis in high prevalence, molecular studies have also detected streptococci and some fastidious anaerobic species—P. alac- tolyticus, P. propionicum, P. micra, F. alocis, and D. pneumosintes—in several cases of root canal- treated teeth with apical periodontitis (Siqueira and Rôças 2004a; Gomes et al. 2008; Rôças and Siqueira 2012). Streptococci were found to comprise 9–99% of the total bacterial counts in root canal-treated teeth (median 75.5%) (Rôças and Siqueira 2012). These are very large numbers in terms of abundance and may suggest an important role for streptococci in treatment failures. Taxa related to the genera Dialister, Eubac- terium, Fusobacterium, Gemella, Mogibacterium, Peptostreptococcus, Prevotella, Propionibacterium, Selenomonas, Solobacterium, Streptococcus, and Veil- lonella and several as-yet-uncultivated bacteria have

also been reported to occur in root canal-treated teeth by studies using broad-range PCR and clone library analysis (Rolph et al. 2001; Sakamoto et al. 2008). In general, molecular methods have demonstrated that the microbiota of root canal-treated teeth with apical periodontitis is more complex than previously anticipated by culture studies.

As-yet-uncultivated phylotypes may correspond to 55% of the taxa detected in treated canals. Collectively, they can also be present in high proportions, repre- senting about half of the 16S rRNA gene sequences retrieved in clone libraries (Sakamoto et al. 2008). Some as-yet-uncultivated phylotypes have been found among the most prevalent taxa in treated canals (Rôças et al. 2008; Sakamoto et al. 2008). As-yet-uncultivated bacteria have been reported to dominate the microbial community in several individual cases (Sakamoto et al. 2008), helping explain why culture studies failed to detect bacteria in some treated root canals.

Molecular methods have also strengthened the asso- ciation of persistent/secondary intraradicular infec- tions with treatment failures; bacteria have been detected in virtually all treated cases with apical peri- odontitis (Rôças et al. 2004b,c; Siqueira and Rôças 2004a; Rôças and Siqueira 2012; Hong et al. 2013). On the other hand, previous culture and histobacteriologic studies have failed to detect microorganisms in some cases of root canal-treated teeth with persistent disease (Molander et al. 1998; Sundqvist et al. 1998; Pinheiro et al. 2003). This discrepancy is better explained by the low sensitivies of culture and microscopic methods and the occurrence of as-yet-uncultivated phylotypes and strains. In addition, many microbial cells can be lost or pass unnoticed as a result of some steps during cultur- ing procedures (e.g., sample transportation, dilutions, short incubation time), or during sample processing for microscopic examinations, including decalcifica- tion, washings, and serial sectioning. The detection of bacteria by staining of demineralized tissue sections is only reliable when large numbers of microorganisms are present in the region under examination (Watts and Paterson 1990). For instance, it has been postulated that for every microorganism detected in histologic sections, 25 000 microorganisms have to be actually present (Stanley 1977). It has been shown that regard- less of the type of organism present in the cerebrospinal fluid, the percentage of positive microscopic smear results is only 25% with <103 colony forming units (CFU)/mL and 60% in the range of 103–105 CFU/mL (La Scolea and Dryja 1984). Where smaller numbers of

116 Endodontic Microbiology

bacteria are expected, bacteria may only be detected if serial sections are examined under high magnification (×400 or above). The microbiologically negative data derived from culture (Molander et al. 1998; Sundqvist et al. 1998; Pinheiro et al. 2003) and microscopic stud- ies (Nair et al. 1990, 1999) require very careful inter- pretation in the light of the techniques’ limitations, to avoid reaching overestimating conclusions about the role of nonmicrobial factors in endodontic treatment failures.

5.16 Extraradicular infections

Studies using molecular techniques (specifically the checkerboard assay, FISH, clone library analysis, and pyrosequencing) have confirmed previous find- ings from some culture studies as to the occurrence of extraradicular bacteria in persistent chronic api- cal periodontitis lesions (Gatti et al. 2000; Sunde et al. 2000, 2003; Handal et al. 2009; Subramanian and Mickel 2009; Saber et al. 2012). The issue of extraradicular infections is discussed in more detail in Chapters 6 and 10.

5.17 Other microorganisms in endodontic infections

5.17.1 Archaea

Archaea comprise a highly diverse group of prokary- otes, distinct from Bacteria in terms of genetic, biochemical, and structural features. For instance, they possess unique flagellins and ether-linked lipids, and lack peptidoglycan in their cell walls. Members of this domain have been tradionally recognized as extremophiles but recently some of these microorgan- isms have also been found to flourish in nonextreme environments, including the human body. To date, no member of the Archaea domain has been described as a human pathogen. Although studies have failed to detect Archaea in primary endodontic infections (Siqueira et al. 2005b; Rôças and Siqueira 2011a,b), others have detected methanogenic Archaea in low prevalences (Vickerman et al. 2007; Ozok et al. 2012; Paiva et al. 2012), except for one study that found these organisms in 25% of the canals of untreated teeth with chronic apical periodontitis (Vianna et al. 2006a).

Archaeal diversity was limited to a Methanobrevibac- ter oralis-like phylotype and the size of the archaeal population accounted for up to 2.5% of the total prokaryotic community (i.e., bacteria plus archaea), as evaluated by real-time PCR (Vianna et al. 2006a).

5.17.2 Fungi

Fungi are eukaryotic microorganisms that have been found only in 2% of primary root canal infections by broad-range PCR using universal fungal primers (Siqueira et al. 2002a). However, a species-specific PCR-based study has reported the occurrence of Can- dida albicans in 21% of the samples from primary root canal infections (Baumgartner et al. 2000). Another study using species-specific PCR has found C. albi- cans in 9% of the root canal-treated teeth with apical periodontitis (Siqueira and Rôças 2004a). (For a more detailed discussion of fungi see Chapter 9.)

5.17.3 Viral infections

Viruses are obligate intracellular parasites in the sense that they are totally dependent on living cells to replicate. Therefore, the necrotic pulp is not a place where viruses can prosper. On the other hand, recent molecular studies have detected some herpesviruses in samples taken from apical periodontitis lesions, where living host cells abound. Evidence of herpesvirus infection has been observed in symptomatic apical periodontitis lesions (Sabeti et al. 2003a,b), abscesses (Chen et al. 2009; Ferreira et al. 2011a,b) and large lesions (Sabeti et al. 2003b; Sabeti and Slots 2004). (For more detailed discussion of viral infections see Chapter 8.)

5.18 Next-generation DNA sequencing analyses of the endodontic microbiome

Recent studies have investigated the diversity of the endodontic microbiome using the pyrosequencing approach. Li et al. (2010) compared the endodontic bacterial diversity as determined by pyrosequencing or the cloning/Sanger sequencing approach. Pyrose- quencing yielded a 600-fold increase in depth of coverage compared to Sanger sequencing. Sanger sequencing and pyrosequencing yielded 8 vs. 13 phyla, 10 vs. 22 classes, 11 vs. 43 orders, 20 vs. 97 families, and 25 vs. 179 genera, respectively. These

Molecular Analysis of Endodontic Infections 117

results showed that pyrosequencing allowed much better characterization of the endodontic microbiome than the traditional Sanger sequencing approach.

In another study of the endodontic microbiome using pyrosequencing, Santos et al. (2011) compared samples from symptomatic (abscesses) with those from asymptomatic endodontic infections. Overall, about 900 bacterial species-level phylotypes belong- ing to 67 genera and 13 phyla were detected (Fig- ure 5.13). The most abundant phyla in symptomatic infections were Firmicutes, Fusobacteria, and Bac- teroidetes, while in asymptomatic infections the dom- inant phyla were Firmicutes, Bacteroidetes, and Acti- nobacteria. Members of Fusobacteria were much more prevalent in symptomatic cases. The most abun- dant/prevalent genera in symptomatic infections were Fusobacterium and Parvimonas. Twenty genera were exclusively detected in symptomatic infections and 18 in asymptomatic infections. Only 18% of the

phylotypes were shared by the two conditions. Symp- tomatic infections were significantly more diverse than asymptomatic infections. Although a high interindividual variation in bacterial communities was observed, many samples tended to group together according to the type of infection, which suggests the existence of patterns related to disease severity.

Lim et al. (2011) also used pyrosequencing to compare the microbiome of symptomatic and asymptomatic endodontic infections. Overall, 141 bacterial genera from 13 phyla were detected. In the genus level, Pyramidobacter, Streptococcus, and Lep- totrichia were more abundant in asymptomatic teeth, while Neisseria, Propionibacterium, and Tessaracoc- cus were more frequent in symptomatic cases. Total bacteria counts were significantly higher in symp- tomatic teeth than in asymptomatic teeth. Hsiao et al. (2012) used pyrosequencing to evaluate the transition of the microbiome from a healthy oral condition to an

Fig. 5.13 Relative abundance of the different bacterial phyla in symptomatic (acute) and asymptomatic (chronic) endodontic infections. (a) Overall data. (b) Data according to the clinical condition. Source: Santos et al., http:// journals.plos.org/plosone/article?id=10.1371/journal.pone.0028088. Used under CC BY 4.0 https://creativecommons.org/ licenses/by/4.0/.

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118 Endodontic Microbiology

endodontic infection, and showed a decreased diversity in root canal and abscess samples when compared with the oral samples. Streptococcus was the most abundant genus in the oral cavity, while Prevotella and Fusobac- terium were most abundant in diseased samples.

Pyrosequencing has been used to unravel the diversity of bacterial communities present specifi- cally in the apical root canal of teeth with pri- mary infections. Siqueira et al. (2011) evaluated DNA extracts from cryopulverized apical root segments of extracted teeth with primary apical periodontitis. The sequences obtained were taxonomically classified into 187 bacterial species-level operational taxonomic units (OTUs) (at 97% similarity), 84 genera, and 10 phyla. The most represented, abundant, and prevalent phyla were Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Actinobacteria. The mean number of species-level phylotypes per sample was 37, rang- ing from 13 to 80. A great interindividual variation in the composition of the apical microbiome was evi- dent. Another study (Ozok et al. 2012) used similar approaches to evaluate the microbiome present in the apical and coronal segments of infected root canals. Overall, approximately 600 taxa were found, repre- senting 24 bacterial phyla and one archaeal phylum. Proteobacteria were more abundant in the apical sam- ples, while Actinobacteria prevailed in the coronal samples. Significantly more taxa were found in the apical samples than in coronal samples.

The bacterial community profiles associated with primary and persistent endodontic infections was sub- ject of a pyrosequencing study by Hong et al. (2013). Analyses of untreated and treated teeth revealed 803 taxa belonging to 148 genera and 10 phyla. The Bac- teroidetes phylum was the most abundant in both pri- mary and persistent infections. No significant differ- ences in bacterial diversity were observed for the two conditions. The mean number of species-level taxa per sample was about 98 in primary infections and 122 in persistent infections. The authors concluded that per- sistent infections have as diverse bacterial community as primary infections.

Extraradicular infection in root canal-treated teeth with symptomatic apical periodontitis lesions was evaluated for bacterial diversity using pyrosequencing (Saber et al. 2012). Lesions were sampled during apical surgery, and 54% of them yielded PCR ampli- cons. Bacteria belonged to 10 phyla and 73 genera. The most abundant genera were Fusobacterium, Streptococcus, Prevotella, Corynebacterium, and

Porphyromonas. (For a more detailed discussion of periapical microbiology see Chapter 6.)

All these studies are in line with many others exam- ining different regions of the human body to show that NGS technologies, especially the pyrosequening approach, have already revolutionized the knowledge of the human-associated microbial diversity (Siqueira et al. 2012). However, there is still a need for improve- ments, such as increasing resolution of identification to the species level, reducing costs, and implementing bioinformatics tools for accurate and high throughput analyses of the data generated by these methods.

5.19 Conclusions

The impact of molecular methods in medical micro- biology is remarkable. Endodontics as a health care discipline that deals with infectious disorders has sig- nificantly benefited from the power of these technolo- gies.

As the breadth of bacterial diversity in endodontic infections has been deciphered by molecular biology methods, the list of candidate endodontic pathogens has expanded to include several cultivable and as-yet- uncultivated species that had been missed or under- estimated by culture-dependent methods. Bacteria are by far the most frequent and most diverse group of microorganisms involved with these infections. Fig- ure 5.2 depicts several bacterial species and phy- lotypes that have been found by different molecu- lar methods in samples taken from distinct clinical conditions. The occasional presence of fungi in endodontic infections has been confirmed by molecu- lar methods. Also, other microorganisms never found previously in association with apical periodontitis— archaea and herpesviruses—have been recently detected by molecular approaches. A role for Archaea and viruses in the etiology of apical periodontitis remains to be clarified.

Another important contribution of molecular meth- ods to the field of endodontic microbiology refers to bacterial community analyses. Studies using community-profiling techniques have given support to the concept that the bacterial biofilm community as a whole is the unit of pathogenicity in endodon- tic infections, which heralds a paradigm shift in the pathogenesis of apical periodontitis. Communi- ties may vary according to the disease type and geo- graphic location of the individuals. Each individual

Molecular Analysis of Endodontic Infections 119

has its own endodontic microbiome in terms of species composition and abundance.

Molecular biology techniques hold the hope of mak- ing the knowledge of endodontic infectious processes still more accurate in the near future. Moreover, molec- ular methods have the potential to make diagnosis more rapid and evidence-based antimicrobial therapy a reality. In fact, the future looks bright for those involved in endodontic microbiology research and for those who will benefit from the refined knowledge (i.e., clinicians and patients). Paraphrasing the great Dutch microbiologist Martinus Beijerink “Happy are those who are starting now.”

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Chapter 6 Extraradicular Endodontic Infections Brenda P.F.A. Gomes and Ericka T. Pinheiro

6.1 Introduction 6.2 Brief review of the endodontic

microorganisms in infected root canals 6.3 Pathways of microbial access to the

dental pulp 6.4 Infection of the root canal space 6.5 Sequelae of pulp infection 6.6 Bacterial invasion of the

periapical tissues 6.7 Microbial factors in periapical lesions

6.8 Bacterial evasion of host defense 6.9 Extraradicular endodontic infections

6.9.1 Acute apical abscesses 6.9.2 Biofilms on the external root

surface (extraradicular biofilms) 6.9.3 Periapical biofilms

6.10 Treatment of endodontic infections 6.11 Conclusions 6.12 Acknowledgments 6.13 References

6.1 Introduction

Microorganisms and their by-products are considered the primary etiologic agents in the development of pulp necrosis and periapical lesions, and are largely responsible for the failure of endodontic treatment (Kakehashi et al. 1965; Möller 1966; Sundqvist 1976; Möller et al. 1981; Nair et al. 2005; Lin et al. 1992; Gomes et al. 1994; 1996b, 2004b; Nair 2006; Siqueira and Rôças 2011).

Bacterial products can act on the host tissues in two basic ways: (i) through a direct toxic effect in the tissues by enzymes (e.g., hyaluronidase, collagenases) or metabolic end products (e.g., short-chain fatty acids, indole acids, sulfur compounds, ammonia, polyamines) and (ii) through indirect toxicity by stim- ulating the inflammatory defense systems through the release of components from the bacterial cells, such as lipopolysaccharide, peptidoglycan, and lipoteichoic acid (Siqueira and Rôças 2007). Lipopolysaccharide (LPS), or endotoxin, is a major constituent of the cell wall of Gram-negative bacteria, and is secreted

in vesicles by growing organisms or released during the disintegration of bacteria after death. Endotoxin is one of the most important virulence factors involved in the development of periapical inflammation and bone destruction, activating immune-competent cells and leading to the release of a variety of proinflammatory mediators. Lipid A is the bioac- tive component of LPS that is responsible for the majority of the host’s immune response (Endo et al. 2012).

Therefore, one of the most important steps to suc- cessful endodontic treatment is microbial elimination from the inside of the root canal system by mechanical and chemical procedures, because the microorganisms cannot be eliminated by defense mechanisms (Gomes et al. 1996c; Nair et al. 2005; Nair 2006).

Six biologic factors lead to, or have been asso- ciated with, asymptomatic radiolucencies persisting after root canal treatment:

1. Intraradicular infection persisting in the complex apical root canal system;

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2. Extraradicular infection, usually identifiable in the form of periapical actinomycosis;

3. Extruded root canal filling or other exogenous mate- rials that cause a foreign body reaction;

4. Accumulation of endogenous cholesterol crystals that irritate periapical tissues;

5. True cystic lesions; and 6. Periapical healing by scar tissue formation (Nair

2006).

However, the main reasons for endodontic failure are found inside the root canal, and are caused by the persistence of intraradicular infection. Such infec- tions can arise from inadequate aseptic control (such as inadequate dental dam isolation), inappropriate access cavity, missed canals or canal ramifications, inade- quate chemomechanical preparation, coronal and api- cal microleakage, as well as other causes (Nair 2006).

Microorganisms surviving disinfection measures may die or remain viable depending on the amount of nutrients available and their ability to survive in con- ditions of nutritional deficiency. Microorganisms that remain viable will result in endodontic failure only if they have access to the periapical tissues, if they are pathogenic, and if they are plentiful enough in number to induce or perpetuate a periapical lesion (Seltzer and Farber 1994; Gomes 1995; Gomes et al. 1996c; Özok et al. 2012).

Even in treated root canals, some bacteria may remain alive in the anatomic complexity of the root canal system, particularly in the apical third (Wada et al. 1998; Nair et al. 2005; Özok et al. 2012), because they are not affected by the measures used in endodon- tic infection control.

The presence of an extraradicular infection is also considered a possible cause of treatment failure. Over- all, its prevalence ranges from 6% to 50%, depend- ing on the technique used for bacterial detection in different studies (Sunde et al. 2003; Ricucci and Siqueira 2010). However, the high success rate of both endodontic treatment (83%) and nonsurgical retreat- ment (80%) of teeth with apical periodontitis (Ng et al. 2011) indicates that the main cause of the infection is inside the root canal. Moreover, failure of endodontic retreatment requiring surgical intervention is mostly because of the presence of microorganisms in the isthmus and apical ramifications (Wada et al. 1998; Nair et al. 2005). Once the apical portion is surgically removed, and the apex reinstrumented and sealed, the success rate averages 88–94% (Setzer et al. 2012).

Extraradicular infection usually originates from an intraradicular infection that has spread to the periradic- ular tissues via apical foramen. It is composed of dif- ferent morphologic types of microorganisms, which adhere to the cementum/dentin near the apex, and are surrounded by a layer of polysaccharide, which acts as a shield. Such a microbial arrangement is known as a biofilm. Biofilms, which can reach 50–100 μm in thickness, are resistant to antibiotics (about 1500–8000 times), and are large enough to thwart the immune sys- tem (Costerton et al. 1987, 1994, 1999; Distel et al. 2002).

Studies have demonstrated the presence of bacte- ria on the external root surface (Tronstad et al. 1990; Molven et al. 1991; Ferreira et al. 2004; Wang et al. 2012) or in the periapical lesion (Sundqvist and Reuter- ving 1980; Nair 1987; Tronstad et al. 1987; Sjögren et al. 1988, 1997; Wayman et al. 1992; Abou-Rass and Bogen 1998; Siqueira 2003; Signoretti et al. 2011, 2013; Wang et al. 2012).

6.2 Brief review of the endodontic microorganisms in infected root canals

Currently, over 1000 bacterial species or taxa have been found in the human oral cavity, belonging to 13 phyla (Wade 2013). All these taxa are able to reach the root canal system and are thus potential endodontic pathogens (Jung et al. 2000). Currently, about 916 microbial taxa have been identified within the root canal system (Santos et al. 2011). Of them, 486 taxa were found in acute endodontic infections, 265 in chronic infections, and 165 in both (Santos et al. 2011). These microorganisms usually live among strict anaerobes (Sundqvist 1992a,b; Gomes et al. 1996a, 2004b; Ribeiro et al. 2011; Özok et al. 2012; Siqueira and Rôças 2013). This fact suggests that a selective process operates inside the canals, where nutritional factors, redox potential variability, pH, temperature, positive interactions, antagonisms between microor- ganisms, and the host defense mechanisms have impor- tant roles (Gomes et al. 1996b; Marsh and Martin 2009; Özok et al. 2012).

As happens in the oral cavity, bacteria are the most commonly found microorganisms in root canals, belonging to 13 phyla according to present knowl- edge. Of the major phyla, Firmicutes, Fusobacteria, and Bacteroidetes are the most abundant in acute infections (Santos et al. 2011), while Firmicutes,

Extraradicular Endodontic Infections 131

Bacteroidetes, and Actinobacteria are the most abun- dant in chronic infections (Santos et al. 2011; Ribeiro et al. 2011). In addition, fungi, yeasts (Molander et al. 1998; Sundqvist et al. 1998; Siqueira et al. 2002; Pin- heiro et al. 2003; Siqueira and Rôças 2004; Anderson et al. 2013), viruses (Slots et al. 2003; Sabeti et al. 2003, 2012), and Archaea (Vianna et al. 2006; Özok et al. 2012) are also detected in the root canals.

6.3 Pathways of microbial access to the dental pulp

It is known that bacteria cannot reach the pulp through the crown as long as the enamel layer is intact. Further- more, the root walls are similarly naturally impervious. Nevertheless, it is clinically apparent that the dental pulp, even in the intact tooth, can become infected (Gomes 1995).

Normally, the enamel and cementum layers protect and isolate the dentin and pulp from bacterial inva- sions. When one of these tissues is lost, the bacte- ria have the opportunity to invade the pulp tissues. The microorganisms’ main routes of access to the pulp cavity are dental caries, exposure of the denti- nal tubules, direct pulp exposure, microleakage of restorative materials, and lateral canals of teeth with periodontal involvement. Other examples of commu- nication can be through root resorptions (internal and external), fractures, and iatrogenic procedures (Gomes 1995; Love 2009). The development of a pulp infection secondary to a bacteremia, also known as anachoresis, has not been supported by scientific evidence (Siqueira and Rôças 2011).

The most common route of bacterial irritation is dental caries, which induces inflammatory responses and eventually bacterial invasion of the pulp tissue that results in pulp necrosis if appropriate therapeutic measures are not adopted (Gomes 1995; Love 2009).

Once the infection overcomes physical and biologic barriers, its expansion depends upon the survival of the microorganisms within the pulp space (Gomes 1995).

6.4 Infection of the root canal space

Once the microorganisms invade the pulp chamber, they shift to the inside of the tissues via cell divi- sion and limited motility, until they reach the apical third of the root canal. In this microsystem, bacterial

communities are formed, favoring the establishment of facultative microorganisms in the coronal and middle third of the canal and the establishment of strict anaer- obic microorganisms in the apical third. However, this does not mean that facultative microorganisms are not found in the apical region or that strict anaerobes are not found in the coronal third. Strict anaerobic bac- teria may be protected from oxygen by the presence of another microorganism (Gomes 1995; Özok et al. 2012).

Once microorganisms reach the root canal space, they can be found suspended in the lumen of the root canal (planktonic form), with a variety of morpho- logic types consisting of cocci, bacilli, and filamen- tous forms, or they might adher to the root canal walls, forming a biofilm (sessile form). Bacteria can be found penetrating the dentinal tubules, colonizing the lateral, secondary, and accessory canals.They can also form biofilms in the external root surface (cementum) or in the periapical region (Figure 6.1). The colonization of these sites is directly related to the duration of infec- tion and the composition of the microbiota (Nair 1987; Nair et al. 1990, 2005; Tronstad et al. 1990; Ricucci et al. 2005; Ricucci and Siqueira 2010).

6.5 Sequelae of pulp infection

Pulpal infection is followed by pulpal disease (rang- ing from reversible to irreversible pulpitis), which will proceed to total necrosis, depending on how the host defense mechanisms cope with the increased num- bers of (virulent) bacteria and their products. If left untreated, pulpal disease will spread beyond the apex of the tooth, leading to periapical disease. Initially, only the periodontal ligament will be involved in the periapical reaction, but, eventually, resorption of cementum (and dentine) and breakdown of the alve- olar bone ensues, so all the tissues of the periodon- tium become affected. Thus, the infection is a cascade reaction that starts with dental caries and then pro- gresses to pulpal disease, pulpal necrosis, and peri- apical disease. Periapical disease may have systemic manifestations with clinical signs, such as high tem- perature, malaise, and leucocytosis; in susceptible patients, where bacterial endocarditis is a potential risk, it can have life-threatening implications (Gomes 1995; Tronstad 2008).

In order to avoid the spread of infection from the root canal to the periapical tissues, root canal treatment

132 Endodontic Microbiology

(a) (b) (e)

Fig. 6.1 Microbial location: (a) adhered to the root canal walls and inside the dentinal tubules; (b) suspended in the lumen of the root canal; (c) between the dentin and the gutta-percha filling material; (d) penetrating the dentinal tubules of root-filled teeth; (e,f) in the external root surface.

is carried out. This therapy should be performed under aseptic conditions so that additional external sources of contamination are avoided. This treatment is essen- tially a debridement and disinfection procedure, and its objective is removing microorganisms, debris, and potential substrate to establish conditions in which infection will not recur. The prepared root canal should be well sealed to prevent further infection. Moreover, if the canals are completely obturated apically and laterally, the microorganisms, assuming any are still present in the main canal, in the dentinal tubules, or between canal wall and root filling, would not be able to survive. Appropriate restoration of endodontically treated teeth will decrease the coronal microleakage and contribute to the success of the treatment (Gomes 1995; Lin and Huang 2011).

In the past decade, regenerative procedures of the dental pulp are considered following an infection, par- ticularly in immature teeth. However, prior to pulp regeneration procedures, the pulp space and dentinal walls need to be sufficiently disinfected to promote regeneration (i.e., physiologic replacement of dam- aged tooth structures, including dentin and root struc- tures, as well as cells of the pulp–dentin complex). The necessary level of disinfection is likely higher than that accepted for traditional endodontic therapy, because the traditional techniques of lowering bac- terial loads and preventing bacterial access to peri- apical tissues are conducive to healing. In contrast, a lack of filling in the canal as the regenerative tissue is developing may be conducive to bacterial prolif- eration. Thus, it is necessary to maintain an aseptic

Extraradicular Endodontic Infections 133

environment in the pulp space following disinfection procedures for a longer period to allow the new tissue sufficient time to establish itself in the root canal envi- ronment. Finally, while the emphasis in regenerative procedures is clearly on resumption of mineralization to achieve completed root formation and maturation of the tooth, it is essential that the regenerative tis- sue contains the elements of a robust immune system, which would allow it to clear any residual infection and combat any coronal, tubular, or periapical sources of new infection (Fouad 2011).

6.6 Bacterial invasion of the periapical tissues

Fish and MacLean (1936) suggested that bacterial tox- ins and noxious products of pulp degradation pass from the root canal into the periapical spaces. These toxins initiate the destructive inflammatory responses observ- able within the alveolar bone. Therefore, in cases of chronic lesions, four well-defined anatomopathologic zones appear in the periapical region as a consequence of the tissue’s reaction to the irritation produced by bacteria and their products inside the canal. Starting from the apex, the zones are as follow:

1. The infected zone, characterized by the presence of bacteria and polymorphonuclear leucocytes;

2. The contaminated zone, characterized by the pres- ence of round cells taking the place of normal cells;

3. The zone of irritation, characterized by the presence of histiocytes and osteoclasts; and

4. The zone of stimulation, characterized by the pres- ence of fibroblasts and osteoblasts (Gomes 1995).

It can therefore be deduced that in necrotic teeth the host defense mechanisms outside the canal are more “equipped” to fight against bacterial attacks, because there is an effective collateral blood circula- tion at the periodontal level. The result of this conflict depends on the number and virulence of the microor- ganisms and on the host defenses. This contest can continue indefinitely or until the bacteria and their by-products are eliminated by some form of therapy (Gomes 1995).

Although the zone of infection is mainly restricted to the root canal system and dentinal tubules, bacteria sometimes overcome the host defense and invade the periapical tissues, resulting in the development of a

periapical inflammatory lesion. According to the type and pathogenicity of the bacteria in the root canal and the status of the host’s defense, three basic kinds of periapical lesions can occur: abscess, granuloma, or radicular cyst. Suppuration and abscess formation are likely to develop if pyogenic organisms, such as Prevotella intermedia and Porphyromas endodon- talis, predominate. On the other hand, if less virulent organisms are present in great number, diffusion of bacterial antigens may result in an infiltration of the periapical tissues by macrophages, lymphocytes, and other inflammatory elements, causing a granuloma to develop (Trowbridge and Stevens 1992).

6.7 Microbial factors in periapical lesions

The development of a periapical lesion is a host- mediated immune response to microbial invasion. Innate immune cells of periapical tissues recog- nize bacterial antigens and produce proinflammatory cytokines, leading to bone resorption (Stashenko et al. 1994). These cytokines include tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-11, and IL-17, which along with other factors are in charge of activation and differentiation of osteoclasts (Stashenko et al. 1994; Taubman et al. 2005). During bone resorp- tion, some cytokines also induce matrix metallopro- teinase (MMP) production, which acts in the degra- dation of bone extracellular matrix (Kusano et al. 1998). Large amounts of proinflammatory cytokines and MMPs have been detected in periapical lesions, and seem to have important roles during the devel- opment of periapical lesions (Safavi and Rossomando 1991; Corotti et al. 2009).

Some bacterial virulence factors, such as LPS (known as endotoxins), are associated with increased production of proinflammatory cytokines and MMPs. LPS liberated by Gram-negative bacteria has been detected in teeth with pulp necrosis (Gomes et al. 2009; Martinho et al. 2010, 2012). Strong evidence correlates the presence of LPS in periapical lesions with inflammatory reactions in these tissues (Schon- feld et al. 1982). In experimental periapical lesions in rats, the level of LPS in periapical tissues gradu- ally increased with increasing time after pulp expo- sure to the oral cavity (Yamasaki et al. 1992). More- over, LPS-stimulated cells expressed higher levels of MMP genes than nonstimulated cells (Letra et al. 2013).

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However, some authors have questioned the signifi- cance of LPS in the development of a periapical lesion, because its progression in LPS hyporesponsive mice was similar to that found in a healthy control ani- mal (Fouad and Acosta 2001). It has been suggested that other bacterial virulence factors also have impor- tant roles in the pathogenesis of periapical lesions. In terms of antigenicity, other cell-wall components, such as peptidoglycan, lipoteichoic acid (LTA) of Gram- positive bacteria and fimbriae, can induce the host immune cells to produce an inflammatory response with significant cytokine production (Draing et al. 2008).

6.8 Bacterial evasion of host defense

Survival in this region, where host defenses have greater access to the infectious agent, is possible only for microorganisms able to override these defenses. Few bacterial species are able to survive within the periradicular tissues, making them therefore respon- sible for the failure of endodontic treatment. Studies have reported the isolation of species of Actinomyces spp. and Propionibacterium propionicum (Sundqvist and Reuterving 1980; Sjögren et al. 1988, 1997; Siqueira 2003; Signoretti et al. 2011, 2013; Wang et al. 2012) in resistant endodontic periapical lesions. These bacteria can form cohesive colonies, with large num- bers of cells, thus escaping the collective phagocytosis that would be performed by defense cells (Fidgor et al. 1992).

Another mechanism for bacterial evasion of host defenses is the arrangement of bacteria in biofilm on the external root surface. The periradicular biofilm is characterized by a population of microorganisms that are attached to the cementum and/or dentin in the apical portion of the root and are surrounded by their extracellular products (outer layer known as a polysaccharide glycocalyx), which form an intermi- crobial matrix. The structure of the polysaccharide matrix surrounding the biofilm limits access of defense molecules (antibodies and complements) and phago- cytic cells (macrophages and neutrophils) (Palmer and White 1997).

The ability of bacteria to survive in the periapical tissues raises the controversy of whether the human defense mechanism is able to identify, ingest, and destroy, or at least weaken, the foreign invaders. How- ever, some microorganisms can survive even after

phagocytosis by polymorphonuclear and mononuclear leukocytes, multiplying and even killing the cell that ingested them. Moreover, even if the microorgan- isms are killed, nonbiodegradable components of their cell may persist for prolonged periods and stimu- late chronic inflammatory responses. The inability of phagocytes to degrade intracellular bacteria or their products may be because of a number of sur- vival mechanisms, such as inhibition of the respira- tory burst, the presence of shielding capsular mate- rial, inhibition of the fusion of the phagolysosome, survival within and escape from the phagolysosome, lack of adequate lysosomal enzymes, and the pres- ence of a virulence gene in certain microorganisms (Seltzer and Farber 1994). Moreover, some microor- ganisms associated with endodontic infections, such as Porphyromonas endodontalis and P. gingivalis, can invade different cell types, including endothelial cells (Dorn et al. 2002; Li et al. 2008). After invasion, they may persist in the tissue because of their abil- ity to transmit among different cell lines (Li et al. 2008).

The fact that microorganisms are able to survive in the periapical tissues and maintain an infectious disease process underlines the importance of hav- ing an adequate level of asepsis in endodontic ther- apy, thus keeping the canal and periapical tissues free from viable bacteria (Wada et al. 1998; Nair et al. 2005).

6.9 Extraradicular endodontic infections

Microbial invasion of apical tissues is usually a con- sequence of intraradicular infection. A typical case of an extraradicular infection is an acute apical abscess, wherein a massive bacterial invasion occurs with a con- sequent accumulation of pus in the periapical region. In the same way, a milder and more continuous micro- bial invasion of apical tissues occurs in chronic api- cal abscesses, with formation of exudate which drains through a sinus tract. Except for this situation, bacteria are generally found in a minority of asymptomatic api- cal periodontitis tissues of untreated teeth. Nair (1987) observed bacteria within the body of periapical lesions in only 5 out of 31 lesions, including one case of peri- apical actinomycosis, one cyst, and three abscesses. Similarly, Ricucci et al. (2006) detected bacteria in apical tissues of untreated teeth only in abscesses or cysts, found in 18 out of 50 periapical lesions. As most

Extraradicular Endodontic Infections 135

invaders usually cannot survive the host’s defense, they may be considered as transient microorganisms in api- cal tissues.

However, some microbial species have the abil- ity to circumvent the host’s innate immune response and establish extraradicular infections, mainly by their capacity to develop biofilms on the external root sur- face or form bacterial aggregates within the inflamed apical tissues.

Extraradicular infections and periapical biofilms have been mainly found in posttreatment apical peri- odontitis (Wang et al. 2012, 2013), and are therefore considered possible causes of endodontic failure (Nair 2006).

6.9.1 Acute apical abscesses

An acute apical abscess is characterized by pus for- mation from microbial invasion of periapical tissues (Siqueira and Rôças 2013; Sousa et al. 2013). Invad- ing bacteria must have high numbers and virulence factors to overcome the host defense and to establish acute infections. In these cases, culture and molecular studies of purulent exudate or the root canal revealed a polymicrobial infection dominated by anaerobic species (De Sousa et al. 2003; Montagner et al. 2010, 2012; Santos et al. 2011; Sousa et al. 2013, 2014; Siqueira and Rôças 2013).

Molecular methods have some advantages over culture-dependent methods for microbial analysis of endodontic infections because of their higher sensitiv- ity and the capability to detect as-yet-uncultivated bac- terial species. Moreover, molecular methods involving 16S rRNA-based bacterial diversity analyses, includ- ing cloning and Sanger sequencing, have made possi- ble the study of microbial communities (Siqueira and Rôças 2005).

For these reasons, molecular analyses of acute api- cal abscesses have disclosed a more complex micro- bial community than that previously shown by culture- based studies. Species of the genera Fusobacterium, Parvimonas, Prevotella, Porphyromonas, Dialister, Streptococcus, and Treponema were prevalent in most studies (Siqueira and Rôças 2013). However, no spe- cific pathogen was associated with acute apical abscess development. On the contrary, the bacterial commu- nity seems to be the unit of pathogenicity of acute infections, where the virulence is mainly a result of microbial interactions in the community (Siqueira and Rôças 2009, 2013).

Knowledge of microbial diversity of endodon- tic infections is improving as molecular techniques develop. Recently, the bacterial diversity of abscesses was disclosed by pyrosequencing analysis (Santos et al. 2011; Hsiao et al. 2012). This high-throughput sequencing technique has a superior degree of detec- tion, commonly referred to as depth of coverage, than traditional Sanger sequencing (Harrington et al. 2013). Therefore, it has revealed much higher bacterial diver- sity than previously reported for acute endodontic infections (Santos et al. 2011; Hsiao et al. 2012). San- tos et al. (2011) found that acute infections were sig- nificantly more diverse than chronic infections. The most abundant phyla in acute infections were Fir- micutes (e.g., genera Parvimonas, Dialister, Eubac- terium, Filifactor, Peptostreptococcus, and Streptococ- cus), Fusobacteria (e.g., genus Fusobacterium), and Bacteroidetes (e.g., genera Prevotella and Porphy- romonas). Moreover, the Fusobacterium genus was more prevalent in acute infections than in chronic cases in this study. Similarly, Hsiao et al. (2012) found that Fusobacterium was the most abundant genus in abscesses, as revealed by pyrosequencing analysis.

The treatment of acute apical abscesses comprises drainage of the pus through the root canal and/or inci- sion of the mucosa/skin to promote relief from pain. In most cases, after the acute symptoms have ceased, the endodontic treatment will control the intraradicu- lar infection, whereas the host defense will eliminate the extraradicular bacteria. Figure 6.2 illustrates the treatment of an acute apical abscess.

6.9.2 Biofilms on the external root surface (extraradicular biofilms)

6.9.2.1 Definition, composition, and prevalence

Microbial biofilms consist of a dynamic community of interacting cells that are attached to a substrate and embedded in a matrix of extracellular polymers (Costerton et al. 1994). From the clinical point of view, one of the major changes in bacterial biofilms is their increased resistance to host defense mechanisms and antimicrobial agents (Mohammadi et al. 2013).

Chronic apical periodontitis is a disease caused mainly by intraradicular bacterial biofilms, which can be found adhered to dentin walls of the main/secondary canals, lateral canals, apical ramifications, and isth- muses. Bacteria derived from biofilms can also pene- trate dentinal tubules. Occasionally, they may reach the

136 Endodontic Microbiology

(a) (b) (c)

(e)

(d)

(f) (g) (h)

Fig. 6.2 Treatment of acute apical abscess. Patient with history of acute pain, presenting pain on palpation and tenderness to percussion in the upper left first premolar tooth. (a) Swelling present in the left side of the patient’s face. (b) The maxillary left second premolar shows a deep carious lesion. (c) Radiograph shows an apical widening in the tooth. (d) Surgical drainage and placement of a drain in the periapical region of the tooth, which remained for 24 hours. (e) Decrease of the patient’s swelling. (f) Drain removal, root canal debridement, placement of a root canal medication for 7 days, coronal restoration. (g) Radiograph showing the root canal filling. (h) Restoration of tooth.

external root apical surface via dentinal tubules/apical foramens, and form extraradicular biofilms (Ricucci and Siqueira 2010).

Several methods have been used to visualize the extraradicular biofilm structure, including scanning electron microscopy (SEM; Tronstad et al. 1990; Lomçali et al. 1996; Siqueira and Lopes 2001; Noiri et al. 2002; Leonardo et al. 2002), fluorescence in situ hybridization (FISH; Sunde et al. 2003), histo- bacteriologic techniques with Brown and Brenn Gram staining (Ricucci et al. 2009; Ricucci and Siqueira 2010), or a combination of techniques (Wang et al. 2012, 2013). These studies have shown that periapi- cal biofilms comprised different morphologic types of microorganisms, including cocci, rods, and filaments, which were adhered to the external apical surface, especially in the cementum resorption areas. These microorganisms were embedded in an extracellular matrix and coated with a smooth structure (bacterial by-products) that provides protection against the host defense cells.

Extraradicular biofilms are mainly related to post- treatment apical periodontitis, which is character- ized by persistent apical inflammation even after

satisfactory endodontic procedures (Noiri et al. 2002; Wang et al. 2012). It is well accepted that extraradicular bacteria are not found in teeth with pulp necrosis when there are no radiographically visible periapical lesions (Leonardo et al. 2002; Wang et al. 2013). However, the data on the prevalence of extraradicular biofilms in untreated teeth with asymptomatic apical periodon- titis show conflicting results. Whereas some studies have frequently observed extraradicular microorgan- isms in teeth with pulp necrosis and radiographically visible periapical lesions (Leonardo et al. 2002, 2007; Ricucci and Siqueira 2010; Wang et al. 2013), others have revealed that it is a rare condition (Siqueira and Lopes 2001; Ricucci et al. 2009; Ricucci and Siqueira 2010; Wang et al. 2013).

Lomçali et al. (1996) found microorganisms in most of the root tips when they conducted an SEM of eight extracted teeth with chronic periapical lesions; similar findings were also observed by Leonardo et al. (2002). In contrast, Siqueira and Lopes (2001) observed extraradicular microorganisms in only one out of 27 extracted teeth analyzed by SEM.

Recent studies using histobacteriologic techniques revealed a low prevalence of extraradicular biofilms

Extraradicular Endodontic Infections 137

in teeth with necrotic pulps and apical periodontitis (Ricucci et al. 2009; Ricucci and Siqueira 2010; Wang et al. 2013). Ricucci and Siqueira (2010) observed that extraradicular biofilms were present in only 6 out of 100 teeth with apical periodontitis, and were usually dependent on intraradicular biofilms. These findings help to explain why periapical repair usually occurs after correct endodontic disinfection procedures of teeth with necrotic pulps.

Although they are a rare condition, extraradic- ular biofilms may represent a constant source of infection, thus resulting in persistent apical inflam- mation. In fact, extraradicular biofilms have been closely associated with failed endodontic treatment. The nature of the biofilm infection is further compli- cated when endotoxin-producing Gram-negative bac- teria are present. Once released, endotoxin elicits the dosage-dependent stimulation of various inflammatory cytokines and signal transduction pathways, leading to an array of symptoms (Jiang et al. 2002). Using both SEM and Brown and Brenn Gram-staining techniques, Wang et al. (2013) detected extraradicular biofilms in all teeth with persistent apical periodontitis (10 cases) and in only three cases of primary chronic apical peri- odontitis.

6.9.2.2 Extraradicular biofilms in posttreatment apical periodontitis

The presence of biofilms on the root tips of teeth with persistent apical periodontitis has been widely reported in the literature. Tronstad et al. (1990) found the presence of periapical biofilms in teeth refrac- tory to endodontic treatment. Using SEM, the authors observed a bacterial plaque adjacent to the apical foramen, which was dominated by cocci and rods held together by an extracellular material. Noiri et al. (2002) also found extraradicular bacterial biofilms in teeth with refractory apical periodontitis, which con- sisted of both bacteria and glycocalyx-like structures. Occasionally, extraradicular biofilms also show areas of mineralization with calculus-like appearances, as reported by some authors (Ricucci et al. 2005; Ricucci and Siqueira 2010).

Besides different bacterial morphologic types, fun- gal shapes have also been observed within extraradic- ular biofilms through SEM examination. Ferreira et al. (2004) reported a clinical case of posttreatment api- cal periodontitis caused by extraradicular infection. They examined the root tip with SEM, and found cocci

and fungal forms covered with an extracellular poly- meric substance in the resorption areas of the root apex (Figure 6.3).

Even though extraradicular biofilms have been implicated in the failure of endodontic treatment, it is important to note that most unsuccessful outcomes can be attributed to persistent intraradicular infection remaining in the inaccessible apical areas of the root canal system. Ricucci et al. (2009) examined histo- logically 24 previously treated teeth with apical peri- odontitis, and found intraradicular bacteria in all cases, whereas extraradicular biofilms were observed in only five teeth. Moreover, it was shown that extraradicular biofilms were more frequent in symptomatic teeth, and were usually dependent on the intraradicular infection (Ricucci and Siqueira 2010).

Signoretti et al. (2011) reported a case of persis- tent apical periodontitis caused by an intraradicular infection associated with an extraradicular biofilm. This report described a case of a previously treated lower first molar associated with the sinus tract and a periapical lesion. Procedural accidents were detected during nonsurgical endodontic retreatment, including zip and overfilling, which might have contributed to the persistence of intraradicular infections. Therefore, endodontic microsurgery was indicated. After surgery, the root apex was analyzed by SEM, and revealed bacterial biofilm surrounding the apical foramen and external radicular surface (Figures 6.4 and 6.5). The bacterial species found on this root surface were Actinomyces naeslundii, Actinomyces meyeri, Pro- pionibacterium propionicum, Clostridium botulinum, Parvimonas micra, and Bacteroides ureolyticus.

The microbial profile of extraradicular biofilms has been revealed by culture and by molecular studies. Fuji et al. (2009) analyzed the microorganisms on the surface of 20 apexes that had been obtained directly from apical lesions during root end surgery and pro- cessed using aerobic or anaerobic culture techniques. The majority of the isolated strains were facultative anaerobes, but they were frequently found in combi- nation with obligate anaerobes, forming a mixed bac- terial consortium on the external root surface. Propi- onibacterium acnes, Staphlylococcus epidermidis, and Fusubacterium nucleatum were the most frequently isolated species, so these three species might have an important role in the formation of polymicrobial biofilms.

Similarly, Noguchi et al. (2005) detected biofilm- forming bacteria in 20 root apexes associated

138 Endodontic Microbiology

(a) (b) (c) (d)

(e) (f) (g) (h)

Fig. 6.3 Resolution of persistent periapical infection by endodontic surgery. (a) Periapical radiograph after root canal treatment showing a large periapical lesion in the upper left first premolar tooth. (b) Radiographic evidence of healing of the periapical reaction 2 months after endodontic surgery. (c) Scanning electron microscopic (SEM) analysis of the removed root apex showing the buccal and lingual surfaces with extruded gutta-percha from the lingual foramen (arrow). (d) Higher magnification of the lingual foramen with extruded gutta-percha surrounded by resorption lacunae. (e) Lingual foramen presenting reproducing fungal forms. (f) Lateral of the extruded gutta-percha in the lingual foramen showing fungal forms attached to the filling material. (g) The surrounding resorptions and dentine of the lingual foramen, totally covered in a kind of “net,” probably an extracellular polymeric substance. (h) Radiographic review at 24 months postsurgery. Source: Ferreira et al. (2004). Reproduced with permission of John Wiley and Sons.

with refractory apical periodontitis using a species- specific-PCR (polymerase chain reaction) identifi- cation of three Gram-negative obligate anaerobes. Fusobacterium nucleatum, Porphyromonas gingi- valis, and Tannerella forsythensis were frequently detected. Moreover, these bacteria were observed in the biofilm structure via immunohistochemical methods.

Recently, a molecular study has revealed a high prevalence of Actinomyces sp. and Propionibacterium sp. in the external root surfaces of teeth with post- treatment apical periodontitis. Wang et al. (2012) investigated the microbial diversity of extraradicular biofilms in persistent apical periodontitis using PCR and denaturing gradient gel electrophoresis (PCR- DGGE) approach with histobacteriologic analysis. The following species were detected from 13 apical root samples: Actinomyces sp. oral, Propionibacterium sp., Prevotella sp. oral, Streptococcus sp., Porphy- romonas endodontalis, and Burkholderia. In summary, the histobacteriologic analysis revealed a complex

community of Gram-positive and Gram-negative bacteria represented mainly by rods and filaments.

Interestingly, the extraradicular biofilms that are located in the root apex seem to be very similar to sub- gingival biofilms. Both biofilms have been observed to contain the initial colonizers, represented by Strep- tococcus spp. and Actinomyces spp.; the species that coaggregate with the others and contribute to biofilm formation, such as Fusobacterium spp. and Propi- onibacterium spp.; and the presumptive pathogens that colonize already formed biofilms, including Gram-negative anaerobic species (Tronstad 2009).

In summary, extraradicular biofilms occur when microorganisms have the ability to bypass the host defense mechanisms and colonize the external root surface. In situ studies have indicated that the presence of extraradicular biofilms are rare conditions, and are usually dependent on intraradicular biofilms. There- fore, intraradicular infections are considered the main cause of endodontic failure, which may be associated with extraradicular infections. However, if present,

Extraradicular Endodontic Infections 139

Fig. 6.4 Persistent extraradicular infection in a root-filled asymptomatic human tooth: scanning electron microscopic analysis and microbial investigation after apical microsurgery. (a) A preoperative periapical radiograph showing the mandibular left first molar with an apparent radiolucency around the distal apex and widening of the periodontal ligament at the apex of the mesial root. (b) A postoperative radiograph after nonsurgical retreatment. (c) An immediate postoperative radiograph showing the resected distal root. (d) The 24-month radiograph follow-up, showing apical healing. Source: Adapted from Signoretti (2011). Reproduced with permission of Elsevier.

extraradicular biofilms may lead to a persistent infec- tion, because they are inaccessible to nonsurgical endodontic treatment. Moreover, intra- and extraradic- ular biofilms can function as a constant source of apical tissue infection.

6.9.3 Periapical biofilms

Extraradicular infection has been found not only in the form of extraradicular biofilm on the root tips

of previously treated teeth, but also in the form of bacterial aggregates within the bodies of persistent inflammatory lesions. The latter usually originate from and are dependent on intraradicular infection. How- ever, independent extraradicular infections may occur in some specific situations; the most typical case is apical actinomycosis (Nair 2006). It has been sug- gested that some bacterial species have the ability to form a biofilm-like structure within periapical lesions as a strategy to circumvent the host’s innate immune

140 Endodontic Microbiology

(a) (b) (c)

(d) (e)

Fig. 6.5 Persistent extraradicular infection in a root-filled asymptomatic human tooth: scanning electron microscopic analysis and microbial investigation after apical microsurgery. (a) Persistent sinus tract on the buccal alveolar mucosa associated with the distal apex of the lower left first molar. (b) Endodontic microsurgery: flap closure. (c) Root fragment for SEM examination. (d) SEM of extruded gutta-percha observed in the root apex was not removed after endodontic retreatment, noting in A the filling material after nonsurgical root canal retreatment and in B the previous root canal filling material that extruded through apical ledging during deobturation. (e) A, Uninstrumented apical foramen; B,C, bacterial colonies adhering to external radicular surface. Source: Adapted from Signoretti (2011). Reproduced with permission of Elsevier.

response, leading to persistent apical periodontitis. However, the status of apical actinomycosis as an inde- pendent entity has been questioned in the literature (Ricucci and Siqueira 2008).

6.9.3.1 Apical actinomycosis

Apical actinomycosis is a chronic disease that does not differ clinically or radiographicallly from other chronic apical periodontitis lesions, being identi- fied mainly by the histopathologic and microbio- logic examinations of surgical material obtained from periapical lesions (Nair 2006). Although it has been argued that apical actinomycosis may not be uncom- mon (Sakellariou 1996), the prevalence of apical acti- nomycosis among all apical lesions seems to be low; less than 5%. Therefore it should not be considered a

common cause of endodontic failure (Hirshberg et al. 2003). Nevertheless, it is important to note that the prevalent data are based on very few studies (Nair and Schroeder 1984; Hirshberg et al. 2003), because most of the publications on apical actinomycosis are case reports (Sakellariou 1996; Ricucci and Siqueira 2008; Al-Hezaimi 2010).

Actinomyces spp. and Propionibacterium propi- onicum are commonly detected in apical actinomy- cosis lesions (Happonen 1986; Sjögren et al. 1988). Actinomyces have special surface structures that enable them to build cohesive colonies, forming microscopic or macroscopic clusters in the periapical tissues (Fig- dor et al. 1992). Macroscopic clusters sometimes exu- date through the sinus tract as yellow granules and, because of this, were called sulfur granules in the older literature. In fact, microscopic analysis revealed that

Extraradicular Endodontic Infections 141

those granules consisted of bacteria that were tightly packed in extracellular material, such as a biofilm-like structure (Sunde et al. 2002). Moreover, they appeared in histologic analysis as a bacterial core with radiating peripheral filaments that result in the typical “ray fun- gus” patterns, which was the origin of the name of the genus Actinomyces (Nair 2006).

Sunde et al. (2002) found granules, similar to the so- called sulfur granules, in 9 out of 36 refractory apical lesions, which were analyzed by an anaerobic culture, SEM, and transmission electron microscopy (TEM). In these granules, Actinomyces israelii, A. viscosus, A. naesludii, and A. meyeri were identified. In addition to Actinomyces, other bacterial species were isolated from the granules. SEM analysis demonstrated rod and spirochete-like cells, and the TEM revealed bac- teria with Gram-positive and Gram-negative cell walls involved in a matrix of extracellular material.

Actinomyces species have normally been found associated with other bacterial species in apical aggre- gates. Actinomyces species might have an important role in biofilm formation by leading to the develop- ment of environmental conditions that attract other bacteria to the biofilm community, such as spirochetes and Gram-negative anaerobic bacteria (Sunde et al. 2002).

A newer Actinomyces species, A. radicidentis, was identified in root canal infections of teeth with per- sistent apical periodontitis (Kalfas et al. 2001). It has been shown that A. radicidentis, like other pathogenic Actinomyces, has the ability to form large aggregates of cells that hold themselves together by embedding themselves in an extracellular matrix. Consequently, this biofilm environment helps the microbial commu- nity to escape the host defense by preventing phagocy- tosis and thus allowing the establishment of an inde- pendent extraradicular infection (Nair et al. 2008).

Actually, most apical actinomycosis case reports found bacterial aggregates in the body of periapical lesions. Therefore, apical actinomycosis has usually been considered an example of an extraradicular cause of persistent periapical lesions. However, whether the latter is dependent on or independent of intraradicu- lar infection has not yet been clarified, because most studies have not evaluated the apical root canal associ- ated with the extraradicular actinomycotic aggregates (Ricucci and Siqueira 2008).

Ricucci and Siqueira (2008) reported a case of extraradicular actinomycosis that was supported by bacterial biofilms organized in apical ramifications of

the root canal. The patient presented with a root-filled tooth associated with persistent apical periodontitis, and periradicular surgery was indicated. The presence of sulfur granules and bacterial filaments arranged in dense aggregates within the apical pathologic tis- sue led to the diagnosis of apical actinomycosis. The histobacteriologic analysis of the apical root tip and apical lesion revealed that intraradicular bacterial biofilms were contiguous to extraradicular bacterial aggregates.

Therefore, it seems that apical actinomycosis may be a consequence of persistent intraradicular infection rather than an independent extraradicular infection.

6.9.3.2 Periapical microbial diversity in posttreatment apical periodontitis

While several studies have shown that Actinomyces species and Propionibacterium propionicum can sur- vive in apical tissues (Happonen et al. 1985; Sjögren et al. 1988), it is controversial whether other bacterial species can live for a long period after invading the apical tissues. In fact, a diversity of bacterial species has been identified in periapical lesions of root-filled teeth via different methods, including culture (Tron- stad et al. 1987; Sunde et al. 2002; Signoretti et al. 2013) and molecular techniques (Sunde et al. 2000; Gatti et al. 2000; Handal et al. 2009; Subramanian and Mickel 2009). Also, bacteria have been visualized in aggregates in some areas of periapical lesions by FISH (Sunde et al. 2003).

Molecular analyses of posttreatment apical peri- odontitis have revealed a more complex microbiota than that previously shown by culture-based studies. The first molecular studies used a checkerboard DNA– DNA hybridization technique to identify targeted bac- terial species from refractory periapical lesions and highlighted the polymicrobial nature of extraradicu- lar infections (Gatti et al. 2000; Sunde et al. 2000). Most lesions harbored species of Fusobacterium, Pep- tostreptococcus, Actinomyces, Campylobacter, Por- phyromonas, Tannerella, Treponema, Aggregatibac- ter, and Streptococcus (Sunde et al. 2000).

Advances in molecular methods have allowed the identification of several novel and as-yet-uncultivated bacterial species from persistent periapical lesions (Subramanian and Mickel 2009; Handal et al. 2009). They revealed that periapical lesions refractory to endodontic therapy might harbor polymicrobial infec- tions with many as-yet-uncultivated bacterial species.

142 Endodontic Microbiology

Handal et al. (2009) used cloning and sequencing of the 16S bacterial ribosomal RNA (rRNA) to study the complexity of the bacterial community of persis- tent apical lesions. Seventy-five taxa were detected, and 36% of them were as-yet-uncultivated bacterial species. All specimens harbored 1–11 different bacte- rial species, with a mean of 7 species per lesion. A high diversity of species was identified, including Fusobac- terium spp., Prevotella spp., Tannerella forsythia, Porphyromonas endodontalis, Treponema denticola, Bacteroidetes spp., Peptostreptococcus spp., and Streptococcus spp. The authors concluded that the microbial etiology of persistent periradicular dis- ease is far more complex than previously thought. Their results are in accordance with the new find- ings of microbial diversity of secondary or persis- tent root canal infections. Microbial communities of root-filled canals with posttreatment apical periodon- titis have been analyzed using 16S rRNA cloning and sequencing (Sakamoto et al. 2008) and pyrosequenc- ing (Anderson et al. 2012; Hong et al. 2013). The latter studies revealed a markedly higher diversity of the microbiota than other molecular techniques. More- over, persistent endodontic infections seem to have a diverse bacterial community similar to the primary infections (Hong et al. 2013).

However, the correlation of persistent root canal and periapical infections has not yet been established. Most studies evaluated only the periapical tissues and did not evaluate the associated root ends, so it is not known whether the bacteria found within the apical tissues are dependent on or independent of intra- or extraradicular biofilms. Moreover, it is possible that parts of radic- ular biofilms may be separated and released into the periapical tissues as bacterial aggregates.

Subramanian and Mickel (2009) analyzed the microbial profile of both root ends and periapical tis- sues using molecular methods for microbial quantifi- cation and diversity analysis. They found that the bac- terial load on root ends was significantly greater than the load in the soft tissue section. Enterococcus fae- calis and Burkholderia cepacia predominated in both samples. Interestingly, although most bacterial species were detected in both sites, several species showed greater association with the root end than with the soft tissue, including Campylobacter gracilis and Strep- tococcus gordonii. Moreover, species from the gen- era Lactobacillus, Propionibacterium, and Bifidobac- terium, which are all Gram-positive rods, were found only on root ends. In a biofilm structure, these bacteria

are considered to be the early colonizers, suggest- ing that they have been present as a biofilm on the external root surface. In contrast, several other species showed association with periapical soft tis- sue, including Atopobium rimae, Peptostreptococcus micros, Streptococcus genomospecies C8, Dialister sp. E2 20 E1, and Eubacterium strain A35MT. Therefore, this study supported the hypothesis that persistent peri- apical lesions may be fed by intra- or extraradicular biofilms.

6.10 Treatment of endodontic infections

Bacteria organized as intraradicular biofilms are the main cause of apical periodontitis in nontreated teeth (Ricucci and Siqueira 2010). Those biofilms are mostly removed by the chemomechanical action of instru- ments and irrigants during the enlargement and shap- ing of the main/secondary root canals of a primary root canal treatment. However, the inaccessibility of bacteria present in the root canal system, includ- ing apical ramifications and isthmuses, represents a formidable challenge for disinfection. Consequently, current endodontic treatment procedures are not able to completely sterilize infected root canals, leading to a posttreatment residual infection with a clinical impli- cation that will depend, among other factors, on the balance between microbial and host defense factors (Wu et al. 2006; Siqueira and Rôças 2008). In most cases, the reduction of microbial and endotoxin levels obtained by endodontic treatment may be enough to promote periapical healing. In a recent study, the pro- portion of roots with complete radiographic healing after primary root canal treatment was 83% (Ng et al. 2011).

Therefore, the aim of endodontic treatment of infected root canals is to provide maximal bacterial/ endotoxin reduction, which is accomplished mainly through mechanical instrumentation and chemical irri- gation. In this context, the complexity of microbial communities of endodontic infections and their biofilm structure organization should be considered when evaluating antimicrobial strategies during endodontic treatment.

Given that microbial biofilms can be removed and disrupted, mainly mechanically and/or physically, the mechanical action of endodontic instruments has a significant role in biofilm removal from the main canal. Studies comparing different instrumentation

Extraradicular Endodontic Infections 143

techniques showed no difference in bacterial reduction between hand and rotary instrumentation techniques when instruments of similar diameters were used to prepare infected root canals (Berber et al. 2006; Lin et al. 2013; Nakamura et al. 2013; Rôças et al. 2013). However, considering the clinical aspects, it is impor- tant to note that rotary nickel–titanium instrumentation can promote greater enlargement of the root canal with less canal transportation when compared to manual instrumentation with stainless steel files. Larger api- cal preparation would thus provide a greater bacterial and endotoxin reduction during endodontic treatment (Martinho et al. 2010; Siqueira 2011; Marinho et al. 2012). Clinical studies have suggested that enlarge- ment of the apical size would result in an increased healing outcome for patients with necrotic pulps and periapical lesions (Saini et al. 2012; Aminoshariae and Kulild 2015).

Although mechanical instrumentation alone can reduce the number of bacteria and endotoxins in infected root canals, irrigating solutions with antimi- crobial action must be used to achieve maximum dis- infection during treatment (Byström and Sundqvist 1981, 1983). The use of antimicrobial solutions pro- motes further bacterial reduction, especially in the lumen of the canal, acting on planktonic bacteria or dis- rupting the biofilm during root canal instrumentation. However, they may have limited action on biofilms in noninstrumented areas. In vitro studies have shown that complex biofilms are more resistant to the antimi- crobial effects of irrigating solutions than previously reported for monocultures or planktonic cells (Shen et al. 2011; Stojicic et al. 2013). Moreover, so far, no endodontic irrigating solution alone can eliminate mature bacterial biofilms (Stojicic et al. 2013).

Recent studies have shown that almost 50% of the teeth remained infected after root canal preparation, which highlights a need to search for new strategies of root canal disinfection (Rôças and Siqueira 2011). In fact, supplementary disinfecting procedures have already been proposed for enhanced disinfection after instrumentation, including passive ultrasonic irrigation (PUI), photodynamic therapy (PDT), and laser irradia- tion. However, to date, the antimicrobial effectiveness of these procedures has mainly been tested in ex vivo studies. Therefore, in vivo studies are still required to demonstrate the superiority of these methods over con- ventional ones in enhancing root canal disinfection.

In order to reach maximal bacterial/endotoxin reduction of infected root canals, some authors have

recommended the use of intracanal medication dur- ing treatment, although this is one of the most con- troversial issues in endodontics (Bergenholtz and Spangberg 2004). Calcium hydroxide paste in an inert vehicle is the most commonly used medica- ment, although its combination with other substances, including chlorhexidine (Gomes et al. 2003, 2009; Vianna et al. 2007; Sousa et al. 2014) or camphorated paramonochlorophenol (Rôças and Siqueira 2011) have been studied as alternatives.

Nevertheless, it is important to note that success- ful endodontic treatment would depend not only on antimicrobial strategies to control infection, but also on procedures that prevent reinfection (Siqueira 2011). In this context, root canal obturation would prevent bac- terial growth by eliminating empty spaces and block- ing the entrance of periapical fluids in the root canal, because sealers alone cannot inhibit microbial growth for long (Gomes et al. 2004a). Also, restoration would prevent coronal microleakage and root canal reinfec- tion, which may contribute to higher rates of success of endodontic treatment (Ray and Trope 1995; Imura et al. 2007; Ng et al. 2011).

After satisfactory endodontic procedures of clean- ing, shaping, obturation, and restoration, periapical healing occurs in most cases. However, posttreatment apical periodontitis will persist if endodontic proce- dures fail to reduce significantly the intraradicular infection (Siqueira and Rôças 2008). It has been shown that most cases of posttreatment apical periodontitis are associated with poor-quality primary treatment, and most of them can be resolved with nonsurgical retreatment (Wu et al. 2006). The outcome of sec- ondary root canal treatment seems to be similar to the primary treatment, with a success rate of approxi- mately 80% (Ng et al. 2011).

However, posttreatment apical periodontitis may also be a consequence of persistent intraradicular infection, usually in inaccessible areas of the root canal system, which may be associated with extraradicular infections. In such cases, a surgical procedure may be required to treat the posttreatment apical periodonti- tis. The success rate of endodontic microsurgery using contemporary methods is >90% (Song and Kim 2012).

In conclusion, studies have shown that apical peri- odontitis development or healing depends on the balance between bacterial load and host defense. Therefore, the aim of the endodontic treatment is to reduce microorganisms to levels that are compati- ble with periapical healing. In cases of posttreatment

144 Endodontic Microbiology

apical periodontitis, a nonsurgical or surgical retreat- ment may be required.

6.11 Conclusions

Extraradicular infection usually originates from an intraradicular infection that has spread to the periradic- ular tissues via the apical foramen. Microorganisms located in the apical portion of the canal are in a strate- gic position to cause harm to the host. Bacterial prod- ucts or components may directly or indirectly activate the host’s immune system, leading to inflammation of periapical tissues (Mattison et al. 1987). The inflam- matory event itself results in a hostile environment, with an exacerbation of the local immune response, in an attempt to contain the invasion of microorganisms. However, regardless of the microbial colonization of the root canals, bacteria are rarely found in periapical granulomatous tissues of untreated teeth, suggesting that a granuloma is not an area in which bacteria live, but where they are destroyed (Kronfeld 1939). There- fore, microorganisms that are able to invade and settle in this environment must be able to develop coping mechanisms to adhere to the tissues, and must support changes in the redox potential and in nutrient demand.

Extraradicular infection is composed of various morphologic types of microorganisms, which adher to the cementum/dentin near the apex and are sur- rounded by a layer of polysaccharide that acts as a shield. Such a microbial arrangement is known as a biofilm. Moreover, bacterial aggregates have been found within the body of periapical lesions, proba- bly as a result of intra- or extraradicular infections. Biofilms on the external root surface or within periapi- cal lesions have been mainly found in posttreatment apical periodontitis (Wang et al. 2012, 2013). Their prevalence varies depending on the technique used for bacterial detection in different studies, ranging from 6% to 50% (Sunde et al. 2003; Ricucci and Siqueira 2010).

The microbial species associated with complex bac- terial communities organized on biofilm possess char- acteristics that differ from their planktonic forms, such as increased metabolic diversity and efficiency; resis- tance to phagocytic cells, antimicrobial agents, and environmental stresses; and enhanced pathogenicity (Costerton et al. 1999; Marsh 2005), which make the extraradicular biofilm a tough challenge to overcome by means of a endodontic nonsurgical approach (Sig- noretti et al. 2013). In such cases, a surgical procedure

may be required to treat posttreatment apical periodon- titis. Nowadays, the success rate of endodontic micro- surgery ranges from 88% to 94% (Setzer et al. 2012).

As it is still not possible to use the endodontic nonsurgical approach to detect the presence of an extraradicular infection, all efforts should be made to promote effective disinfection of the root canal by mechanical and chemical procedures. As microbial biofilms can only be removed or disrupted physically, the mechanical action of endodontic instruments has a significant role in removing biofilm from the main and secondary canals. Because of the instruments’ inability to reach every part of the root canal system, the con- comitant use of antimicrobial substances is necessary to increase the effectiveness of endodontic procedures. However, the development of new antimicrobial strate- gies that can improve root canal disinfection is neces- sary, given the difficulty of removing the biofilms.

6.12 Acknowledgments

We would like to thank all members and postgraduate students of the Endodontics Division of the Piracicaba Dental School, State University of Campinas, UNI- CAMP, especially Drs. Carlos Vieira Andrade Junior, Iadasa de Quadros, Fernanda Graziela Corrêa Sig- noretti, Flaviana Bombarda de Andrade, Neylla Teix- eira Sena, Marlos Barbosa Ribeiro, and Vanessa Bel- lochio Berber for providing the figures, and Miss Eloá Cristina Bı́cego Pereira for her technical assistance. We would like also to thank the Brazilian agencies FAPESP, CNPq, and CAPES.

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Chapter 7 Virulence of Endodontic Bacterial Pathogens Christine Sedgley

7.1 Introduction 7.1.1 Virulence and pathogenicity 7.1.2 Biofilms

7.2 Genetic aspects of bacterial virulence 7.2.1 Horizontal gene transfer 7.2.2 Accessory genetic elements 7.2.3 Quorum sensing

7.3 Virulence factors 7.3.1 Lipopolysaccharide 7.3.2 Peptidoglycan 7.3.3 Lipoteichoic acids 7.3.4 Fimbriae 7.3.5 Capsules 7.3.6 Extracellular vesicles 7.3.7 Exotoxins

7.3.8 Extracellular proteins 7.3.9 Metabolic by-products

7.4 Virulence associated with endodontic microorganisms 7.4.1 Porphyromonas and Prevotella 7.4.2 Fusobacterium 7.4.3 Parvimonas 7.4.4 Streptococcus 7.4.5 Lactobacillus 7.4.6 Enterococcus 7.4.7 Actinomyces 7.4.8 Propionibacterium 7.4.9 Combinations of microorganisms

7.5 Conclusions and future directions 7.6 References

7.1 Introduction

Humans have coevolved with a multitude of diverse microbial species that constitute the normal or com- mensal microflora, typically existing in biofilm com- munities. This flora populates the mucosal surfaces of the oral cavity, upper respiratory tract, gastrointesti- nal tract, urogenital tract, and the surface of the skin. In the healthy host, the absence of a constant state of inflammation indicates that a balance has developed between bacteria and the epithelial and mucosal sur- faces, allowing both bacterial survival and prevention of the induction of inflammation that cause damage (Henderson and Wilson 1998; Sansonetti 2011). How- ever, pathogenic microorganisms causing disease in a susceptible host must be able to adhere, colonize,

survive, propagate, and invade, while at the same time evade host defense mechanisms (Cross 2008). When the invading microorganisms are sufficiently virulent and the host is sufficiently compromised, damage can occur.

7.1.1 Virulence and pathogenicity

Microbial virulence generally refers to the degree of pathogenicity or disease-producing ability of a microorganism. In turn, pathogens are commonly dis- tinguished from nonpathogens by their expression of intrinsic characteristics called virulence factors. The expression of virulence factors can enable a microorganism to establish itself on or within a host, thereby enhancing its potential to cause disease, the

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response to which will be dependent on host–microbe interactions.

Traditionally, pathogenesis was often described in terms of a predominantly microbial etiology, with lit- tle, if any, emphasis on host factors and the immune response. This approach was in part influenced by Koch’s landmark studies showing a direct cause and effect relationship between specific culturable micro- bial species and diseases, for example Mycobac- terium tuberculosis and tuberculosis, and Bacillus anthracis and anthrax (Koch 1884). However, not all “pathogens” cause disease in all hosts, and “non- pathogens” in healthy hosts can become “pathogens” in immunocompromised hosts. Also, the ability of bac- teria to cause disease has been described in terms of the number of infecting bacteria, yet small numbers of highly virulent microorganisms can damage the immunocompromised host, whereas large numbers of low-virulence microorganisms may be countered by the healthy host (Walker et al. 2006). As such, it may be difficult to distinguish virulence traits from com- mon traits in commensals and opportunistic pathogens

(Casadevall and Pirofski 1999) and in biofilm commu- nities (Kuboniwa et al. 2012). Virulence is therefore understood to be multifactorial (Moine and Abraham 2004), with the susceptibility of the host playing a critical part (Casadevall and Pirofski 2001).

7.1.2 Biofilms

Adding to the complexity of the virulence and pathogenicity relationship is that bacterial infections typically exist as complex biofilm communities (Costerton et al. 1999). These are highly heteroge- neous structures possessing a variety of mechanisms to evade host defenses and enable survival under challenging conditions (Fux et al. 2005; Stewart and Franklin 2008; Lewis 2010; Poole 2012). Biofilm communities are highly successful etiologic agents in endodontic infections (Svensater and Bergenholtz 2004; Ricucci and Siqueira 2010) (Figure 7.1).

Biofilms are sessile or pedunculated microbial com- munities composed of tower- or mushroom-shaped microcolonies containing cells irreversibly attached to

(a) (b)

Fig. 7.1 Examples of intracanal biofilms with different bacterial cell morphologies. (a) The predominance of cocci. Note the high concentration of cells in contact with the root canal wall (Taylor’s modified Brown and Brenn, original magnification ×1000). (b) Predominance of filamentous forms. Note the irregular distribution of bacterial cells within the extracellular material (original magnification ×1000). Source: Adapted from Ricucci and Siqueira (2010). Reproduced with permission of Elsevier.

Virulence of Endodontic Bacterial Pathogens 151

a substratum, an interface, or each other. Microbial cells occupy only a small proportion of biofilms. A highly heterogeneous matrix composed of water and extracellular polymeric substances (EPS) produced by cells within the biofilm account for the largest propor- tion of most biofilms. EPS provide multiple functions: provision of a scaffold for the biofilm, nutrient source, water retention, an energy sink, ionic exchange, sorp- tion of organic and inorganic compounds, protec- tion, and exchange of genetic information (Flemming and Wingender 2010). Channels exist throughout the biofilm structure that facilitate the inflow of nutrients and the outflow of waste materials (Stewart 2012). Other important components of the EPS include extra- cellular polysaccharides, proteins, and DNA (eDNA) (Flemming and Wingender 2010; McDougald et al. 2011). eDNA has a critical role in the initial establish- ment of biofilms and is involved with adhesion, aggre- gation, cohesion, and exchange of genetic information (Whitchurch et al. 2002; Flemming and Wingender 2010; Barnes et al. 2012).

Microbial cells in the heterogeneous biofilm com- munity encounter many diverse stresses and environ- mental challenges, for example, exposure to nutrient limitation, reactive oxygen and nitrogen species, mem- brane damage, pH variations, and elevated temperature (Kuboniwa et al. 2012; Poole 2012). Oxygen levels and metabolic rates at the center of a microcolony are reduced compared with near its surface (de Beer et al. 1994). Subsequently, nutrient availability and access to electron acceptors can vary considerably throughout the biofilm, resulting in variations in the growth stage of microbial cells, ranging from rapidly growing to dormant. The latter dormant “persister cells” have been associated with recalcitrant biofilm infections (Lewis 2010). In addition, deep in the biofilm, microbial cells can be induced to enter a stationary phase and thus lose susceptibility to killing by antimicrobials that target dividing cells (Borriello et al. 2004). Mechanisms that cells can use to cope with these variations in metabolic activity, gene expression patterns, and potential phe- notypic variations include turning certain genes on or off, by random gene switching, or by the creation of fitter mutants that can arise as part of a natural selection process (Stewart and Franklin 2008).

Microorganisms possess a diverse arsenal of global stress responses that can facilitate their survival in challenging circumstances (Boles et al. 2004; Kuboniwa et al. 2012). These include the alternative sigma factors RpoS (Hengge-Aronis 2002) and RpoH

(Guisbert et al. 2008), gene repressor LexA (Kelley 2006) and small molecule effectors, such as (p)ppGpp that induce the stringent response (Potrykus and Cashel 2008; Chavez de Paz et al. 2012). Adap- tation and survival can be enabled by the sub- sequent modulation of intracellular metabolic pro- cesses which include horizontal gene transfer of mobile genetic elements, downregulation of error- correcting enzymes, and upregulation and activation of error-prone DNA polymerases (Foster 2007). In addition, two-component toxin–antitoxin (TA) sys- tems are involved in stress responses, with the sta- ble protein toxins directed against specific intracellu- lar targets, and small RNAs or degradable proteins (antitoxins) neutralizing the toxin or inhibiting toxin synthesis (Hayes and Van Melderen 2011). TA sys- tems are involved with quorum sensing (Belitsky et al. 2011), as well as persister cell formation (Kim et al. 2010), and may regulate the switch from plank- tonic to biofilm lifestyles (Wang et al. 2011). As part of the biofilm life cycle, dispersal of microcolony cells (Figure 7.2) provides the opportunity for the formation of new biofilms containing parental and genetic variants (McDougald et al. 2011), as such providing an avenue for the dissemination of virulent cells.

Microorganisms in biofilm communities have sev- eral mechanisms by which they can resist the detri- mental effects of antimicrobial therapy (del Pozo and Patel 2007). For example, the concentration and abil- ity of an antimicrobial to penetrate the EPS matrix, and the depth of the biofilm, will affect the extent of exposure of cells. The outer cells of the biofilm will be rapidly exposed to the highest concentration of the antimicrobial, while exposure of deeper parts will depend on the ability of the antimicrobial to diffuse through the EPS matrix (Stewart and Costerton 2001). Multispecies biofilms demonstrate greater resistance to antimicrobial agents than single-species biofilms, which may be attributable to the cooperative behavior of the community which enables survival upon expo- sure to the agent (Burmolle et al. 2006; Elias and Banin 2012).

Variations in physicochemical factors in root canals could potentially influence the virulence and pathogenicity of the microbial community within the biofilm structure. These include the availability of exogenous and endogenous nutrients, the degree of anaerobiosis (oxygen tension) and pH level, as well as the surfaces available for adherence and their

152 Endodontic Microbiology

Fig. 7.2 Active biofilm dispersal and variant formation. Active dispersal of the parental strain and genetic variants, represented by different cell types, leads to subsequent attachment and colonization that can be initiated by either single variants, to generate clonal biofilms, or multiple variants, to form mixed-variant biofilms. Source: McDougald et al. (2011). Reproduced with permission of Macmillan.

characteristics (e.g., cells, dentin, medicament rem- nants, and root filling materials). Further, biofilms can persist despite the deprived nutritional conditions found in the necrotic or previously treated root canal systems and subsequent harsh conditions imposed by root canal treatment procedures (Ricucci and Siqueira 2010).

7.2 Genetic aspects of bacterial virulence

Bacterial virulence factors are encoded by genes usu- ally located on chromosomal DNA. The predominant means by which chromosomal genes are inherited is vertical, in other words via replication, segregation, and cell division. Mechanisms of genetic variability between generations can include point mutations and genetic rearrangements.

7.2.1 Horizontal gene transfer

Virulence factors may also be encoded by genes found on accessory genetic elements such as plasmids. These can profoundly influence genome plasticity and evolution by allowing movement of genetic information both within and between species and conferring traits facilitating survival under atypical conditions. For example, interactions between geneti- cally distinct bacteria are involved in the establishment and maintenance of multispecies biofilms (Sedgley et al. 2008; Kolenbrander et al. 2010). This form of dissemination of genes occurs via horizontal gene transfer (HGT), whereby genes may move between bacterial cells that are otherwise genetically unrelated. HGT of DNA in bacteria occurs by three basic meth- ods: transformation, transduction, and conjugation, and provides pathogens with the means to adapt rapidly.

Virulence of Endodontic Bacterial Pathogens 153

Transformation of bacteria involves the active uptake by a cell of eDNA and its subsequent incor- poration into the recipient genome. Natural transfor- mation has been observed in the oral Gram-positive bacteria Streptococcus mutans in biofilms (Li et al. 2001), Streptococcus gordonii Challis (Wang et al. 2002), and in Streptococcus pneumoniae (Morrison and Lee 2000).

Transduction involves gene transfer whereby bacte- rial viruses (also termed bacteriophages) carry genetic material to recipient cells. Bacteriophages have been isolated from Actinobacillus (now Aggregatibacter) actinomycetemcomitans (Haubek et al. 1997) and Actinomyces spp. (Yeung and Kozelsky 1997) in den- tal plaque and from Enterococcus faecalis in saliva samples (Bachrach et al. 2003).

Conjugation is the most efficient HGT phenomenon in bacteria. The requirement for cell–cell contact dis- tinguishes conjugation from transduction and trans- formation. DNA is transferred between cells that are in physical contact, allowing unidirectional trans- fer of genetic information from donor to recipient (Figure 7.3). Conjugation can involve the crossing of species barriers and can also occur between bac- teria and eukaryotic cells (Waters 2001). Chromoso- mal DNA segments, plasmids, and transposons can be transferred by conjugation.

In contradistinction, the “health” of a microor- ganism is also dependent on the presence of its own protective “immune” system. For example, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and genes encoding CRISPR- associated (cas) proteins, or CRISPR-cas module, is a prokaryotic immune system widespread among Archaea and Bacteria that confers resistance to HGT of exogenous mobile genetic elements such as viruses (phages), plasmids, and transposons (Barrangou et al. 2007). Evidence for the presence of CRISPR-cas in endodontic and oral E. faecalis strains raises intrigu- ing questions as to how prokaryotic immune systems might modulate interactions within the polymicrobial endodontic biofilm environment (Burley and Sedgley 2012) and modulate expression of virulence factors.

7.2.2 Accessory genetic elements

Accessory genetic elements include plasmids, bac- teriophages, transposons, insertion sequences, and pathogenicity islands (Figure 7.4). Plasmids are extra- chromosomal autonomously replicating elements important for bacterial adaptability and survival by the provision of functions that might not be encoded by the chromosome. Plasmids are found in bacteria (Clewell and Francia 2004a), Archaea (Brugger et al.

Fig. 7.3 Conjugative plasmid transfer in Gram-negative bacteria.

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Fig. 7.4 Virulence factors associated with bacteria.

2002), and yeasts (Jayaram et al. 2004). They are typically covalently closed circular, double-stranded DNA molecules that replicate independently of chromosomal DNA, and range in size from ∼1 to

>200 kilobase pairs (kb) (Sedgley and Clewell 2004). A simplified “map” of pAD1, a conjugative plasmid originally isolated from a clinical isolate of E. faecalis, is shown in Figure 7.5. The copy number of plasmids

Virulence of Endodontic Bacterial Pathogens 155

Fig. 7.5 Simplified map of the conjugative plasmid pAD1 originally isolated from E. faecalis DS16. Segments are described according to the functions encoded by genes contained within: (1) replication and maintenance; (2) regulation of pheromone response; (3) structural genes relating to conjugation; (4) unknown; (5) cytolysin biosynthesis; (6) unknown; (7) resistance to UV light; oriV, origin of replication; oriT, origin of transfer. Source: Sedgley and Clewell (2004). Reproduced with permission of John Wiley and Sons. Copyright © 2004, John Wiley and Sons.

in the bacterial cell is generally characteristic for the particular plasmid, and can range from >30 copies per cell for a small plasmid (e.g., <10 kb) to 1–2 copies for larger plasmids (e.g., >25 kb).

Some plasmids in E. faecalis transfer copies of themselves by conjugation from one bacterial cell to another using small peptides called sex pheromones as essential signals in the process (Clewell et al. 2002) (Figure 7.6). The pheromones are secreted by a potential recipient cell which activates the trans- fer system of a potential donor cell. The plasmid is then transferred from the donor to the recipient cell. Once the recipient cell has acquired the plasmid, it assumes a phenotype of the original donor and shuts down the production of endogenous pheromone. How- ever, the recipient cells that receive the donated DNA (transconjugants) continue to produce pheromones specific for donors harboring different classes of plasmids.

From a clinical perspective, plasmids are particu- larly important because they are involved in the dis- semination of antibiotic resistance, thereby enabling the survival of the strain, as well as a diverse range of products that may potentially contribute towards virulence” which may adversely affect the host. These products include cytotoxins, adhesins, and certain metabolic enzymes. For example, cytolysins encoded by genes on plasmids in E. faecalis, often in association with clinical isolates (Huycke and Gilmore 1995), can lyse erythrocytes and other eukaryotic cells (Martinez and Baquero 2002).

Fig. 7.6 Pheromone initiated conjugative plasmid transfer. The pheromone induces the appearance of a surface adhesin (aggregation substance) that facilitates the attachment of the donor and recipient cells. Aggregates give rise to conjugal channels through which the plasmid is transferred from the donor to the recipient cell. Source: Sedgley and Clewell (2004). Reproduced with permission of John Wiley and Sons. Copyright © 2004, John Wiley and Sons.

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Information relating to plasmids associated with endodontic microbiology appears to be limited to those associated with Enterococcus species. Interestingly, The E. faecalis MC4 strain used in root canal infection studies in primate models (Moller et al. 2004) harbors

a 130 kb conjugative, pheromone (cCF10) responding plasmid, pAMS1, conferring chloramphenicol, strep- tomycin, and tetracycline resistances (Flannagan et al. 2008). Plasmid DNA was isolated from 25 of 33 endodontic enterococcal isolates (31 E. faecalis and two Enterococcus faecium strains) recovered from patients in Sweden, with up to four plasmids per strain (Sedgley et al. 2005b). Interestingly, several strains, which on initial screening appeared to be clones based on pulsed field gel electrophoresis analyses of total DNA, were actually shown to have distinct plasmid types (Figure 7.7). Further, phenotypic studies showed that 16 of the 25 plasmid-positive strains exhibited a clumping response (characteristic of a response to pheromone) when exposed to a culture filtrate of a plasmid-free strain, suggesting the potential for con- jugative transfer of genetic elements in these endodon- tic isolates.

It is conceivable that if endodontic strains contain conjugative plasmids with genes that could enhance virulence during or after endodontic treatment, such properties might be transferrable to other strains remaining in the root canal system. Indeed, bidirec- tional transfer between S. gordonii and E. faecalis of an erythromycin resistance gene on the conjugative plasmid pAM81 in root canals was shown in an ex vivo model (Sedgley et al. 2008) (Figure 7.8), suggesting a capacity for the species to modulate virulence expression and antibiotic resistance acquisition under varying environmental conditions in the root canal system.

Transposons, sometimes also called jumping genes, are segments of DNA that can move (“jump”) from

� Fig. 7.7 (a) Total DNA and (b) plasmid DNA analysis of endodontic E. faecalis. (a) Pulsed field gel electrophoresis (PFGE) of SmaI-digested genomic DNA. Note similarities between GS3–GS7, GS12, and GS21. Reference standard: lambda phage DNA. (b) Plasmid analysis of the same E. faecalis isolates. Lane M, molecular size marker (1 kb Plus DNA Ladder, Invitrogen); –, undigested; H, digested with HindIII. Strain designations are shown above the lane designations. Isolates classified based on PFGE pattern as clonal, GS3, GS12, and GS21 are similar in plasmid content. GS4 and GS5 appear to be alike in plasmid content. GS6 and GS7 each contain two similar small plasmids; however, GS6 has two additional plasmids. Source: Sedgley et al. (2005b). Reproduced with permission of John Wiley and Sons. Copyright © 2005, John Wiley and Sons.

Virulence of Endodontic Bacterial Pathogens 157

one DNA molecule to another—for example, from the chromosome to a resident plasmid (Hayes 2003). Elements similar to the conjugative transposon Tn916 were detected in 4 of 15 tetracycline-resistant bac- teria isolated from root canals (Rossi-Fedele et al. 2006). Insertion sequences are short DNA sequences that usually encode the ability to transpose but do not carry accessory genes like transposons do. Virulence

(a)

(b)

(c)

genes can also be transferred via pathogenicity islands, or horizontally transferable genomic islands that are located on the bacterial chromosome or may be a part of a plasmid (Coburn et al. 2007). More information is required about the role of accessory genetic elements encoding virulence genes in endodontic infections.

7.2.3 Quorum sensing

In order to regulate physiologic activities, some bacte- ria communicate and coordinate behavior via signaling molecules using a intercellular signaling system called quorum sensing (Parsek and Greenberg 2005; Keller and Surette 2006). Virulence gene expression is con- trolled by quorum sensing in numerous microorgan- isms such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa (Antunes et al. 2010). When a high density population reaches a certain threshold (quorum), the concentration of normally low levels of certain diffusible signal molecules becomes high enough to act as autoinducers that trigger a synchronized response. Gram-negative bacteria use N-acyl homoserine lactone-based signaling while Gram-positive bacteria utilize small peptides; autoinducer-2 (AI-2) signaling is used by both Gram- negative and Gram-positive bacteria (Fuqua and Greenberg 2002; Clewell and Francia 2004a; Li and Nair 2012).

� Fig. 7.8 Horizontal gene transfer (HGT) in the root canal. Scanning electron micrographs showing accumulations of E. faecalis JH2-2/pAM81 and S. gordonii Challis-Sm (a) 24 hours and (b) 72 hours after inoculation into the root canal. (c) Bi-directional HGT of the plasmid pAM81 was confirmed by purification of the plasmid in transconjugants. pAM81 plasmid DNA from donor and transconjugant strains, digested with HindIII. Lanes 1 and 14, molecular size marker; lane 2, E. faecalis JH2-2/pAM81 (donor); lane 3, S. gordonii Challis-Sm (plasmid-free recipient); lane 4, S. gordonii Ch24RC; lane 5, S. gordonii Ch24F; lane 6, S. gordonii Ch72RC; lane 7, S. gordonii Ch72F; lane 8, S. gordonii Challis-Sm/pAM81 (donor); lane 9, E. faecalis JH2-2 (plasmid-free recipient); lane 10, E. faecalis J24RC; lane 11, E. faecalis J24F; lane 12, E. faecalis J72RC; lane 13, E. faecalis J72F. Plasmid DNA restriction fragments were separated by electrophoresis on 0.8% agarose gels in TBE buffer (3.5 hours at 50 V), stained with ethidium bromide, and visualized under ultraviolet light. Source: Adapted from Sedgley et al. (2008). Reproduced with permission of Elsevier.

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7.3 Virulence factors

It is well established that a diverse array of virulence factors can modulate bacterial adherence and invasion by the avoidance of host defense mechanisms, or by indirectly or directly causing host damage. However, the specific application of this knowledge to endodon- tic bacterial pathogens remains sparse at this time. Some virulence factors with established or poten- tial relevance to endodontic infections are listed in Table 7.1 and depicted in Figure 7.4.

While both Gram-negative and Gram-positive bac- teria have cytoplasmic membranes of similar lipid bilayer structure, considerable structural differences are evident peripherally (Figure 7.4). In addition, within the periplasm a number of important processes and regulatory functions occur that are essential to the viability and growth of the cell. Molecules within the periplasm provide a buffer against the changes occurring in the local surroundings. For many bac- terial species, proinflammatory cytokine induction is a major virulence mechanism related to stimulation by certain components associated with the bacterial cell wall.

7.3.1 Lipopolysaccharide

Lipopolysaccharide (LPS), also termed endotoxin, is an integral component of the bacterial envelope of Gram-negative bacteria. It is composed of three parts: the lipid A portion of the molecule (the endotoxin component) serves as an anchor in the outer membrane while the LPS core (an oligosaccharide) and side chain

(the polysaccharaide O-antigen) projects from the surface.

LPS varies among different bacterial species or strains and has numerous biologic effects. For exam- ple, LPS can activate the Hageman factor (factor XII) (Bjornson 1984) which in turn can activate several plasma protease cascades both directly and indirectly. Diversity in structure of the lipid A component may facilitate the evasion of recognition by Toll-like recep- tor 4 (TLR4) of immune cells. LPS is recognized by TLR4 and promotes the secretion of proinflamma- tory cytokines (Trent et al. 2006). For example, the expression of the proangiogenic vascular endothelial growth factor (VEGF) in odontoblast-like cells and macrophages was upregulated by LPS (Botero et al. 2003), in part mediated by TLR4 signaling (Botero et al. 2006). LPS can also trigger the mobilization of immunosurveillance mechanisms in the pulp. In vitro studies showed that LPS from clinical isolates acti- vated complement (Horiba et al. 1992) and stimulated interleukin-1 beta (IL-1β) release from human den- tal pulp cells in a time- and dose-dependent manner (Hosoya and Matsushima 1997). In postnatal human dental pulp stem cells, the expression of IL-8 was rapidly induced by LPS (Chang et al. 2005). LPS can also bind to CD14, a receptor that can be both solu- ble and membrane bound (Gioannini and Weiss 2007; Jerala 2007), with subsequent stimulation of inflam- matory cytokines.

In the endodontic literature, endotoxin is the micro- bial virulence factor that has received the most atten- tion. The presence of endotoxin was associated with periapical bone destruction in monkeys and cats

Table 7.1 Bacterial virulence factors that may contribute to endodontic infections

Gram-negative Gram-positive Effect

Structural components Lipopolysaccharide + – Proinflammatory Peptidoglycan + + Proinflammatory Lipoteichoic acid – + Proinflammatory Fimbriae + + Adherence

Products Capsules + + Protection Extracellular vesicles + – Secretory products Exotoxins + + Diverse Extracellular proteins/enzymes + + Diverse Short-chain fatty acids + + Proinflammatory Superoxide anions + + Proinflammatory

Virulence of Endodontic Bacterial Pathogens 159

(Dwyer and Torabinejad 1980; Dahlen et al. 1981). In humans, endotoxin was positively correlated with pulpal pain and periapical inflammation (Schein and Schilder 1975; Schonfeld et al. 1982). Similarly, endo- toxin was more likely to be detected in root canals of symptomatic teeth than that of asymptomatic teeth (Horiba et al. 1991) and at a higher concentration (Jacinto et al. 2005).

7.3.2 Peptidoglycan

Peptidoglycan (PG) is the major component of Gram- positive cell walls, providing cell wall shape and strength, while counteracting cytoplasmic osmotic pressure. PG consists of peptides and polysaccharides that form a homogeneous cross-linked layer outside the plasma membrane. In Gram-negative bacteria, the PG layer is considerably thinner. PG is released upon cell lysis and can induce the upregulation of both proinflammatory and antiinflammatory cytokines. For example, PG from Staphylococcus aureus induced IL-6 and IL-10 mRNA accumulation in monocytes and T cells, and stimulated IL-6 production in human dental pulp cells (Wang et al. 2000). PG-induced cytokine expression was mediated by TLR2 in fibrob- lasts (Yoshimura et al. 1999). The production of IL-6 was stimulated by PG from Lactobacillus casei in a time- and dose-dependent manner in human den- tal pulp cells (Matsushima et al. 1998). An adaptive immune response via macrophages may be facilitated by PG (Myhre et al. 2006). PG potency is strongly boosted in the presence of LPS (Wang et al. 2001; Myhre et al. 2006); this conceivably has broad impli- cations for endodontic infections which are charac- teristically polymicrobial with significant numbers of Gram-negative anaerobes.

7.3.3 Lipoteichoic acids

Lipoteichoic acids (LTA) are an integral cell wall component of Gram-positive bacteria. They are amphiphilic polymers composed of glycerol phos- phates and complex glycolipid. LTA can induce a diverse range of inflammatory diseases in animals (Costa et al. 2003) including nephritis, arthritis, sep- tic shock, and multiorgan failure (Ginsburg 2002). The binding of Gram-positive bacteria to fibronectin in cell membranes and to neutrophils and lymphocytes may be facilitated by the lipid component of LTA (Court- ney et al. 1988). In odontoblasts, LTA upregulated the

expression of the cell-surface receptor TLR2, and the production of proinflammatory chemokines CCL2 and CXCL10 (Durand et al. 2006). When released upon cell lysis, LTA can bind to target cells either specifi- cally to TLRs and CD14 or nonspecifically to mem- brane phospholipids (Wang et al. 2000). Thereafter, LTA can interact with circulating antibodies and acti- vate the complement cascade. While LTA and LPS share many pathogenic properties, LTA is the less active, on a weight-for-weight basis (Myhre et al. 2006). LTA can trigger the release of many molecules from neutrophils and macrophages which include acid hydrolases, highly cationic proteinases, bactericidal cationic peptides, growth factors, reactive oxygen and nitrogen species, and cytotoxic cytokines (Ginsburg 2002). Alone or in combination, these molecules can subsequently amplify damage. LTA has both proin- flammatory (Telles et al. 2003) and antiinflammatory (Plitnick et al. 2001) effects. In macrophages and pulp cells, LTA can induce the expression of VEGF (Telles et al. 2003). However, LTA also inhibits the func- tion of IL-2, an autocrine growth factor for T cells (Plitnick et al. 2001). A recent study showed that LTA from E. faecalis induced apoptosis of the human osteoblast cell line MG63 (Tian et al. 2013) (Fig- ure 7.9). It has been hypothesized that LTA may pro- vide a selective advantage to Gram-positive bacteria by interfering with the immune response to infection (Hahn and Liewehr 2007).

7.3.4 Fimbriae

Fimbriae are thin, filamentous macromolecules made of protein subunits of up to 10 nanometers (nm) in diameter and between approximately 100 nm and sev- eral micrometers in length. They are distinct from flag- ella, which are longer and involved in cell motility. Fimbriae are involved in the attachment of bacteria to surfaces and interactions with other bacteria. Fim- briae are found on the surface of Gram-positive and Gram-negative species, including species recovered from endodontic infections, for example, Actinomyces israelii (Figdor and Davies 1997) and Actinomyces naeslundii (Wu and Fives-Taylor 2001). There are sev- eral “types” of fimbriae. For example, type 1 fimbriae of uropathogenic E. coli mediate adherence to urinary epithelium (Capitani et al. 2006). Type IV fimbriae, which can aggregate into bundles, have been detected on Eikenella corrodens (Hood and Hirschberg 1995), a periodontal pathogen that has also been detected in

160 Endodontic Microbiology

(a) (b)

Fig. 7.9 Observation of human osteoblast-like MG63 cell apoptosis by Hoechst 33258 staining under a fluorescence microscope (original magnification, ×200). After MG63 cells were treated with different concentration of LTA from E. faecalis for 48 hours, Hoechst 33258 staining was used to assess apoptotic cells (arrows). The number of apoptotic cells was increased in a LTA–dose-dependent manner with marked morphologic changes found in cell apoptosis: condenser chromatin and disintegration of the nuclear membrane. (a) Control group; (b) 25 mg/mL LTA-treated group; (c) 50 mg/mL LTA-treated group; (d) 100 mg/mL LTA-treated group. Source: Adapted from Tian et al. (2013). Reproduced with permission of Elsevier.

Virulence of Endodontic Bacterial Pathogens 161

root canal samples from teeth with acute periapical abscesses (Rôças and Siqueira 2006). The fimbriae of a periodontal clinical isolate of Prevotella interme- dia were shown to induce hemagglutination (Leung et al. 1996). Fimbriae can also differentially facili- tate bacterial adherence and invasion (Wu and Fives- Taylor 2001). For example, type II fimbriae of Por- phyromonas gingivalis were more highly adherent to epithelial cells than type I fimbriae and thus had a greater potential to contribute towards virulence (Kato et al. 2007).

7.3.5 Capsules

Capsules are generally composed of predominantly polysaccharides and form a well-organized layer coat- ing the outside of the cell wall. Microorganisms with capsules can evade or counteract host immune defenses by enabling avoidance of opsonin-mediated phagocytosis, or the recognition of the underlying cell by complement and antibodies and subsequent phagocytosis (Abeyta et al. 2003). In Gram-negative black-pigmented bacteria, capsules were seen to facil- itate the avoidance of, or survival after, phagocyto- sis (Sundqvist et al. 1982). Capsule formation by a pathogenic strain of S. pneumoniae was shown to facil- itate efficient transfer from their initial site in a host, the lumenal mucus, to the epithelial surface, a capability not shared by capsule-deficient mutant strains (Nelson et al. 2007). Capsules can also provide protection of the microorganism against host defense factors, desic- cation, bacterial viruses, and hydrophobic toxic mate- rials (e.g., detergents). For example, in P. gingivalis the capsule is a key virulence determinant in mul- tiple capacities: evading phagocytosis, reducing the host inflammatory response, and enhancing bacterial survival (Singh et al. 2011).

7.3.6 Extracellular vesicles

Extracellular vesicles develop from evagination of the outer membrane of Gram-negative bacteria. They con- tain proteins and lipids derived from the periplasm and have an average diameter of 50–250 nm (Beveridge 1999). The contents of the vesicles are released into the extracellular environment where they can partici- pate in a diverse array of virulence-associated activ- ities involving both prokaryotic and eukaryotic cells (Kuehn and Kesty 2005): bacterial adhesion, pro- teolytic activities, hemagglutination, and hemolysis

(Kinder and Holt 1989). Extracellular vesicles can modulate interactions between adjacent bacteria. For example, vesicles from P. gingivalis induced aggre- gation among Streptococcus spp., Fusobacterium nucleatum, A. naeslundii, and Actinomyces visco- sus (Kamaguchi et al. 2003). Extracellular vesicles can also provide their own “protection” by binding chlorhexidine (Grenier et al. 1995). Leukotoxin pro- duced by A. actinomycetemcomitans (Kato et al. 2002) and the cysteine proteases Arg- and Lys-gingipain pro- duced by P. gingivalis (Duncan et al. 2004) are specific virulence factors associated with vesicles produced by oral bacteria. The presence of outer membrane vesicles in lesions associated with teeth with refractory apical periodontitis was shown using transmission electron microscopy (Sunde et al. 2002).

7.3.7 Exotoxins

Exotoxins are a diverse array of toxins secreted by a living microbial cell or released during cell lysis. They can target eukaryotic cells and other microor- ganisms, as well as the extracellular matrix. Bacte- rial cytotoxins act on eukaryotic cells by targeting the cell cytostructure, either directly by modifying actin or indirectly by targeting regulators, in partic- ular Rho GTPase regulators which are essential for the functional integrity of the immune system (Akto- ries and Barbieri 2005). Exotoxins can trigger exces- sive and aberrant activation of T cells. Some exotox- ins are extremely potent, for example, toxic shock syndrome toxin-1 (TSST-1) (Dinges et al. 2000) and enterotoxins associated with food poisoning produced by certain strains of S. aureus (Balaban and Rasooly 2000) and pathogenic E. coli strains such as O157:H7 (Gyles 2007). The superantigens of Streptococcus pyo- genes can result in multiorgan failure (Sriskandan et al. 2007). Synergism among pathogenic factors can also be involved as demonstrated in methicillin-resistant S. aureus (MRSA) which causes haemorrhagic necro- tizing pneumonia by utilizing a bacterial toxin that destroys respiratory tissue and immune cells. The exo- toxin works in synergy with other factors expressed by the strain: the leukotoxin Panton–Valentine leukocidin (PVL) and the proinflammatory Protein A in combi- nation enhance strain virulence (Labandeira-Rey et al. 2007). Based on the elevated expression of proinflam- matory cytokines by T cells obtained from periodon- titis sites, it has been hypothesized that superantigens produced by periodontitis-associated bacteria may be

162 Endodontic Microbiology

contributory to the disease process (Sriskandan et al. 2007).

Bacterial toxins that target other microorgan- isms are called bacteriocins. These are protein or peptide “antibiotics” produced by some strains of Gram-positive and Gram-negative bacteria that are bacteriostatic or bactericidal to other, often closely related, bacterial strains as well as other species and genera. The production of bacteriocins may provide the producer strain with a selective advantage over other strains, especially those closely related to the bacteriocin-producing strain (Tomita et al. 1997). The capacity for bacteriocin production was shown in 14 of 33 Enterococcus species recovered from infected root canals (Sedgley et al. 2005a).

7.3.8 Extracellular proteins

Extracellular proteins are produced by bacteria or released during bacterial cell lysis. Many of this diverse group are enzymes with the potential to con- tribute to the spread of infection. The products may directly interact with TLRs to activate cells of the innate and adaptive immune systems resulting in the production of cytokines. In monkeys, different strains recovered from infected root canals were shown to vary in their ability to produce different histolytic enzymes, several of which contributed to tissue dis- integration, including hyaluronate lyase, chondroitin- sulfatase, beta-glucuronidase, Dnase, and acid phos- phatase (Dahlen et al. 1983).

Extracellular proteinases demonstrating col- lagenolytic, elastinolytic, and serinolytic activities were identified in Bacillus pumilus strains recovered from two teeth with necrotic pulps and apical peri- odontitis (Johnson et al. 2008). The authors postulated that collagen and elastin degradation during apical periodontitis could be attributed to the combination of these proteolytic activities with host proteinases and peptidases. Posttreatment apical periodontitis may also be associated with the presence of extracellular proteins produced by S. gordonii, Streptococcus angi- nosus, and Streptococcus oralis (Chavez de Paz et al. 2005). Endodontic P. gingivalis isolates demonstrated evidence of the collagenase gene (Odell et al. 1999) which may explain why increased collagenase was found in association with larger periapical lesions (Hashioka et al. 1994).

In the enterococci, important extracellular virulence-related proteins are cytolysin, serine pro- tease, gelatinase, aggregation substance, enterococcal surface protein (Esp), Ace (an adhesin to collagen of E. faecalis), and EfaA (E. faecalis antigen) (Gilmore et al. 2002). The expression of the protein serine protease contributed to the binding of E. faecalis to dentin (Hubble et al. 2003). The production of gelatinase, observed in more than 70% of E. faecalis strains recovered from infected root canals (Sedgley et al. 2005b), was associated with extended survival in root-filled teeth (Sedgley 2007) (Figure 7.10).

7.3.9 Metabolic by-products

The process of metabolism results in various metabolic by-products, many of which are released into the extracellular environment by living organisms or following lysis. The production of highly reactive free radicals and biologically toxic superoxide anion pro- duction is common among cells of the immune system and some bacterial species. For example, E. faecalis (Huycke et al. 1996) can produce extracellular superoxide that causes lysis of erythrocytes (Falcioni et al. 1981). Interspecies interactions may also be modulated by superoxides; extracellular superoxide production by E. faecalis enhanced its survival in a mixed infection with Bacteroides fragilis in mice (Huycke and Gilmore 1997).

Short-chain fatty acids such as butyric and propi- onic acids are fermentation by-products of obligate anaerobes that can stimulate the release of cytokines (Niederman et al. 1997). In vitro investigations showed that penetration of butyric acid could occur in root canals of tooth sections obturated with gutta-percha and AH26 (Kersten and Moorer 1989). Short-chain fatty acids might have a role in the periapical infection process, perhaps via stimulation of monocyte IL-1β production, a cytokine associated with bone resorption (Eftimiadi et al. 1991) and increased T-lymphocyte cell apoptosis (Kurita-Ochiai et al. 2006).

7.4 Virulence associated with endodontic microorganisms

The relationship between the microflora in advancing caries and the histopathology of pulpitis involves irreversible tissue damage, healing, and repair that

Virulence of Endodontic Bacterial Pathogens 163

Fig. 7.10 Survival of gelatinase-positive E. faecalis OG1RF in dentinal tubules 8 months after obturation with gutta-percha and RoekoSeal. Negative control, no bacteria (A1 and inset in A2), absence of gelatinase-negative E. faecalis TX5128 (B1 and inset in B2) and presence of gelatinase-positive E. faecalis OG1RF (C1 and inset in C2). TX5198 is a gelatinase-negative mutant of OG1RF. Brown and Brenn stain. Source: Adapted from Sedgley (2007). Reproduced with permission of Elsevier.

164 Endodontic Microbiology

is modulated by both innate and adaptive immune responses (Hahn and Liewehr 2007). The microflora of carious dentin that has been strongly implicated in endodontic infections subsequent to pulpitis includes significant numbers of lactobacilli (Chhour et al. 2005) and Gram-negative bacteria (Martin et al. 2002). Many species recovered from infected root canals have also been identified as commensals in the oral cavity. The transition from oral commensal to root canal pathogen may reflect an innate ability to switch on virulence genes that enable survival and propagation in the root canal environment. However, the toll-like receptor response is tightly controlled to avoid an inflammatory response to commensals (Sirard et al. 2006).

7.4.1 Porphyromonas and Prevotella

These genera of nonmotile Gram-negative obligately anaerobic rods belonging to the Bacteroidetes phy- lum have been shown in pyrosequencing analysis to be the most abundant phylum in both primary and persistent infections (Hong et al. 2013). Within the endodontic literature, these species have sometimes been generically identified as black-pigmented Bac- teroides (BPB) based on the fact that some species form brown or black pigments when cultured on blood- containing media. They are frequently detected in root canal samples from symptomatic and asymptomatic root canal infections, and aspirates from acute peri- apical abscesses (Haapasalo et al. 1986; Siqueira and Rôças 2005). In vitro studies have shown that LPS from Prevotella and Porphyromonas stimulated IL-1β release (Hosoya and Matsushima 1997), IL-8 expres- sion (Chang et al. 2005), and VEGF expression (Botero et al. 2003). P. intermedia fimbriae induced hemag- glutination activity in vitro (Leung et al. 1996). Super- natants of Porphyromonas endodontalis, P. gingivalis, and P. intermedia stimulated the expression of VEGF and IL-6 in human pulp fibroblasts (Yang et al. 2003, 2004).

Multiple virulence factors are associated with Por- phyromonas gingivalis (Holt et al. 1999; Cugini et al. 2013), including VimA, a virulence modulating gene with multiple functions for virulence regulation (Aruni et al. 2013). P. gingivalis FimA fimbriae are classi- fied into six genotypes. FimA fimbriae were found in approximately one-third of 50 primary endodontic infection samples (from 25 root canals and 25 acute apical abscess aspirates), with no correlation between fimbriae type and the presence of symptoms (Rôças

and Siqueira 2010). In another study, FimA fimbriae were found in a similar proportion of samples (63 of 158 root canal samples); however, types II and IV were more frequent in symptomatic cases (Wang et al. 2010).

It has been hypothesized that P. gingivalis might behave as a keystone pathogen in periodontal disease by using sophisticated strategies to disrupt the nor- mally symbiotic microflora, thereby creating a dys- biotic microbial environment that can evade the host immune system (Hajishengallis et al. 2012). An exam- ple of such a strategy might be associated with the aforementioned gingipain proteases located in extra- cellular vesicles (Duncan et al. 2004) providing pro- tection. Immunization of nonhuman primates with a gingipain-based vaccine resulted in a reduction of overall microbial load and P. gingivalis in subgingi- val plaque, and the inhibition of alveolar bone loss (Page et al. 2007). Whether this species similarly has a keystone pathogen role in endodontic infections is an intriguing possibility that remains to be established.

7.4.2 Fusobacterium

Fusobacterium is a genus of Gram-negative anaer- obic spindle-shaped rods or filaments belonging to the Fusobacteria phylum. Clinically, the recovery of F. nucleatum has been associated with the most severe flare-ups pain and swelling (Chavez de Paz Vil- lanueva 2002). They are nonmotile with the major end-products of metabolism being butyric acid as well as lesser amounts of acetic, lactic, formic, and propi- onic acids. The release of inflammatory cytokines from neutrophils can be stimulated by butyric acid produced by F. nucleatum (Niederman et al. 1997). Virulence factors released by fusobacteria can stimulate numer- ous biologic effects. LPS from F. nucleatum induced a rapid immune response when applied to pulp tissues in rats (Dahlen 1980), and produced a large array of biologic effects in macrophage-like cells (U937 cells) by the upregulation of proinflammatory cytokines IL- 1β, IL-6, tumor necrosis factor α (TNF-α), and IL-8 (Grenier and Grignon 2006). In vitro investigations have shown that F. nucleatum may induce aggregation and apoptosis of immune cells (Huynh et al. 2011).

7.4.3 Parvimonas

Parvimonas (previously Peptostreptococcus) is a genus of Gram-positive, asporogenous, anaerobic

Virulence of Endodontic Bacterial Pathogens 165

cocci from the Clostridiaceae family, of the phylum Firmicutes. The species Parvimonas micra (previously Peptostreptococcus micros) is commonly associated with periodontal disease and has been recovered from endodontic abscesses in children (Brook et al. 1981). P. micra can bind human plasminogen on their cell sur- face which can be activated into plasmin. This activ- ity, along with proteolytic capabilities, may facilitate dissemination of bacterial cells (Grenier and Bouclin 2006). Positive associations were described between F. nucleatum and P. micra in teeth with apical peri- odontitis (Sundqvist 1992), suggesting that this is a synergistic association that might enhance pathogenic- ity (Bolstad et al. 1996).

7.4.4 Streptococcus

Streptococcus is a genus of Gram-positive, asporoge- nous, facultatively anaerobic, catalase-negative cocci or coccoid bacteria, which belong to the phylum Fir- micutes. They are nonmotile and approximately 1 μm in diameter, occurring in pairs or chains. Several strep- tococcal species can form capsules. The cell walls of streptococci contain PG and LTA. Greater production of extracellular LTA by S. mutans was observed when grown in low pH (6.0 or 6.5) conditions at low dilu- tions (Jacques et al. 1979). Upon lysis of the bacterial cell (inducible by lysozyme, cationic peptides from leukocytes, or beta-lactam antibiotics), PG and LTA are released. These can bind to cell surface receptors and induce the release of proinflammatory cytokines. In mice, the production of proinflammatory cytokines was induced by extracellular products of Streptococcus sanguis and Streptococcus mitis (Takada et al. 1993). Contact with Streptococcus salivarius was associated with increased expression of IL-6, IL-8, and TNF-α by oral epithelial cells (Mostefaoui et al. 2004). Both LTA and PG from Streptococcus spp. induced expression of TNF-α in human monocytes (Heumann et al. 1994). Extracellular proteins produced by root canal isolates of S. gordonii, S. anginosus, and S. oralis may con- tribute to posttreatment apical periodontitis (Chavez de Paz et al. 2005).

Streptococci have cell surface adhesins that facil- itate binding to various substrates, including dentin, as well as other bacterial cells and epithelial cells (Jenkinson 1994). They may also recognize compo- nents present within dentinal tubules, such as col- lagen type I, which stimulates bacterial adhesion and intratubular growth. Cell surface adhesin proteins

SspA and SspB, members of the antigenI–II family of streptococcal polypeptides, are involved in the binding and growth of streptococci into dentinal tubules (Love et al. 1997). S. gordonii has been shown to invade apical dentin up to 60 μm into dentinal tubules and up to 200 μm at the cervical and midroot level (Love 1996). S. gordonii amylase-binding protein A func- tions as an adhesin to amylase-coated hydroxyapatite (Rogers et al. 2001). A putative cell-surface collagen- binding (CbdA) protein identified in S. gordonii with similar amino acid sequence to the Ace protein found in E. faecalis was associated with increased survival of S. gordonii in instrumented root canals ex vivo (Moses et al. 2013).

7.4.5 Lactobacillus

Lactobacillus is a genus of Gram-positive rods or coccobacilli that grows under anaerobic, facultatively anaerobic, or microaerophilic conditions and belongs to the phylum Firmicutes. Lactobacilli convert sugars, including lactose, to lactic acid. They are generally considered nonpathogens (Brouqui and Raoult 2001), apart from their association with dental caries (Brook 2003). Along with streptococci, lactobacilli are impor- tant microorganisms in the caries process, comprising up to 50% of bacterial species in advanced carious lesions (Chhour et al. 2005). Oral Lactobacillus spp. bind to collagen type I, the major collagen of dentin (McGrady et al. 1995). The expression of TNF-α through TLR2 in mouse immune cells was induced by Lactobacillus LTA (Matsuguchi et al. 2003). In teeth with apical periodontitis undergoing root canal treat- ment, Lactobacillus spp. (and Olsenella uli, a member of the Olsenella genus which was originally separated out from the Lactobacillus genus) predominated over other Gram-positive rods (Chavez de Paz et al. 2004).

7.4.6 Enterococcus

Enterococcus is a genus of Gram-positive, faculta- tively anaerobic, coccoid bacteria, which belongs to the phylum Firmicutes. Enterococcal cells are ovoid and occur singly or in pairs or short chains, and can grow at temperatures ranging 10–45◦C. E. faecalis and E. faecium are the most common enterococcal species found in humans. E. faecalis is a common causative agent of infective endocarditis (Hill et al. 2007). Enterococci, predominantly E. faecalis, are fre- quently recovered from previously treated root canals

166 Endodontic Microbiology

(Rôças et al. 2004; Sedgley et al. 2006a). Interestingly, in studies on primate models, the addition of E. faecalis to a four-strain collection (Streptococcus anginosus, Peptostreptococcus anaerobius, Prevotella oralis, and Fusobacterium nucleatum) resulted in higher survival of the complete combination than the same bacte- rial combination without E. faecalis (Fabricius et al. 2006). When selected bacterial strains were inocu- lated into the root canals of these animals, E. faecalis was the only species to be reisolated from all 24 root canals and, together with other organisms, was able to produce radiographic evidence of apical periodonti- tis after 8–12 months, even in the one canal where E. faecalis survived by itself (Moller et al. 2004). E. faecalis can survive for extended periods in the root canal system in vitro (Sedgley et al. 2005a), in a man- ner that may be dependent on treatment conditions and the phenotype, such as presence of gelatinase activity (Sedgley 2007) (Figure 7.10). Gelatinase production by E. faecalis recovered from root canals undergoing retreatment was significantly associated with the pres- ence of periapical radiolucency (Wang et al. 2011).

The persistence of E. faecalis in treated root canals has been attributed to resistance to the high pH of antimicrobial agents used during treatment, but the specific mechanisms are not clear. The ability for E. faecalis to survive over an extended period at pH

10 was associated with a 37-fold increase in gene transcripts of ftsZ, a gene involved in cell division (Appelbe and Sedgley 2007). Virulence factors iden- tified in enterococci recovered from the oral cav- ity and infected root canals (Sedgley et al. 2004, 2005b, 2006b; Duggan and Sedgley 2007) are pre- sented in Table 7.2. These include those with the potential to promote adaptation and survival in differ- ent environments: enterococcus surface protein (Esp), collagen-binding protein (Ace), and aggregation sub- stance (AS), as well as factors that enable secretion of proteases (e.g., gelatinase) and toxins (e.g., cytolysin) (Sedgley et al. 2005b; Reynaud af Geijersstam et al. 2007). Serine protease and Ace contribute to the abil- ity of E. faecalis to bind to dentin (Kowalski et al. 2006), and resistance to killing by human neutrophils is promoted by AS (Rakita et al. 1999).

Proteomic analysis of samples from seven infected root canals reported that the majority of the vir- ulence and antibiotic resistance proteins present in the samples analyzed were secreted by enterococci, with evidence of enterococcal derived AS, extracellu- lar serine proteinase, hemolysin, and proteins associ- ated with pheromone response and conjugative transfer (Nandakumar et al. 2009). Multilocus sequence typing (MLST) analysis of E. faecalis genotypes from canals of root-filled teeth with periapical lesions showed that

Table 7.2 Oral and endodontic Enterococcus faecalis with phenotypic and genotypic characteristics of virulence traits

Phenotypic tests Virulence genesa

Source n Gelatinase Bacteriocin Haemolysin Pheromone responseb gelE ef184/fsr esp asa asa373 ace cylA efaA

Endodontic Primary 14 13 6 0 7 14 1 8 14 0 14 3 14 Retreatment 8 3 3 0 3 7 4 3 6 0 8 2 8 Unknown 11 8 4 0 6 11 5 8 11 0 11 1 11 Combined 33 24 13 0 16 32 10 19 31 0 33 6 33

% 73 39 0 48 97 30 58 94 0 100 18 100 Oral Oral rinse 17 5 8 5 4 13 5 4 12 0 17 2 17 Tongue swab 3 0 2 2 1 2 1 2 3 0 3 2 3 Saliva 1 1 1 0 0 1 0 0 0 0 1 0 1 Combined 20 5 10 7 5 15 6 6 15 0 20 4 20

% 25 50 35 25 75 30 30 75 0 100 20 100

aagelE, gelatinase; ef184/fsr, gelatinase-negative phenotype; esp and ace, surface adherence factors; asa and asa373, aggregation substance; cylA, cytolysin activator; efaA, endocarditis antigen. bProduction of aggregation substance in response to E. faecalis pheromones. Source: Combined data from Sedgley et al. (2004, 2005b, 2006a) and Duggan and Sedgley (2007).

Virulence of Endodontic Bacterial Pathogens 167

approximately one-third of strains belonged to lin- eages associated with capsule expression and produc- tion of multiple virulence factors (Pinheiro et al. 2012).

While the above data, combined with the high prevalence and a capability for extended survival of E. faecalis in the root canal system, suggest a sig- nificant role for the species in the pathogenesis of human root canal infections, this has never been con- clusively established. E. faecalis as a single species in primate root canals caused only low-grade peri- apical reactions, and only when present with other species within an “eight-strain collection” was lesion size larger (Fabricius et al. 1982). In addition, Ente- rococcus spp. were equally prevalent in previously treated root canals of teeth with or without a periapi- cal lesion (Kaufman et al. 2005, Zoletti et al. 2010). It has been hypothesized that the tissue damage found in periapical infections that involve E. faecalis may be more to do with the host response than direct dam- age from bacterial products (Kayaoglu and Orstavik 2004). Indeed, the virulence and pathogenic character- istics of E. faecalis from nosocomial infections appear to differ from strains recovered from endodontic and oral sources. For example, determinants in E. faecalis for the prokaryotic adaptive immune system CRISPR- cas were found in proportionally more endodontic (25 of 34) and oral (15 of 21) strains than multidrug- resistant hospital-acquired (9 of 23) strains (P = 0.01 and 0.04, respectively), indicating that the hospital strains were “more susceptible” to invasion by for- eign DNA carrying antibiotic resistance determinants (Burley and Sedgley 2012). Similarly, E. faecalis iso- lates from endodontic infections and nonoral hospital- ized patients showed different genetic and virulence profiles in MLST analysis investigations (Penas et al. 2013).

7.4.7 Actinomyces

These nonspore-forming Gram-positive bacteria belong to the phylum Actinobacteria and occur as branched rods or filaments, or as rudimentary mycelia. All species can grow anaerobically. Many Actino- myces species are commensals in the oral cavity but can become opportunistic pathogens in humans and other mammals (Yeung 1999; Mardis and Many 2001). Occasionally, they cause actinomycosis. A. israelii has been implicated in osteoradionecrosis of the jaw (Happonen et al. 1983). Actinomyces species can be fimbriated or nonfimbriated. Higher cell-surface

interactive forces associated with fimbriated than non- fimbriated Actinomyces may contribute to modulation of their coaggregation properties and adhesion (Tang et al. 2004). A. israelii and A. naeslundii injected into mice resulted in the production of suppurative lesions (Coleman and Georg 1969). Actinomyces species have been recovered from primary root canal infections and secondary root canal infections nonresponsive to conventional treatment (Borssen and Sundqvist 1981; Siqueira et al. 2002; Chavez de Paz et al. 2004). There are several case reports of Actinomyces species being isolated from persistent lesions following root canal filling (Sakellariou 1996; Ricucci and Siqueira 2008), sometimes several years after completion of treatment (Sjogren et al. 1997; Hancock et al. 2001). This may be in part caused by the ability of the branching filamen- tous organisms to evade elimination by host phagocytic cells (Figdor et al. 1992), or the formation of biofilms that overwhelm the host response (Figure 7.11).

7.4.8 Propionibacterium

Propionibacterium species belong to the phylum Acti- nobacteria, and are slow-growing, nonsporulating, Gram-positive anaerobic rods with propionic acid an end-product of fermentation. They are normal inhabi- tants of the skin and are usually nonpathogenic (Roth and James 1988) but can be frequent contaminants of blood and body fluid cultures. The human cutaneous propionibacteria include Propionibacterium acnes and Propionibacterium propionicum (also called Propioni- bacterium propionicus, and formerly Arachnia propi- onica). These species can be opportunistic pathogens, causing diverse infections that include acnes vulgaris (Vowels et al. 1995), central nervous system infec- tions (Mory et al. 2005), and infective endocarditis (Delahaye et al. 2005). The species produce proin- flammatory enzymes (lipases, neuraminidases, phos- phatases, and proteases) with the capacity to contribute to direct damage to the host (Perry and Lambert 2006). The production of cytokines IL-1α, IL-1β, IL-8, and TNF-α by monocytes was induced by P. acnes (Vowels et al. 1995). P. acnes was recovered from 10 of 11 root canals lesions without periapical abscesses and from 8 of 9 root canals with periapical abscesses; the phylo- types of all strains differed from P. acnes skin isolates from the same patient, indicating that contamination during sampling was not responsible for the presence of these isolates in the root canal samples (Niazi et al. 2010).

168 Endodontic Microbiology

(a)

(e)

(i)

(f) (g) (h)

(b) (c) (d)

Virulence of Endodontic Bacterial Pathogens 169

P. propionicum has been implicated as a causative agent of a disease process similar to actinomycosis (Happonen et al. 1985). The species has been cultured from deep layers of infected root canal dentin (Ando and Hoshino 1990) and has the ability to penetrate into dentinal tubules (Siqueira et al. 1996). In teeth with apical periodontitis undergoing root canal treatment, the most frequent Propriobacterium spp. species cul- tured was P. propionicum (Chavez de Paz et al. 2004).

7.4.9 Combinations of microorganisms

In addition to quorum sensing communication, inter- actions among different strains and species—both antagonistic and synergistic—would be expected in endodontic infections. For example, the production of a bacteriocin by a producer strain that has an inhibitory activity against other strains may provide the pro- ducer with a selective advantage (Riley and Wertz 2002), thereby modulating the infectious process. In contrast, virulence might be enhanced by synergis- tic interactions among species. For example, P. micra enhanced the pathogenicity of Bacteroides melanino- genicus (Prevotella melaninogenica) or Bacteroides asaccharolyticus (Porphyromonas spp.) in experimen- tal infections in guinea pigs (Sundqvist et al. 1979).

Other examples of beneficial interactions uti- lize coaggregation interactions. Using confocal microscopy, coaggregation interactions were observed in association with Prevotella, Streptococcus, and Fusobacterium species isolated from acute endodontic infections (Khemaleelakul et al. 2006). Coaggre- gation has been also observed among F. nucleatum and E. faecalis, S. anginosus, Peptostreptococcus anaerobius, and Prevotella oralis (Johnson et al. 2006). F. nucleatum may provide a specific link or connection for other coaggregating microorganisms (Kolenbrander et al. 1989). Interactions among geneti- cally distinct bacteria are involved in the establishment

and maintenance of biofilms (Kolenbrander et al. 2010) and provide an accommodating environment for HGT (Sorensen et al. 2005).

It has been proposed that the combination of F. nucleatum, Prevotella spp., and Porphyromonas spp. may provide a risk factor for endodontic flare- ups by acting in synergy to increase the intensity of the periapical inflammatory reaction (Chavez de Paz Villanueva 2002). In mice, the combination of F. nucleatum and either P. gingivalis or P. intermedia induced more severe pathologic subcutaneous lesions in mice in mixed compared to pure culture (Baumgart- ner et al. 1992). Similarly, subcutaneous injections of the combination of P. intermedia and P. micra resulted in a more severe response in mice in combination rather than separately (Araki et al. 2004).

Specific interactions between streptococci and other bacteria may facilitate their invasion into dentin. For example, coinvasion of dentinal tubules by P. gingi- valis and S. gordonii, but not with S. mutans, was facilitated by the streptococcal antigen I–II polypep- tide (Love et al. 2000). Significant associations were reported to occur between specific combinations of species and clinical symptoms: swelling and the com- bination of P. micra and Prevotella spp., wet canals and the combinations of Eubacterium spp. with either Prevotella spp. or Peptostreptococcus spp., and pain and the combination of Peptostreptococcus spp. and Prevotella spp. (Gomes et al. 1996). In six of nine patients, the combination of F. nucleatum and Strep- tococcus spp. was associated with symptoms (Fouad et al. 2002). In root canals of teeth with apical peri- odontitis receiving treatment there may be an associ- ation between Lactobacillus spp. and Gram-positive cocci (Chavez de Paz et al. 2004).

Endodontic infections are typically polymicrobial biofilm communities (Siqueira and Rôças 2009). Two recent studies have applied proteomic analyses to evaluate the infected root canal microflora as a

� Fig. 7.11 (a) Preoperative and (b) postoperative radiographs. (c) The patient presented 1 year and 8 months later with a sinus tract. (d) A gutta-percha point was inserted in the sinus tract, and a radiograph was taken. The radiolucent area was considerably increased. (e) Periradicular surgery was performed, and pathologic lesion specimen was obtained in its original relationship with the root apex. The circle indicates a likely communication between the center of the lesion and the sinus tract. (f,g) Overviews of the two sulfur granules (Taylor’s modified Brown and Brenn; original magnification, ×50). (h) Higher power view from the center of the sulfur granule in (f). Concentration of intertwining bacterial filaments (original magnification, ×1000). (i) Periphery of the sulfur granule in (f). Bacterial filaments are arranged in dense aggregates at the periphery, surrounded by layers of an amorphous material. Concentrations of neutrophilic leukocytes appear on the outer surface, some of which are in close contact with the bacterial matrix (original magnification, ×400; original magnification of the inset, ×1000). Source: Adapted from Ricucci and Siqueira (2008). Reproduced with permission of Elsevier.

170 Endodontic Microbiology

community (Nandakumar et al. 2009; Provenzano et al. 2013). The main advantage of this approach over genomic techniques is that by directly characterizing the virulence-associated proteins present in endodon- tic infections, a more global insight into the phys- iology and pathogenicity of the infected root canal microbial community can be obtained. In the ear- lier study, bacterial proteins in preoperative samples collected from teeth with primary (n = 4), persis- tent (n = 2), or unassigned (n = 1) endodontic infec- tions were identified using reverse-phase nano-liquid chromatography-tandem mass spectrometry (nLC- MS/MS) (Nandakumar et al. 2009). The majority of proteins identified were of enterococcal origin, with several proteins involved in virulence and pathogenic- ity. Other nonenterococcal proteins identified included those with functions in tissue invasion and virulence (microbial collagenase metalloprotease from Bacil- lus sp., a putative virulence-associated protein from Corynebacterium sp.), adhesion (fibronectin-binding A domain protein from Exiguobacterium sp., coagu- lation factor 5/8 type domain protein from Paenibacil- lus sp., fibrinogen-binding protein from S. agalac- tiae, putative lipoprotein from S. gordonii, bacterial immunoglobulin-like domain protein from Treponema denticola), LPS synthesis (N-acylneuraminate cytidy- lyltransferase from F. nucleatum), and conjugation (TraG family protein from P. gingivalis). The more recent study identified bacterial proteins in preoper- ative samples from the root canals of 12 teeth with necrotic pulps and asymptomatic apical periodontitis and in pus aspirants from two cases of acute apical abscess using two complementary mass spectrome- try platforms: nanoflow liquid chromatography cou- pled with linear ion trap quadrupole Velos Orbitrap and liquid chromatography-quadrupole time-of-flight (Provenzano et al. 2013). In this study, it was notewor- thy that the number of proteins identified in pooled abscess samples was higher than in pooled root canal samples (173 and 88 proteins, respectively). While the majority of proteins were related to metabolic and housekeeping processes, microbial proteins involved in pathogenicity and virulence were detected: adhe- sion (n = 3; glycosyltransferase 1, tight adherence protein G, and coagulation factor 5/8 type domain pro- tein), proteolytic activity (n = 8; collagenase, metal- loprotease, serine protease, extracellular protease, and endopetidases), exotoxin activity (Streptopain, SPE B, a cysteine protease-like exotoxin), and tissue invasion (a putative invasin). Taken together, although these

data are from small samples, they provide an intriguing snapshot of the types of proteins expressed by microor- ganisms during the endodontic infection process.

7.5 Conclusions and future directions

Recent studies using pyrosequencing techniques have established that root canal infections are more complex multispecies microbial communities than previously considered (Li et al. 2010; Santos et al. 2011; Ozok et al. 2012; Siqueira et al. 2012; Hong et al. 2013). While many key mechanisms remain to be elucidated, it is clear that root canal infections are adaptable and heterogeneous biofilm communities under the influ- ence of variable local environmental selective pres- sures, synergistic and antagonistic microbial cell–cell interactions, and a range of host defense responses. Further, within these communities, there are many “putative” species that have yet to be formally iden- tified and whose significance is not known (Siqueira and Rôças 2013).

In general terms, microbial virulence factors enable the microorganism to replicate and disseminate within a host by subverting or eluding host defenses (Cross 2008). A diverse arsenal of microbial virulence factors are available in endodontic infections. It is reasonable to expect that the possession of certain virulence traits by one or a combination of species would provide a selective advantage over other species in terms of survival and propagation of the infectious process. In particular, LPS has been long recognized as a bacterial virulence factor of significance to endodontic infec- tions. However, at this time many other putative viru- lence factors and their relationship with actual clinical signs and symptoms are mostly speculative. In addi- tion, the contribution of virulence factors associated with as-yet-uncultivated organisms to the pathogene- sis of endodontic infections is not at all clear.

Considering that complex multispecies biofilms are implicated in endodontic infections, future stud- ies building on the “global” genomic–proteomic approaches described in recent publications are likely to provide the most valuable insight into mecha- nisms of virulence and pathogenicity during endodon- tic infections. The design and implementation of such studies is expected to present considerable chal- lenges but the information obtained has the poten- tial to be rewarding beyond the scope of endodon- tics (Kuboniwa et al. 2012; Han and Wang 2013). A

Virulence of Endodontic Bacterial Pathogens 171

better understanding of the mechanisms by which the host regulates virulence gene expression by endodon- tic microorganisms may help explain different clini- cal presentations of endodontic infections and in turn ultimately contribute to the identification of effective therapeutic targets.

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Chapter 8 Viruses in Endodontic Pathosis Mohamed Sabeti

8.1 Introduction 8.2 General description of herpesviruses 8.3 Human cytomegalovirus 8.4 Epstein–Barr virus 8.5 Herpes simplex virus types 1 and 2 8.6 Varicella-zoster virus 8.7 Human herpesvirus-6 8.8 Human herpesvirus-7 and -8

8.9 Association between herpesviruses and apical disease

8.10 Pathogenesis of herpesvirus- associated apical disease

8.11 Model for herpesvirus-mediated apical disease

8.12 References

8.1 Introduction

Viruses are the simplest and smallest microorganisms that infect humans. They consist of either DNA or RNA surrounded by a protein coat termed a capsid. The most commonly known viruses within oral cavity are the herpesviruses. These are the most important DNA viruses that cause oral disease in humans. The hallmark of herpesvirus infection is immune system impairment.

Herpesviruses cause disease in humans in two ways: herpesvirus infections may result at the site of entry or they may enter the circulation and infect distant organs. The mode of release of the virions can deter- mine the pattern of infection from the infected cell (Tucker and Compams 1992; Bergelson 2009; Contr- eras et al. 2014). If the virion is released from the api- cal part of the cell, the infection will become localized; however, if the virion is released from the basolateral side of the cell, the infection becomes a disseminat- ing infection (Tucker and Compams 1992; Bergelson 2009; Contreras et al. 2014). It has also been shown that the outcome of the viral infection depends upon the

cellular immune responses to block viral DNA replica- tion and interference from herpesvirus infection with host defense (Jones and Sun 1997; Rolzman et al. 2005; Contreras et al. 2014). Herpesviral replication takes place in the nucleus of the host cell. The viral replication and the production of infectious virions involve activations of three sets of genes: the expres- sion of immediate-early, early, and late classes of genes. Late (structural) genes are expressed during the productive (lytic) phase of herpesviral infections. Figure 8.1 describes the infection process of her- pesviruses.

In past two decades, new viruses have been iden- tified that have expanded our knowledge and under- standing of viral infections and their pathogenicity. Human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV) seem to be important putative pathogens of human periodontitis and symptomatic periapical lesions, causing pathosis either by inducing immuno- suppression with a subsequent risk of aggressive bacte- rial infections or by infecting periodontal cells directly. Apical periodontitis and its etiopathogenesis, espe- cially the molecular events preceding and causing

179

180 Endodontic Microbiology

Capsid Nonstructural proteins Attachment and penetration by fusion

Immediate early Protein synthesis

Early Protein synthesis and genome replication

Exocytosis and release

ACTIVE

LATENT

Protein

Nucleus

Late Protein synthesis (structural protein)

Lysis and release

Assembly and release

DNA

DNA genome

mRNA

Fig. 8.1 The replication of herpesviruses. A virion initiates infection by fusion of the viral envelope with the plasma membrane after attachment to the cell surface. The capsid is transported to the nuclear pore, where viral DNA is released into the nucleus. Viral transcription and translation occur in three phases: immediate early, early, and late. Immediate early proteins shut off cell protein synthesis. Early proteins facilitate viral DNA replication. Late proteins are structural proteins of the virus that form empty capsids. Viral DNA is packaged into preformed capsids in the nucleus. Viral glycoproteins and tegument protein patches in cellular membranes and capsids are enveloped. Virions are transported via endoplasmic reticulum and released by exocytosis or cell lysis. Source: Slots et al. 2002. Reproduced with permission of John Wiley and Sons.

disease onset, are associated with a wide range of bac- teria and viruses and interfere with innate and adap- tive cellular, humoral immune response, and affect cytokine networks. Pulpal and periapical infections exhibit complex microbial ecologies involving syner- gistic, antagonistic, and commensal interrelationships among resident microorganisms. Sundqvist (1992) found strong positive associations between Fusobac- terium nucleatum and Parvimonas micra, Porphy- romonas endodontalis, Selenomonas sputigena, and Campylobacter rectus, and negative or neutral associ- ations with streptococcal species, Propionibacterium propionica, Capnocytophaga ochracea, Veillonella parvula, and other bacteria in root canals of teeth with periapical lesions. Significant relationships may

also exist between endodontic Porphyromonas gingi- valis and Tannerella forsythia or Treponema species (Jung et al. 2000). P. endodontalis may cooperate with Prevotella intermedia or Prevotella nigrescens in the formation of abscesses (Siqueira et al. 1998). A marked shift toward a more anaerobic microbiota has been demonstrated during the development of experimental endodontic infections in monkeys (Fabri- cius et al. 1982). Varying nutritional demands and anaerobic requirements of infecting organisms are important determinants of microbial interrelationships and population changes in the endodontic microbiota (Sundqvist 1994). Differing levels of host resistance may also significantly influence the composition of the periapical microbiota.

Viruses in Endodontic Pathosis 181

Current hypotheses on the pathogenesis of periapi- cal pathosis include both bacterial and host factors, but the pathogenic events that trigger the conversion of a stable, asymptomatic endodontic lesion to a pro- gressive or a symptomatic lesion remain obscure. A commonly held idea regarding apical pathosis as a bacterial disease could not fully explain the pathogen- esis of the disease, site-specificity, and tissue tropism. The pathogenic events that trigger the conversion of a stable, asymptomatic endodontic lesion to a progres- sive or a symptomatic lesion remain obscure. How- ever, hypothetically a combined viral–bacterial infec- tion could explain the major features associated with the disease (Slots 2005). Viral association with bac- teria and apical disease is consistent with pathologic role of both infectious agents. Acute exacerbation of periapical disease may be caused from a combination of herpesviral and bacterial causes. This possibility is consistent with the majority of studies that have observed presence of active herpesvirus infections in symptomatic periapical lesions and the proinflamma- tory potential of herpesviruses (Mogensen and Paludan 2001).

To date, eight human herpesviruses have been iden- tified: herpes simplex virus types 1 and 2 (HSV 1 and 2), varicella-zoster virus (VZV), EBV, HCMV, human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV- 7), and human herpesvirus-8 (HHV-8). Humans are the only source of infection for these eight herpesviruses.

Human herpesviruses are classified into three groups (α,β,δ) based upon details of tissue tropism, pathogenicity, and behavior in the laboratory (Table 8.1). In most individuals, primary infection by herpesviruses occurs early in life and exhibits few or no overt disease symptoms. Herpesviruses remain in infected hosts for a lifetime in a prolonged state of latency but retain their capacity for renewed or episodic

Table 8.1 Classification of human herpesviruses

Herpesviruses Herpes group

HSV-1 α HSV-2 α VZV α EBV δ HCMV β HHV-6 β HHV-7 β HHV-8 δ

reactivated replication. In the latent phase of infection, herpesviruses reside in the following cells:

1. HSV 1 and 2 in sensory nerve ganglia and mono- cytes;

2. EBV in B lymphocytes and salivary gland tissue; 3. VZV in sensory nerve ganglia; 4. HCMV in monocytes, macrophages, lymphocytes,

and salivary gland tissue; 5. HHV-6 in lymphocytes and ductal epithelium of

salivary gland; 6. HHV-7 in lymphocytes and salivary gland tissue;

and 7. HHV-8 in lymphocytes and macrophages.

Reactivation of latent herpesviruses is involved in driving the pathologic process of some types of symp- tomatic periapical disease. Physical trauma, stress, immunosuppression, immune dysfunction, and radio- therapy can trigger herpesvirus activation.

8.2 General description of herpesviruses

Membership in the family Herpesviridae is based on the structure of the virion (Roizman and Pellett 2001). The prototypical structure of herpesviruses consists of a double-stranded DNA genome ranging in size from 120 to 250 kilobase pairs (kb) encased within an isos- apentahedral capsid and an amorphous proteinaceous tegument, which is surrounded by a lipid bilayer enve- lope derived from the host cell membrane. Herpesvi- ral replication takes place in the nucleus of the host cell and involves the expression of immediate-early, early, and late classes of genes. Late (structural) genes are expressed during the productive (lytic) phase of herpesviral infections. After primary exposure, her- pesviruses establish latency in various host cell reser- voirs, from which they may reactivate periodically (Sissons et al. 2002). Box 8.1 summarizes some of the common characteristics of herpesviruses.

Box 8.1 Summary of common characteristics of herpesviruses

� A single double-stranded DNA molecule rang- ing 120–250 kb

� An isosapentahedral capsid containing 162 capsomers

182 Endodontic Microbiology

� An amorphous proteinaceous tegument and, surrounding the capsid and tegument

� A lipid bilayer envelope derived from host cell membrane

� Tissue tropism � The viral productive phase is followed by a

latent phase in host cells, which ensures survival of the viral genome throughout the lifetime of the infected individual

� Latent herpesvirus can undergo sporadic reac- tivation and reenter the productive phase

Most herpesviruses are ubiquitous agents that often are acquired early in life and infect individuals from diverse geographic areas and economic backgrounds (Britt and Alford 1996; Rinckinson and Kueff 1996). Herpesvirus transmission occurs by intimate contact with infected secretions including saliva, blood, and genital secretion (Gautheret-Dejean et al. 1977; Britt and Alford 1996; Ikuta et al. 2000). Acquisition of herpesviruses takes place from an early age and some- times in the uterus. A notable exception is HSV-8 which is contracted in adulthood. Clinical manifes- tations of herpesvirus infections are highly diverse and range from mild or subclinical disease in most healthy individuals to encephalitis, pneumonia, and other potentially lethal infections, and various types of cancer including lymphoma, sarcoma, and carcinoma in immunocompromised hosts. Herpesviruses are the most important DNA viruses that affect the oral cavity.

8.3 Human cytomegalovirus

HCMV is a ubiquitous agent that is often acquired early in life. HCMV is an important pathogen, which causes infection in several ways. HCMV can cause infection either by virus inducing immunosuppression of the host as a primary infection or when the virus is reactivated from a latency stage in an infected individ- ual as an endogenous infection or when a previously infected individual with HCMV is reinfected as exoge- nous reinfection (Contreras et al. 2014).

Transmission of HCMV can happened prenatally from mother to infant. HCMV can be found in blood and in many body secretions including milk, semen, and saliva (Gaitheret-Dejean et al. 1977; Britt and

Alford 1996). It is estimated that asymptomatic secre- tion shedding of HCMV into saliva, cervical secre- tions, semen, and breast milk occurs in 10–30% of infected individuals (Britt and Alford 1996). Most primary infections are asymptomatic. HCMV can infect and replicate in endothelial cells (Grefte et al. 1993a,b), ductal epithelial cells (Mocarski and Stin- ski 1979; Grundy 1990; Sinzger et al. 1995), smooth muscle cells (Platcher et al. 1995; Sinzger et al. 1995; Mocarski 1996), and fibroblasts (Myerson et al. 1984; Platcher et al. 1995; Sinzger et al. 1995; Mocarski 1996). HCMV target peripheral blood monocytes and lymphocytes during active infection (Myerson et al. 1984; Kapasi and Rice 1988; Dankner et al. 1990; Gerna et al. 1992; Meyer et al. 1995; Sinzger et al. 1995; Mocarski 1996). The site of latent HCMV is peripheral monocytes (Maciejowski et al. 1992; Taylor-Wiedeman et al. 1993) and may be found in bone marrow-derived progenitor cells (Maciejewski et al. 1992; Sinzger et al. 1995; Sinzger and Jahn 1996). HCMV is emerging as an important oppor- tunistic pathogen in immunocompromised patients. HCMV is the most common life-threatening infection in patients with HIV(Griffiths and Emory 1997) and is a significant risk factor in transplant complications and rejection (Mutimer et al. 1997; Nowzari et al. 2003).

HCMV can cause serious infectious diseases. Cytomegalovirus infection is of great clinical sig- nificance in pregnant women, newborn infants with congenital or perinatal infection, immunosuppressed transplant patients, and patients with AIDS. HCMV infection can be categorized under three clinical con- ditions: acquired HCMV infection, HCMV inclusion disease, and perinatal disease. Acquired HCMV infec- tion can be observed in individuals with compromised immune systems and patients who have had tissue or organ transplants. HCMV infection can aggravate and enhance the immunosuppressiveness of HIV oppor- tunistic infections. Necrotizing retinitis is a relatively common HCMV-induced complication in untreated HIV-infected individuals (Sweet 1999). HCMV is a potential cause of salivary gland dysfunction in patients with HIV. HCMV activation and resulting dis- ease has become a major clinical problem in transplant recipients. HCMV infection is the most common rea- son for the transplant rejection, including bone mar- row or stem cells grafts (Clark et al. 2003). HCMV inclusion disease is similar to infectious mononucle- osis. HCMV perinatal infection is the major cause of pregnancy complications and birth defects (Alford

Viruses in Endodontic Pathosis 183

et al. 1979). HCMV-infected newborn babies may show low birth weight, jaundice, hepatosplenomegaly, skin rash, microcephaly, or chorioretinitis (Bale et al. 2002). HCMV infection is the cause of mental retar- dation and sensorineural deafness (Revello and Gerna 2004). Approximately one-third of newborn babies with symptomatic congenital HCMV infection born to mothers with recurrent HCMV infection or to mothers with primary HCMV infection during pregnancy may be premature (Boppana et al. 1999).

8.4 Epstein–Barr virus

There are two types of EBV: types 1 and 2. The primary route of EBV acquisition is through salivary exchange in the oropharynx (Rickinson and Kueff 2001). EBV infection occurs in epithelial cells of the orophar- ynx (Sixbey et al. 1984), and EBV DNA, RNA, and viral antigens have recently detected in T lympho- cytes of Kawasaki-like disease (Kikuta et al. 1988), in nasal T-cell lymphomas (Tao et al. 1995), and in the epithelial cells of various human carcinomas (Raab-Traub et al. 1991; Luqmani and Shousha 1995). Latent EBV infection resides in B lymphocytes (Klein 1989).

EBV is a known cause of infectious mononucle- osis and almost certainly has a role in the etiology of nasopharyngeal carcinoma, Burkitt’s lymphoma, and lymphoproliferative disorders in the presence of immunosuppression. Less certain is the role of EBV in rheumatoid arthritis, Hodgkin’s disease, and chronic fatigue syndrome. EBV may be involved in the devel- opment of aggressive types of non-Hodgkin’s lym- phomas affecting gingiva (Yin et al. 1999). EBV is the main causative agent of infectious mononucleosis, which is a common clinical manifestation of primary EBV infection in adolescents and young adults. Symp- toms of this disease included fever, lymphadenopa- thy, malaise, and sore throat. Oral ulcers and multi- ple palatal petechiae have been reported. Patients with HIV experience frequent EBV-2 infection and dual EBV1–EBV2 infections. EBV has also been impli- cated in multiple sclerosis and has a role in the devel- opment of oral hairy leukoplakia as evidenced by EBV replicating within epithelial cells (Walling et al. 2004b), EBV-encoded nuclear antigen 2 (EBNA-2) protein function (Walling et al. 2004a) and an EBV- related decrease in oral epithelial Langerhans’ cells (Yamazaki and Nakajima 2004).

8.5 Herpes simplex virus types 1 and 2

Herpes simplex virus (HSV) infects keratinocytes, monocytes, macrophages, and establishes latency in neurons (Laskaris 1996). Infected patients experience an initial primary infection followed with a period of latency. HSV-1 causes mainly oral infections and HSV-2 causes anogenital infections. HSV-1 is respon- sible for most cases of herpetic gingivostomatitis with clinical presentation of multiple shallow ulcers throughout the keratinized and gland-bearing intraoral surface. Patients with latent herpes simplex infection develop episodes of recurrent oral herpes labialis char- acterized by the occurrence of a cluster of vesicles and shallow ulcers localized to the lateral aspects of the lips. HSV-1 and HSV-2 are also implicated in recur- rent erythema multiforme, Behçet’s syndrome, some oral ulcers, and oral squamous carcinoma (Scully et al. 1991).

8.6 Varicella-zoster virus

Primary infection of VZV (chickenpox) acquired during childhood produces minor lesions throughout the oral cavity, whereas VZV reactivation in adults causes herpes zoster (shingles). Varicella is the initial infection of VZV and is a highly infectious disease transmitted by direct contact with lesions or by inhalation of infective droplets. Oral lesions include vesicles on the lips, and the hard and soft palates (Millar and Troulis 1994; Miller 1996). Both primary and secondary VZV infection can produce gingival lesions (Laskaris 1996; Scully 1996). Following primary infection, VZV remains latent in the dorsal root ganglion cells for possible later reactivation. Her- pes zoster (shingles) develops from reactivation of a varicella infection. It involves the trigeminal nerve and forms ulcerated lesions with prominent red borders, resembling aphthous ulcers. Lesions are unilaterally distributed along the infected nerve (Millar and Troulis 1994; Miller 1996).

8.7 Human herpesvirus-6

HHV-6 exhibits tropism for CD4+ T lymphocytes (Ablashi et al. 1992), oral epithelium, and gingival or sulcular epithelium in periodontitis. It has also been reported to infect a wide variety of cell types (Ablashi

184 Endodontic Microbiology

et al. 1992). HHV-6 infects ductal epithelium of salivary glands and is isolated from the saliva of most individuals (Yadav et al. 1997). HHV-6 infects mainly T lymphocytes and occasionally B lympho- cytes (Lusso et al. 1988). As EBV has been reported to be prevalent in periapical pathosis, molecular inter- action between HHV-6 and EBV may be important in the pathogenesis of periapical infection. HHV-6 is fre- quently detected and reactivated in epithelial tumors of the oral cavity (Shanavas et al. 1992; Yadav et al. 1994). HHV-6 may be involved in oral squamous car- cinoma (Yadav et al. 1997). Yadav et al. (1997) stud- ied 51 squamous cell carcinomas, 18 non-malignant lesions, and 7 normal mucosa samples. HHV-6 was detected in 79% of malignancies, in 67% of lichen planus lesions and leukoplakia, but was absent in nor- mal mucosa. HHV-6 variant B was detected in 60% of the squamous carcinoma lesions.

HHV-6 can be implicated in the pathogenesis of infectious mononucleosis, pneumonia, meningitis, and encephalitis. It has been implicated as a cofactor of accelerated immunosuppression in HIV-infected individuals. It can proliferate CD4+ and CD8+ lym- phocytes and natural killer cells, thereby increas- ing the severity of HIV infection (Yoshikawa et al. 1992). HHV-6 may be the cause of multiple sclerosis (Soldan et al. 1997). HHV-6 is present in the nuclei of brain oligodendrocytes associated with multiple scle- rosis plaques (Challoner et al. 1995).

8.8 Human herpesvirus-7 and -8

Salivary glands are a major site for a persistent and pro- ductive infection by HHV-7 (Sada et al. 1996). HHV-7 infection occurs primarily in early childhood (Wyatt et al. 1991; Clark et al. 1993), and infectious virus is readily isolated from saliva (Hidaka et al. 1993). Studies have revealed HHV-7 and HHV-6 are beta her- pesviruses that are closely related and exhibit serologic cross-reactivity with each other (Levy 1997). HHV-7 infection usually occurs in childhood (Wyatt et al. 1991), and most adults are HHV-7 seropositive. HHV-7 is found in saliva (Hidaka et al. 1993), which presents the major mode of transmission, and is secreted for many years following the initial infection (Takahashi et al. 1997). Minor labial salivary glands often har- bor HHV-7 and may sometimes be the site of viral replication (Kimberlin 1998). In a study of more than 100 specimens from major salivary glands, Sada et al.

(1996) detected HHV-7 in 100% of submandibular, in 85% of parotid, and in 59% of minor lip salivary gland samples. HHV-7 has also been detected in periodontal pocket and gingival biopsy samples (Contreras et al. 2000).

HHV-8 is believed to be associated with develop- ment of Kaposi’s sarcoma. This is the most commonly encountered angiosarcoma within the oral cavity. It is associated with HIV and is rare in the absence of HIV infection. HHV-8 DNA has also been identified in AIDS-related oral Kaposi’s sarcoma (Flaitz et al. 1997) and in non-Hodgkin’s lymphoma, Castleman’s disease, and antiimmunoblastic lymphadenopathy (Luppi et al. 1994; Moore and Chang 1995; Kemeny et al. 1997). HHV-8 has also been detected in periodontal pocket and gingival biopsy samples (Contreras et al. 2000). HHV-8 is present in 25% of the adult US population and in about 8% of children (Lennette et al. 1996). Kaposi’s sarcoma is a unique form of angiosarcoma that occurs mainly in elderly men of Mediterranean, Eastern European, or Middle Eastern descent and in HIV-positive patients. It has a predilection for the palate. HIV-associated Kaposi’s sarcoma has become relatively prevalent and commonly seen on the skin and within the oral cavity in 60% of patients and may later progress to extraoral sites (Flaitz et al. 1997). The disease can also occur with dermal bullous pemphigoid in HIV- negative immunosuppressed patients (Gaspari et al. 1997). Immunosuppression serves to activate a latent HHV-8 infection in Kaposi’s sarcoma. It may become symptomatic in 25% of patients (Di Alberti et al. 1996).

8.9 Association between herpesviruses and apical disease

Several studies have investigated the occurrence of herpesviruses in periapical lesions (Slots 2002; Sabeti et al. 2003a,b,c; Sabeti and Slots 2004; Yildirim et al. 2006; Andric et al. 2007; Saboia-Dantas et al. 2007; Sunde et al. 2008; Li et al. 2009; Hernadi et al. 2010, 2012; Ozbek et al. 2013). cDNA identifica- tion of genes transcribed late during the infectious cycle of herpesviruses was used to indicate herpesvirus active infection (Sabeti et al. 2003a,b,c). The findings obtained from various studies with different samples of patients revealed a strong association of HCMV and EBV with symptomatic periapical lesions (Table 8.2).

Ta b le

8 .2

C yt

o m

eg al

o vi

ru s

(C M

V ) an

d Ep

st ei

n –B

ar r

vi ru

s (E

B V

) ac

ti ve

in fe

ct io

n in

p er

ia p ic

al p at

h o si

St u dy

To ta

l n

o .

o f

p er

ia p

ic al

le si

o n

s (s

it es

) st

u d

ie d

Sy m

p to

m at

ic le

si o n s;

n o

. (%

) in

fe ct

ed a

A sy

m p to

m at

ic le

si o n s

(s it es

); n

o .

(% )

in fe

ct ed

La rg

e si

ze le

si o

n s

(5 ×

7 m

m o

r la

rg er

); n

o .

(% )

in fe

ct ed

Sm al

l si

ze le

si o

n s

(s it

es );

n o

. (%

) in

fe ct

Sa b

et i

et al

. (2

0 0 3 )

1 4

le si

o n

s, 2

h ea

lt hy

p er

ia p

ic al

si te

s n =

1 3

C M

V +

/E B

V −

: 5

(3 8 %

) EB

V +

/C M

V −

: 0

C M

V +

/E B

V +

: 8

(6 2 %

) C

M V −

/E B

V :

n =

1 C

M V +

/E B

V −

: 1

(3 3 %

) EB

V +

/C M

V −

: 0

C M

V +

/E B

V +

: 0

C M

V −

/E B

V −

: 2

(6 6 %

)

C M

V +

/E B

V −

: 1

(1 4

% )

EB V +

/C M

V −

: 0

C M

V +

/E B

V +

: 6

(8 6

% )

C M

V −

/E B

V : 0

C M

V +

/E B

V −

: 4

(4 4 %

) EB

V +

/C M

V −

: 0

C M

V +

/E B

V +

: 2

(2 2 %

) C

M V −

/E B

V −

: 3

(3 3 %

)

Sa b

et i

et al

. (2

0 0 3 )

5 le

si o

n s

w it

h ca

lc ifi

ed n

ec ro

ti c

p u

lp C

M V +

/E B

V +

: 5

(1 0 0 %

) N

o t d o n e

C M

V +

/E B

V +

: 5

(1 0 0 %

) N

o t d o n e

Sa b

et i

et al

. (2

0 0 3 )

1 4

le si

o n s

n =

7 a

C M

V +

/E B

V −

: 1

(1 4 %

) EB

V +

/C M

V −

: 0

C M

V +

/E B

V +

: 6

( 8 6 %

) C

M V −

/E B

V −

: 0

n =

7 C

M V +

/E B

V −

: 0

EB V +

/C M

V −

: 0

C M

V +

/E B

V +

: 1

(1 4 %

) C

M V −

/E B

V −

: 6

(8 6 %

)

C M

V +

/E B

V −

: 0

EB V +

/C M

V −

: 0

C M

V +

/E B

V +

: 7

(5 8

% )

C M

V −

/E B

V −

: 5

(4 2

% )

C M

V +

/E B

V −

: 1

(5 0 %

) EB

V +

/C M

V −

: 0

C M

V +

/E B

V +

: 0

C M

V −

/E B

V −

: 1

(5 0 %

)

Sl o ts

et al

. (2

0 0 4 )

4 4

le si

o n s

n =

2 5 a

EB V +

: 7 5 5

C M

V +

: 1 0 0 %

EB V +

/C M

V +

: 7 6 %

n =

1 9

EB V +

: 3 7 %

C M

V +

: 2 6 %

EB V +

/C M

V +

: 2 5 %

Sa b

et i

an d

Sl o

ts (2

0 0 4 )

3 4

le si

o n s

n =

2 3 a

C M

V +

/E B

V −

: 6

(2 6 %

) EB

V +

/C M

V −

: 1

(4 %

) C

M V +

/E B

V +

: 1 6

(7 0 %

) C

M V −

/E B

V −

: 0

n =

2 3

C M

V +

/E B

V −

: 1

(9 %

) EB

V +

/C M

V −

: 0

C M

V +

/E B

V +

: 4

(3 6 %

) C

M V −

/E B

V −

: 6

(5 5 %

)

C M

V +

/E B

V −

: 3

(1 3

% )

EB V +

/C M

V −

: 0

C M

V +

/E B

V +

: 1

9 (7

9 %

) C

M V −

/E B

V −

: 2

(8 %

)

C M

V +

/E B

V −

: 4

(4 0 %

) EB

V +

/C M

V −

: 1

(1 0 %

) C

M V +

/E B

V +

: 1

(1 0 %

) C

M V −

/E B

V −

: 4

(4 0 %

)

Y il

d ri

m et

al .

(2 0 0 6 )

1 2

le si

o n s

n =

1 2

C M

V +

/E B

V −

: 5

8 %

/0 EB

V +

/C M

V −

: 6

7 %

/0 C

M V +

/E B

V +

: 3 3 %

C M

V −

/E B

V −

: 0

A n

d ri

c et

al .

(2 0 0 7 )

3 3

p er

ia p

ic al

cy st

s n =

1 6

C M

V +

/E B

V −

: 1 6 /0

C M

V +

/E B

V −

: 1 7

(6 1 .1

% )/ 0

Sa b

o ia

-D an

ta s

et al

. (2

0 0

7 )

2 6

H IV

se ro

n eg

at iv

e gr

an u lo

m at

o u s

(n =

2 2 ) cy

st s

(n =

4 )

G ra

n u lo

m a

C M

V +

/E B

V −

: 1 8 .7

5 %

EB V +

/C M

V −

: 4 3 .7

5 %

EB V +

/C M

V +

: 0

Ya zd

i et

al .

(2 0 0 8 )

5 0

le si

o n s

n =

2 8

C M

V +

/E B

V −

: 5 3 .6

% EB

V +

/C M

V −

: 3 .6

% C

M V +

/E B

V +

: 0

C M

V −

/E B

V −

: 0

n =

2 2

C M

V +

/E B

V −

: 2 2 .7

% EB

V +

/C M

V −

: 0

C M

V +

/E B

V +

: 4 .5

% C

M V −

/E B

V −

(c o n ti n u ed

)

Ta b le

8 .2

(C o n ti n u ed

)

St u dy

To ta

l n

o .

o f

p er

ia p

ic al

le si

o n

s (s

it es

) st

u d

ie d

Sy m

p to

m at

ic le

si o n s;

n o

. (%

) in

fe ct

ed a

A sy

m p to

m at

ic le

si o n s

(s it es

); n

o .

(% )

in fe

ct ed

La rg

e si

ze le

si o

n s

(5 ×

7 m

m o

r la

rg er

); n

o .

(% ) in

fe ct

ed Sm

al l

si ze

le si

o n

s (s

it es

); n

o .

(% )

in fe

ct ed

Su n

d e

et al

. (2

0 0 8 )

4 0

le si

o n s

n =

1 8

C M

V +

/E B

V −

: 0

EB V +

/C M

V −

: 7 2 %

C M

V +

/E B

V +

: 0

C M

V −

/E B

V −

: 0

n =

2 2

C M

V +

/E B

V −

: 0

EB V +

/C M

V −

: 2 8 %

C M

V +

/E B

V +

: 0

C M

V −

/E B

V −

: 0

Sa b

et i et

al .

(2 0 0 9 )

1 5

le si

o n s

n =

1 5

C M

V +

/E B

V −

: 6 7 %

EB V +

/C M

V −

: 0

C M

V +

/E B

V +

: 0

C M

V −

/E B

V −

Li et

al . (2

0 0

9 )

5 3

le si

o n

s %

n =

3 2

C M

V D

N A

/m R

N A +

: 1 5 .7

/2 7 .5

% )

EB V

D N

A /m

R N

A +

: 4 3 .1

% /2

1 .6

n =

2 1

C M

V D

N A

/m R

N A +

: 1 6 .1

% /3

2 .3

% EB

V D

N A

/m R

N A +

: 4 5 .2

% /3

2 .3

C M

V D

N A

/m R

N A +

EB V +

: 1 3 .9

% /3

3 .3

% EB

V D

N A

/m R

N A +

: 5 0 %

/3 6 .1

% C M

V D

N A

/m R

N A +

: 0 %

/2 9 .4

% EB

V D

N A

/m R

N A +

: 4 7 .1

% /3

5 .3

H er

n ad

i et

al .

(2 0 1 0 )

4 0

le si

o n s

n =

1 7

C M

V D

N A

/m R

N A +

: 6 /0

% )

EB V

D N

A /m

R N

A +

: 8 2 %

/7 1

n =

1 3

C M

V D

N A

/m R

N A +

: 1

3 %

/0 %

) EB

V D

N A

/m R

N A +

: 6 5 %

/3 5 %

C M

V D

N A

/m R

N A +

: 1

4 %

/0 %

) EB

V D

N A

/m R

N A +

: 9 1 %

/7 6 %

C M

V D

N A

/m R

N A +

: 0 %

/0 %

) EB

V D

N A

/m R

N A +

: 5 3 .3

% /2

1 %

Sa b

et i et

al .

(2 0 1 2 )

1 5

le si

o n s

n =

9 C

M V +

/E B

V −

: 5 5 .3

% EB

V +

/C M

V −

: 8 8 .9

% C

M V +

/E B

V +

: 0

C M

V −

/E B

V −

: 0

n =

6 C

M V +

/E B

V −

: 0 %

EB V +

/C M

V −

: 0 %

C M

V +

/E B

V +

: 0

C M

V −

/E B

V −

: 0

H er

n ad

i et

al .

(2 0 1 2 )

5 8

le si

o n s

n =

2 8

C M

V +

/E B

V −

: 1 4 .3

% EB

V +

/C M

V −

: 8 9 .3

% C

M V +

/E B

V +

: 0

C M

V −

/E B

V −

n =

3 0

C M

V +

/E B

V −

: 1 0 %

EB V +

/C M

V −

: 6 3 .3

% C

M V +

/E B

V +

: 0

C M

V −

/E B

V −

n =

3 1

C M

V +

/E B

V −

: 1 6 .1

% EB

V +

/C M

V −

: 9 3 .5

% C

M V +

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: 0

n =

2 7

C M

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V −

: 1 5 %

EB V +

/C M

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: 7 .4

% C

M V +

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: 0

V er

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al .

(2 0 1 3 )

3 3

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: 1 5 %

EB V +

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: 7 0 %

C M

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: 1 5 %

C M

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: 0

n =

1 3

C M

V +

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: 0

EB V +

/C M

V −

: 3 8 .5

% C

M V +

/E B

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: 0

C M

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/E B

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et al

. (2

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2 8

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% EB

V +

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: 1 8 .7

% C

M V +

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C M

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1 2

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EB V +

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V −

: 8 .3

% C

M V +

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V +

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Sy m

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Viruses in Endodontic Pathosis 187

Herpes simplex virus infection demonstrated no relationship to periapical disease. Periapical lesions harboring HCMV–EBV dual infection tended to show elevated occurrence of anaerobic bacteria, be symp- tomatic, and exhibit large-sized radiographic bone destruction (Sabeti and Slots 2004).

HCMV and EBV in cooperation with specific bac- terial species have also been associated with various types of advanced marginal periodontitis (Slots 2002) and several nonoral infectious diseases (Slots et al. 2002; Brogden and Guthmiller 2003). Most anaer- obic bacteria were isolated from periapical lesions that showed HCMV–EBV dual infection, were symp- tomatic, or were large (Sabeti and Slots 2004). In some older studies, P. gingivalis and P. endodon- talis were recovered only from symptomatic peri- apical lesions, supporting the notion that this group of organisms is capable of inducing acute endodon- tic infection (Sundqvist 1976). However, most of the symptomatic periapical lesions studied failed to yield black-pigmented anaerobic rods. Acute exacerbation of periapical disease may be caused by unique con- stellations of pathogenic bacteria or, alternatively, may result from a combination of herpesviral and bacte- rial causes. The latter possibility is consistent with the observed uniform presence of active herpesvirus infections in most symptomatic periapical lesions and the proinflammatory potential of herpesviruses (Mogensen and Paludan 2001). Herpesviruses pos- sess several virulence factors of potential impor- tance for periapical pathosis, including the ability to induce immune impairment (Michelson 1999; Boeckh and Nichols 2003), and subsequent overgrowth of pathogenic microorganisms (Kimberlin 1998). In peri- odontitis, presence of subgingival HCMV or EBV is related to elevated bacterial load and occurrence of the periodontal pathogens P. gingivalis, T. forsythen- sis, D. pneumosintes, P. intermedia, P. nigrescens, T. denticola, and A. actinomycetemcomitans (Slots 2002, 2005). Herpesviruses seem also to cooperate with pathogenic bacteria in producing a variety of medical diseases, including inflammatory bowel disease, ente- rocolitis, esophagitis, pulmonary infections, sinusi- tis, acute otitis media, dermal abscesses, and pelvic inflammatory disease (Brogden and Guthmiller 2003). Additionally, herpesviruses may give rise to periapical pathosis by inducing cytokine and chemokine release from inflammatory and noninflammatory host cells (Mogensen and Paludan 2001). Periapical sites hav- ing inadequate antiviral immune response may be

particularly prone to tissue breakdown (Sabeti et al. 2003a,b,c). Viruses that infect mammals, other than HCMV and EBV, alone or in cooperation with her- pesviruses, may also have a role in the pathogenesis of pulpal and periapical pathosis (Elkins et al. 1994; Sigurdsson and Jacoway 1995).

The presence of cytomegalovirus in symptomatic periapical pathosis is consistent with the notion that inflammatory cells are the source of the virus. Indeed, latent cytomegalovirus resides in various myeloid progenitor cell types and in more differentiated hematopoietic cell lineages, and cytomegalovirus translocation in the body occurs in monocyte– macrophages and dendritic cells (Mocarski et al. 2007). Also, cytomegalovirus in marginal periodonti- tis lesions exists in macrophages and T lymphocytes (Contreras et al. 1999). CD178, or the Fas ligand, is a member of the tumor necrosis factor (ligand) superfamily. The Fas/Fas ligand system, which is an important cellular pathway mediating apoptosis (Chaudhuri et al. 1999), can potentially eliminate cytomegalovirus-infected cells (Liles et al. 1996). CD178 was expressed in both cytomegalovirus- infected and non-infected cells; however, the mag- nitude of expression of CD178 and cytomegalovirus tended to be inversely related. Sabeti et al. (2012) detected 47% of the lesions studied had a high CD178 expression level and low or barely detectable cytomegalovirus expression, and 33% of the lesions demonstrated a low level of CD178 expression and a high level of cytomegalovirus expression. Sabeti et al. (2012) clearly demonstrated the significance of active HCMV and EBV infection (high RNA expression) in key cytokines production. In this context, the compar- ative analysis exhibited a parallel marked increased in the cytokines and HCMV and EBV expression in symptomatic periapical lesions when compared with asymptomatic lesions. In fact, our findings are in agreement with other current studies that demonstrate there are various but not specific bacterial types, which indiscriminately were actively present in normal, asymptomatic, as well as symptomatic periapical lesions. On this basis, bacterial infection may serve as cofactor in lymphoid transvascular migration, and cytokine expression be involved in the pathogenesis of periapical lesions. IL-12 promotes Th1 population and suppresses Th2 cell type. Therefore, continuous production of IL-12 may have a role in the chronic inflammatory process. In parallel, HCMV and EBV have shown to exert antiapoptotic activity.

188 Endodontic Microbiology

It may be that a periapical cytomegalovirus infec- tion can inhibit CD178 expression that is involved in the regulation of apoptosis. The inhibition of apop- tosis may further result in continuous inflammation and cytokine production and the establishment of a chronic inflammatory stage. The effect of which may be a failure of the host to control or eliminate the viral infection, and subsequently the bacterial infection.

In conclusion, the present flow cytometric analysis and previous histopathologic and poly- merase chain reaction-based findings have iden- tified cytomegalovirus as a frequent inhabitant of symptomatic periapical lesions. As an active cytomegalovirus infection induces a multiplicity of interconnected immune reactions, incorporating cytomegalovirus and other herpesviruses into studies on infectious causes and causal mechanisms of peri- apical pathosis may provide important new insights into the pathogenesis of the disease.

8.10 Pathogenesis of herpesvirus- associated apical disease

Herpesviruses can cause disease as a direct result of viral infection and replication, or because of virally induced impairment of the host defense. Herpesvirus- mediated pathogenicity takes place through several mechanisms, operating alone or in combination, and involving both cellular and humoral host responses (2000 (Table 8.3).

Herpesviruses can cause direct cytopathic effects on periapical fibroblasts, endothelial cells, and bone cells, the results of which are impaired tissue turnover and repair, and ultimately loss of tissue.

HCMV and EBV can infect and alter func- tions of monocytes, macrophages, lymphocytes, and polymorphonuclear leukocytes. Impairment of host defense cells can predispose to overgrowth of

endodontic pathogenic bacteria. Herpesvirus activa- tion can induce significant immunosuppressive and immunomodulatory effects in periapical sites. Her- pesviruses can trigger an array of host responses that include dysregulation of macrophages and lympho- cytes and downregulate the antiviral host immune response (Boeckh and Nichols 2003). Host impair- ment includes silencing of natural killer cells, inhibi- tion of apoptosis, and destruction of components of major histocompatibility complex (MHC) class I and II pathways within macrophages, markedly impairing their principal role in antigen presentation (Michel- son 1999). In addition, HCMV encodes a unique homolog of interleukin-10 (IL-10), a Th2 cytokine that antagonizes Th1 responses, and its immunosuppres- sive properties may help HCMV circumvent detection and destruction by the host immune system (Kotenko et al. 2000). HCMV has also the ability to inhibit the expression of macrophage surface receptors for lipopolysaccharide, which impairs responsiveness to Gram-negative bacterial infections (Hopkins et al. 1996).

Herpesvirus infections elicit proinflammatory cytokine and chemokine release from inflammatory cells. IL-1β and tumor necrosis factor α (TNF-α) are present in significant levels in periapical lesions (Lim et al. 1994; Wang et al. 1997; Kawashima and Stashenko 1999; Márton and Kiss 2000) and prostaglandin E2 (PGE2) concentration is higher in acute than in chronic periapical lesions (McNicholas et al. 1991). These inflammatory mediators, which are most likely produced locally by periapical macrophages (Miyauchi et al. 1996; Lin et al. 2000), are potent bone resorption-stimulating agents (Page et al. 1997; Márton and Kiss 2000). Previous studies have focused on lipopolysaccharide as an inducer of macrophage cytokine production (Page et al. 1997) but HCMV infection may possess higher potential to upregulate IL-1β and TNF-α gene expression in

Table 8.3 Features of cellular and humoral immunity

Item T cells Lymphocytes

Major histocompatibility complex Cytokines

Cellular immunity Th1 CD4+ and CD8+ MHC1 TNF-α, IL-1β, IL-17, prostaglandin E2

Humoral immunity Th2 CD4+ and B cells MHC2 IL-4 and IL-10

Viruses in Endodontic Pathosis 189

monocytes and macrophages. It might be that the relationship of macrophages and their products to periapical pathosis is in part caused by HCMV- mediated cytokine release from periapical macrophages. EBV is a potent polyclonal B- lymphocyte activator, capable of inducing prolifera- tion and differentiation of immunoglobulin secreting cells. Periapical EBV infection may in part be responsible for the frequent occurrence of B cells in periapical lesions (Hernadi et al. 2010). Herpesvirus infections also affect cytokine networks (Mogensen and Paludan 2001). Cytokines and chemokines have important roles in the first line of defense against human herpesvirus infections and contribute significantly to regulation of acquired immune responses. However, by a diverse array of strategies, herpesviruses are able to interfere with cytokine pro- duction or divert potent antiviral cytokine responses, which allow the viruses to survive throughout the lifetime of the host (Alcami and Koszinowski 2000; Tortorella et al. 2000). HCMV infection typically induces a proinflammatory cytokine profile, with production of IL-1β, IL-6, IL-12, TNF-α, interferon (IFN-α/β), IFN-γ (Mogensen and Paludan 2001), and PGE2 (Mocarski 2002). EBV infection stimulates the production of IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-6, IL-8, IL-18, TNF-α, IFN-α/β, IFN-γ, monokine induced by IFN-γ (MIG), IFN-γ-inducible protein 10 (IP-10), and granulocyte–macrophage colony-stimulating factor (GM-CSF) (Mogensen and Paludan 2001). Proinflammatory activities normally serve a positive biologic goal by aiming to overcome infection or invasion by infectious agents, but can also exert detrimental effects when a challenge becomes overwhelming or with a chronic pathophysiologic stimulus. In an effort to counteract ongoing inflammation, the initial proinflammatory response triggers the release of antiinflammatory mediators, such as transforming growth factor-β and IL-1 (Haveman et al. 1999). Also, viruses display great uniqueness when it comes to diverting the potent antiviral cytokine responses to their benefit (Tortorella et al. 2000). PGE2, which is a key mediator of the periapical inflammatory response (Márton and Kiss 2000), increases rapidly in response to exposure of cells to HCMV, bacterial lipopolysac- charide, and the cytokines IL-1β and TNF-α (Sabeti et al. 2003a,b,c), and PGE2 may under certain circumstances support HCMV replication (Takayama et al. 1996). Undoubtedly, a periapical HCMV

infection can induce a multiplicity of interconnected immunomodulatory reactions, and various stages of the infection may display different levels of specific inflammatory cells and mediators, underscoring the complexity of HCMV–host interactions in periapical disease.

Herpesviruses can produce periapical tissue injury as result of immunopathologic responses. Th1 cells, which predominate in periapical lesions (Brogden and Guthmiller 2003), are mediators of delayed type hyper- sensitivity (Seymour et al. 1996). HCMV has the potential to induce cell-mediated immunosuppression by downregulating cell surface expression of MHC class I molecules, thereby interfering with cytotoxic T-lymphocyte recognition. EBV can induce prolifer- ation of cytotoxic T lymphocytes, the main purpose of which is to recognize and destroy virally infected cells, but may secondarily also inhibit various aspects of the immune response.

Control of herpesviral replication and prevention of pathosis depend on both innate and adaptive immune mechanisms. Antiviral antibodies can help control infectious virions and cytotoxic T lymphocytes have an important role in limiting the proliferation of herpesvirus-infected cells. The frequent presence of natural killer cells and CD8 (cytotoxic) T lymphocytes in chronic periapical lesions (Kettering and Torabine- jad 1993; Márton and Kiss 2000) is consistent with an antiherpesviral host response. However, while antiher- pesviral immune responses may be able to protect from disease, they are insufficient to eliminate reservoirs of persistent viral gene expression.

8.11 Model for herpesvirus-mediated apical disease

Figure 8.2 describes an infectious disease model for the development of periapical pathosis based on herpesvirus bacteria–host interactive responses. Her- pesvirus infection of periapical sites may be impor- tant in a multistage pathogenesis by altering local host defenses. Initially, bacterial infection or mechanical trauma of the pulp cause inflammatory cells to enter pulpal and periapical tissues. In infected individuals, latent HCMV resides in periodontal macrophages and T lymphocytes and latent EBV in periodontal B lym- phocytes (Contreras et al. 2000). Reactivation of her- pesviruses from latency can occur spontaneously or during periods of impaired host response, resulting

190 Endodontic Microbiology

Bacteria

Pulpitis/Periapical Inflammation

Macrophages with HCMV

& EBV

B-cells with EBV

Immunosuppression

Infection InflammationHerpesvirus Activation

Endodontopathic Property T-cells with

HSV & HCMV

Trauma

Immunosuppression Cytokines/Chemokines

Others

Overgrowth of gram-negative anaerobic species and other microorganisms

Interleukin-1β Tumor necrosis factorα

Prostaglandin E2 Matrix metallo-proteinases

Cytotoxicity

Inflammation Bone resorption

Collagen degradation

Destructive Periapical Disease

Pulpal Necrosis

Fig. 8.2 Herpesviruses in symptomatic endodontic pathosis.

from immunosuppression, infection, physical trauma, hormonal changes, and so on. Herpesviral–bacterial interactions may help explain various clinical charac- teristics of periapical infections. Alteration between prolonged periods of herpesvirus latency interrupted by periods of activation may partly be responsible for intermittant episodes of periapical disease flareup. Frequent reactivation of periapical herpesviruses in some patients may result in rapid disease progres- sion. Absence of herpesviral infection or reactiva- tion and lack of endodontic pathogenic bacteria may explain why some teeth having necrotic pulp can main- tain periapical health or minimal disease for extended

periods of time. Necrotic teeth have a higher incidence of flareup (Genet et al. 1987; Walton and Fouad 1992; Sim 1997). An abscessed tooth with swelling, pain with a large redographic lesion has a higher rate of flareup (Genet et al. 1987; Trope 1991; Walton and Fouad 1992; Imura and Zuolo 1995). An incidence of flareup in a chronic apical abscess with presence of a sinus tract is unlikely (Torabinejad et al. 1988; Walton and Fouad 1992). Sabeti et al. (2003b,c) have proposed that a symptomatic lesion with swelling and pain has a large load of viruses and will likely lead to flareup. In contrast, an asymptomatic lesion ensures that the flareup will not occur.

Viruses in Endodontic Pathosis 191

Perhaps not coincidentally, acute exacerbation of periapical disease may be caused by unique con- stellations of pathogenic bacteria or, alternatively, may result from a combination of herpesviral and bacterial causes. The latter possibility is consistent with the observed uniform presence of active her- pesvirus infections in symptomatic periapical lesions and the proinflammatory potential of herpesviruses (Mogensen and Paludan 2001). Herpesviral activation leads to increased inflammatory mediator responses in macrophages and probably also in resident con- nective tissue cells within the periapical lesion. After reaching a critical viral load, activated macrophages and lymphocytes may trigger a cytokine/chemokine “storm” of IL-1β, TNF-α, IL-6, prostaglandins, inter- ferons, and other multifunctional mediators, which in an enclosed area have the potential to propagate states of pain (Vane et al. 1998; Rittner et al. 2002; Rutkowski and DeLeo 2002; Zhu et al. 2002) and bone resorption (McNicholas et al. 1991; Mogensen and Paludan 2001; Brogden and Guthmiller 2003). Several of the herpesvirus-associated cytokines and chemokines are prominent in periapical lesions (Nair 1997; Wang et al. 1997; Lader and Flanagan 1998; Kawashima and Stashenko 1999; Márton and Kiss 2000; Radics et al. 2003). Herpesvirus-induced immune impair- ment may also cause an upgrowth of resident Gram-negative anaerobic bacteria (Kimberlin 1998) whose lipopolysaccharide can induce cytokine and chemokine release from various mammalian cells and may act synergistically with HCMV in stimulating IL- 1β gene transcription (Wara-Aswapati et al. 2003). Moreover, in a vicious cycle, triggering of cytokine responses may activate latent herpesviruses and in so doing further aggravate periapical disease.

In conclusion, endodontic inflammation can be ini- tiated by a variety of infectious agents and is medi- ated by cellular components, such as neutrophils, macrophages, and lymphocytes, as well as molecu- lar components, including cytokines and chemokines. These responses possess pro- and/or anti-inflammatory properties, with harmful or beneficial effects. Reacti- vation of latent herpesviruses is involved in driving the pathologic process of cases of symptomatic periapi- cal disease. Herpesvirus reactivation and herpesviral– bacterial interactions may help explain various clini- cal characteristics of periapical infections. Alteration between prolonged periods of herpesvirus latency interrupted by periods of activation may partly be responsible for intermittent episodes of exacerbation

of periapical disease. Frequent reactivation of peri- apical herpesviruses in some patients may result in rapid disease progression. Perhaps not coincidentally, herpesvirus-activating factors are also associated with acute endodontic disease (Torabinejad 1994). Detec- tion of herpesvirus DNA in periapical lesions has brought a new dimension to our knowledge of peri- apical infections and calls for the inclusion of her- pesviruses in studies on the pathogenesis of periapical pathosis, and the findings may have future therapeutic relevance.

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Chapter 9 Fungi in Endodontic Infections Bilge Hakan Sen and B. Güniz Baksi

9.1 General characteristics of fungi 9.2 Oral yeasts and carriage 9.3 Oral candidosis 9.4 Virulence factors and pathogenicity

9.4.1 Morphogenesis and morphologic transition

9.4.2 Adherence 9.4.3 Enzymes 9.4.4 Biofilm formation 9.4.5 Phenotypic switching

9.5 Presence and pathogenicity of yeasts in different dental tissues 9.5.1 Yeasts and dental caries 9.5.2 Dentin colonization and infection

by yeasts

9.5.3 Yeast infection of periodontal tissues

9.5.4 Yeasts in root canals 9.5.5 Extraradicular yeast infections

9.6 Antifungal activity of endodontic irrigating solutions and disinfectants 9.6.1 Sodium hypochlorite 9.6.2 Ethylene diamine tetraacetic acid 9.6.3 Chlorhexidine 9.6.4 MTAD 9.6.5 Calcium hydroxide 9.6.6 Other antifungal measures

9.7 Conclusions 9.8 References

9.1 General characteristics of fungi

Unlike bacteria, which are prokaryotes, fungi are eukaryotic organisms. Most fungi are microscopic molds or yeasts. Molds are tangled masses of filaments of cells. Yeasts are typical unicellular fungi. Yeast cells have a cell wall, containing glucan, mannan, and chitin. Inside the cell wall, cell membrane, nucleus, a large vacuole, and membrane-bound organelles (mitochon- dria, endoplasmic reticulum) comprise other parts of a yeast cell.

The body of a fungus consists of tiny filaments called hyphae. Hyphae are tiny tubes filled with cytoplasm and nuclei. A mat of hyphae visible to the unaided eye is a mycelium. Some hyphae are divided by cross- section segments (walls) called septa. The septa have

holes through which cytoplasm and organelles can move from segment to segment. In certain conditions, yeast cells can grow true hyphae or pseudohyphae (see section 9.4.1 for more information).

9.2 Oral yeasts and carriage

Most of the clinically important oral yeasts belong to the genus Candida. Taxonomy of Candida is as fol- lows: kingdom, Fungi; phylum, Ascomycota; subphy- lum, Ascomycotina; class, Ascomycetes; order, Sac- charomycetales; family, Saccharomycetaceae; genus, Candida (Waltimo et al. 2003b).

Among more than 300 cultivated microbial species or types in the oral cavity, there are many Candida

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species. These benign, commensal, or opportunistic species have important roles in the development of oral as well as dental diseases. There are 150–200 species of Candida. Candida albicans is the most pathogenic type among the seven species most commonly found in the oral cavity (C. albicans, C. glabrata, C. tropicalis, C. pseudotropicalis, C. guilliermondii, C. krusei, and C. parapsilosis). Recently, Candida dubliniensis, Candida famata, and Candida lipolytica have been isolated in patients with human immunodeficiency virus (HIV) infection. Although the latter two do not cause oral candidosis, C. dubliniensis is particulary important because it is resistant to antifungal treatment and is associated with severe candidiasic mucositis of the oral cavity, esophagus, and other locations (López-Martinez 2010; Coronado-Castellote and Jiménez-Soriano 2013).

C. albicans is frequently isolated from the human mouth, yet few carriers develop clinical signs or symp- toms of candidosis. The prevalence of the yeasts tends to be lower in healthy individuals than in hospital patients of any kind. Isolation rates of yeasts from healthy mouths in nine different studies range 2–37% compared to 13–76% in nine other studies with hospi- tal patients (Odds 1988).

9.3 Oral candidosis

Oral candidosis is an opportunistic infection asso- ciated with alteration in local and systemic defense mechanisms. Oral candidosis clinically manifests itself in several different forms, which have been classified as pseudomembranous candidosis (thrush), erythematous (atrophic) candidosis, hyperplastic candidosis, and Candida-associated lesions (denture stomatitis, angular stomatitis, median rhomboid glossitis).

C. albicans is the most prevalent causative agent of oral candidosis when the host’s physical and immuno- logic defenses have been undermined. In general terms, the severity and extent of Candida infections tend to increase with the number and severity of predisposing factors. Depressed host defenses (Odds 1988; Heimdahl and Nord 1990), endocrine disorders (Lamey et al. 1988; Darwazeh et al. 1991; Rajendran et al. 2010), mucosal lesions (Wilborn and Montes 1980; Krogh et al. 1987; Muzyka and Glick 1995), ill-fitting dentures (Olsen and Birkeland 1977; Sama- ranayake and MacFarlane 1980; Budtz-Jorgensen et al.

1983; Beighton et al. 1990), poor oral and denture hygiene (Budtz-Jorgensen 1990) are components that markedly increase the host’s susceptibility to oral can- didosis. Nevertheless, it has been proposed that a major increase in the incidence of candidosis over the past two to three decades can be attributed to iatrogenic causes. Use of broad-spectrum antibiotics, corticos- teroids, drugs that induce neutropenia and xerosto- mia, psychoactive drugs, and, particularly, immuno- suppressive agents are established to be responsible for iatrogenic predisposition (Budzt-Jorgensen 1990; Heimdahl and Nord 1990; Oksala 1990; Peterson 1992; Narhi et al. 1993; Scully et al. 1994; Navazesh et al. 1995; Soysa et al. 2008; Rautemaa and Ram- age 2011). As the numbers of patients who receive immunosuppressive therapy tend to increase annually, and because conditions such as AIDS add further to the number of “immunocompromised” hosts, increase in the incidence of oral candidosis is inevitable (Sama- ranayake 1992; Powderly et al. 1993; Egusa et al. 2008).

9.4 Virulence factors and pathogenicity

C. albicans expresses a repertoire of activities that con- tribute to virulence. Total effect of many Candida fac- tors leads to the establishment of infection in a suitably compromised host. Among these factors are the mor- phogenesis of C. albicans yeast cells to a filamentous growth, the production of phospholipases and secreted proteinases, and host cell recognition by cell surface adhesions (Calderone et al. 2000; Mane et al. 2011; Silva et al. 2011).

9.4.1 Morphogenesis and morphologic transition

C. albicans and other types of Candida are aerobic yeasts that can reproduce in anaerobic conditions. This fungus has been demonstrated to grow in a num- ber of morphologic forms such as yeast (blastospore), true hyphae, pseudohyphae, and chlamydospores. The organism can grow in either yeast or hyphal form, or physically intermediate forms such as pseudohy- phae. Yeast cells grow as round (sometimes oval), single cells and through the process of budding give rise to colonies of physically separate cells. In hyphal growth form, an initial germ tube resembling a bud is extended into a long, unconstricted filament within

Fungi in Endodontic Infections 199

which the individual cells are separated by septae. In between these two extremes, the fungus can exhibit a variety of growth forms that are referred to as pseu- dohyphae (Sudbery et al. 2004). Pseudohyphal growth demonstrates elongated cells connected in chains lead- ing to filaments that resemble hyphae but are made up of yeast-like individual cells. The elongation of buds in pseudohyphae can be so extreme that these fila- ments can superficially resemble hyphae. Because of this paradox, the term filamentous is adopted to refer both to pseudohyphae and hyphae in recent Candida literature (Kumamoto and Vinces 2005a).

Chlamydospores represent another functionally dis- tinct cellular form, exhibiting complex combination of cell types. Chlamydospores are round, refractile spores with a thick cell wall. All growth patterns except chlamydospores show interconversion to each form of growth depending on the environmental conditions such as pH, temperature, and nutritional source.

Morphogenesis is believed to be important for vir- ulence and has been the subject of many studies (Kobayashi and Cutler 1998; Brown 2002; Gow et al. 2002; Liu 2002). The potential role of hyphae forma- tion in virulence has been reviewed in detail as well. Although there are contradictory reports regarding this subject, recent studies support the conclusion that the hyphal form is important for virulence. The hyphal tip is the site of apical secretion of enzymes that are able to degrade proteins, lipids, and other cellular com- ponents that further facilitate infiltration into tissues, presumably by liquefying the substrate in front of the advancing cell (Hube and Naglik 2001). The hyphae of pathogenic fungi also exhibit the phenomenon of con- tact sensing, or thigmotropism, which enables them to navigate according to underlying surface topogra- phy and accordingly locate the points of weakened surface integrity, thereby gaining access to vulnerable sites for invasion (Gow et al. 2002). Although there is still no genetic basis to establish a role for yeast– hyphae morphogenesis as a virulence factor for C. albi- cans, the evidence has accumulated that formation of hyphae is one of the primary components of the over- all virulence strategies of C. albicans (Kumamoto and Vinces 2005b). Recently, authors recommend evaluat- ing the impact of morphogenesis at different disease stages such as colonization, penetration, dissemina- tion, invasion, and necrosis (Gow et al. 2002). How- ever, the fundamental question of whether yeast or hyphal forms are more virulent still remains to be answered.

9.4.2 Adherence

Adherence is the initial step in the process of tis- sue colonization and invasion. C. albicans has been shown to be significantly more adhesive than other Candida species, which rarely cause oral mucosal infections (Ray et al. 1984). Factors promoting the extent and strength of adherence to epithelial cells depend on the initial surface properties of both the organism and the substratum involved and can be influenced by several factors. These include factors related to yeast and host cells as well as the envi- ronmental factors affecting adhesion. Adherence of C. albicans to host tissue is considered a crucial event in the pathogenic process, and is a prerequi- site for colonization and subsequent infection of the host. Adherence to host tissue is achieved by combi- nation of specific and nonspecific mechanisms. Spe- cific mechanisms include ligand–receptor interactions, while nonspecific mechanisms include electrostatic forces, aggregation, and cell surface hydrophobicity. It has been advocated that, although nonspecific interac- tions are involved in adherence, their overall contribu- tion is less than that provided by specific mechanisms (Klotz 1994). An extensive body of literature exists on adherence of C. albicans to various oral mucosal tissues (Willis et al. 2000; Blanco et al. 2006), caries lesions (Marchant et al. 2001), periodontal pockets (Urzúa et al. 2008) as well as dental materials such as denture base acrylics and silicone-based resilient liner materials (Nevzatoglu et al. 2007; Kang et al. 2013; Lazarin et al. 2013), resilient denture-lining materials (Yilmaz et al. 2005), resin composite restorative dental materials (Maza et al. 2002; Bürgers et al. 2009), root canal-filling materials and sealers (Senges et al. 2011), denture base materials (Radford et al. 1998, 1999), orthodontic brackets (Gokdal et al. 2002; Saloom et al. 2013; Silva et al. 2013), and dental implants (Bürgers et al. 2010; Li et al. 2012); however, the mechanism of adherence to many cell types and surfaces may have different prospects (Enache et al. 1996; Cotter and Kavanagh 2000).

C. albicans shows a significant ability to adhere to cells, tissues, extracellular matrix, and abiotic sur- faces (Biasoli et al. 2010; Romeo et al. 2011). Numer- ous studies that have compared candidal adhesion to human cells and tissues suggested that growth tem- perature of the isolates is one of the important fac- tors affecting adherence (Hazen et al. 2001; Sama- ranayake et al. 2003). In addition, it was demonstrated

200 Endodontic Microbiology

that collagen type IV, which is the major component of dermal–epidermal junctions, is a target for candidal adhesion (Jordan et al. 2014). Laminin, fibronectin, and vitronection are large extracellular matrix pro- teins found in basement membrane and are respon- sible for morphogenesis, homeostasis, tissue healing, and structural integrity. Pärnänen et al. (2008), Dono- hue et al. (2011), and Santoni et al. (2001) revealed that these proteins increased the adherence of C. albi- cans to macrophages. In a recent study, it was shown that C. albicans and C. dubliniensis adhere to proline- rich peptides (PRPs) which constitute approximately 10% of whole saliva (Levine 2011; Jordan et al. 2014).

It was advocated that any foreign material in the mouth, whether fixed or removable such as prosthe- ses or orthodontic appliances, alter the microbial envi- ronment and provide favorable surfaces for candidal adherence (Hibino et al. 2009). Surface properties such as surface free energy, surface charge, roughness, and hydrophobicity have been shown to influence the adhe- sion of Candida. Candidal adhesion on biomaterial surfaces depends on the structure and composition of biomaterials as well as on the physicochemical prop- erties of the cell surface, including surface charge and hydrophobicity (Pereira-Cenci et al. 2008; Ali et al. 2013). As is apparent from the above data, the mech- anism of adhesion of Candida species is determined by a number of factors related to the fungal cells, host cells, and environmental conditions (Modrzewska and Kurnatowski 2015).

In general, pathogenicity correlates positively with adherence. Therefore, knowledge of the mechanism by which C. albicans attaches itself to such surfaces may aid the development of treatment strategies that inhibit adherence of the fungus (San Millan et al. 1996). Many antifungal agents display an ability to retard adherence (Cotter and Kavanagh 2000; Al-Dwairi et al. 2012). Many other methods of inhibiting adherence, which are nonantimicrobial, have been proposed. Among these methods were the disruption of surface-bound salivary protein, which promotes the adherence of C. albicans to surfaces (Nair and Samaranayake 1996), and monoclonal antibodies directed against extracel- lular matrix proteins collagen types I and IV (Cotter et al. 1998), the use of gelatin fragments for blocking the adherence of C. albicans to extracellular matrix proteins (Lee et al. 1996), and the use of antibodies and sugar amines to reduce the adherence to buccal epithe- lial cells (Collins-Lech et al. 1984; Lee et al. 1996).

The change in hydrophobicity has also been reported to lead to a significant reduction in susceptibility to fungal adherence. A number of agents including the use of polyhexamethylene biguanides and quaternary ammonium compounds decrease the adhesion (Jones 1995; Schep et al. 1995). The removal of calcium by ethylene diamine tetraacetic acid (EDTA) and ethy- lene glycol tetraacetic acid (EGTA) is suggested to decrease the adherence of C. albicans to various extra- cellular matrix proteins (Klotz et al. 1993). Accord- ingly, the antifungal and fungicidal activity of calcium chelating or binding agents (particularly of EDTA) on C. albicans has been clearly demonstrated by Ates et al. (2005). Recent literature provides evidence that the various plant extracts demonstrated antiadherence activities by modifying the hydrophobicity of the cell wall and the characteristics of the pellicle of the yeast cells (Nordin et al. 2013; Tyagi et al. 2013). How- ever, further long-term in vivo research is warranted to support these preliminary findings.

The importance of adherence of C. albicans to host tissue can be illustrated by its ability to adhere to var- ious mucosal surfaces and to withstand forces that may lead to its removal, such as washing action of saliva or body fluids. Moreover, its ability to adhere to a variety of oral surfaces including buccal epithelial cells, teeth, and saliva molecules as well as adher- ence to inert polymers and coaggregation with several species of oral bacteria including Streptococcus gor- donii, S. mutans, S. oralis, S. sanguis, S. salivarus, and Actinomyces species (Richards and Russell 1987; Branting et al. 1989; Jenkinson et al. 1990; Holmes et al. 1995; Millsap et al. 1998) makes its clearance a complex multifactorial process.

9.4.3 Enzymes

The penetration of the surface epithelial cell by the candidal hyphae is probably brought about by an enzy- matic process in combination with mechanical forces. The secreted aspartyl proteinases (SAPs) degrade many human proteins at lesion sites, such as albu- min, hemoglobin, keratin, and secretory IgA (Hube et al. 1998). To date, nine SAP genes have been iden- tified in C. albicans. The proteolytic activity of SAPs has been associated with tissue invasion (Yang 2003; Meiller et al. 2009). Miranda et al. (2015) investigated the differential patterns of SAPs among C. albicans strains isolated from root canals or lingual dorsum. The endodontic strains demonstrated a significantly

Fungi in Endodontic Infections 201

higher proteinase production and a distinct pattern of SAP expression. Therefore, it was concluded that C. albicans might have a pathogenic role in endodontic infections.

Other than SAPs, the proteolytic enzymes include collagenase, glucosaminidases, acid and alkaline phos- phatases, aminopeptidases, hyaluronidase, and chon- droitin sulfatase, which act on the degradation of extracellular matrix proteins (Scully et al. 1994; Calderone and Fonzi 2001). Salivary proteins, includ- ing immunoglobulin A (IgA), can be degraded by acidic proteinases of Candida, particularly at low pH conditions (Samaranayake et al. 1994). It has been shown that a collagenolytic enzyme produced by C. albicans can digest the human dentine collagen (Kaminishi et al. 1986; Hagihara et al. 1988).

It has also been shown that phospholipases are con- centrated at the tips of fungal hyphae and localized in the vicinity of host cellular compartments where active invasion is occurring (Pugh and Cawson 1977; Ghannoum 2000). These enzyme activities were found in most C. albicans strains, but not in other less viru- lent Candida species (Samaranayake et al. 1984), and cause membrane damage to the host cells resulting in cell lysis (Ghannoum 2000).

9.4.3.1 Evasion

In order to maintain Candida populations in the oral cavity, cells must grow and multiply at a rate at least equal to that of clearance. Cannon et al. (1995a) suggested that a major factor influencing the bal- ance among clearance, colonization, and candidosis is the interaction between C. albicans cells and host defenses. Immune system defects are a major risk fac- tor for candidosis. The presence of Candida species and the candidal overgrowth in the oral cavities of medically compromised patients have been demon- strated in many longitudinal studies (Arendorf and Walker 1979; Hauman et al. 1993; Swerdloff et al. 1993; Grimoud et al. 2003; Golecka et al. 2006; Li et al. 2006). C. albicans can evade host defenses as a result of multiple mechanisms (Luo et al. 2013). Innate primary defense mechanisms have a key role in preventing yeast colonization of the oral cavity. Pri- mary innate defenses include the epithelial barrier and anticandidal compounds of saliva such as lysozyme (Tobgi et al. 1988), histatins (Xu et al. 1991), lacto- ferrin (Nikawa et al. 1993), and calprotectin (Chal- lacombe 1994). The major immunoglobulin in saliva

is secretory IgA (SIgA), which aggregates yeasts and assists in clearance (Scully et al. 1994).

9.4.4 Biofilm formation

Biofilms are structured microbial communities that are attached to a surface. Microorganisms in biofilms are embedded within a matrix of extracellular poly- mers, and characteristically display a phenotype that is markedly different from planktonic cells (Douglas 2003). The first example of a biofilm to be recog- nized in medical systems was dental plaque on tooth surfaces. But, according to the estimates of National Institutes of Health (NIH) and the Centers for Dis- ease Control and Prevention (CDC), more than 60% of microbial infections involve biofilms (Lewis 2001; Wolcott and Ehrlich 2008). These three-dimensional structures are frequently composed of yeast and hyphal cells embedded in an extracellular matrix. Biofilms constitute an important challenge in the management of disseminated Candida infections because of their intrinsic resistance to almost all antifungals in clinical use. Candida biofilms are especially resistant to azoles and amphotericin B, but remain sensitive to the newly introduced echinocandins that target cell wall β-glucan biosynthesis (d’Enfert 2006).

Biofilm infections can be caused by a single micro- bial species or by a mixture of bacterial or fungal species (Costerton 1999). C. albicans has the ability to form biofilms on different surfaces, which is proposed to be one of the major reasons for its increased pathogenicity (Haynes 2001; Wady et al. 2012; Villard et al. 2015). Furthermore, the phenomenon of coaggregation and coadhesion between Candida and different bacteria and the effect of modulating factors such as saliva, sugars, and pH enhance the biofilm formation and colonization of oral mucosal and dental tissues (Jenkinson et al. 1990; Grimaudo et al. 1996; Ganguly and Mitchell 2011). Ning et al. (2013) inves- tigated the biofilm formation capacity of C. albicans on different surfaces, in different growth phases and under anaerobic conditions. One of the important findings of this study was that C. albicans survived and formed biofilms in anaerobic and nutrient-limited environments. However, the cells in starvation phase showed significantly lower biofilm formation than the cells in exponential or stationary phases. In relation to the substratum used for biofilm formation, C. albicans demonstrated more abundant growth on human dentine than on polystyrene and glass slides. It was concluded

202 Endodontic Microbiology

that resistance of C. albicans to growth conditions and survival and biofim formation capacity might explain the high prevalence of C. albicans in root canals with persistent periapical infections.

Microbial biofilms are resistant to a variety of antimicrobial agents, including antibiotics, antisep- tics, and industrial biocides. For example, when fungi exist in the biofilm form, they are five to eight times more resistant to clinically important antifungal agents such as amphotericin B, fluconazole, flucytosine, itra- conazole, and ketaconazole than are planktonic cells (Hawser and Douglas 1995). The mechanisms of biofilm resistance to antimicrobial agents have not been fully understood, but several mechanisms have been suggested:

1. Restricted penetration of drugs through the biofilm matrix;

2. Phenotypic changes resulting from a decreased growth rate or nutritional limitation; and

3. Expression of resistance genes induced by contact with a surface (Mah and O’Toole 2001; Donlan and Costerton 2002).

Bacteria are often found with Candida species in polymicrobial biofilms in vivo, and extensive interac- tions are demonstrated in these adherent populations (Douglas 2003). It has been reported that Candida resistance to fluconazole was enhanced in the pres- ence of slime-producing staphylococci, but unaffected by the presence of a slime-negative mutant (Douglas 2003).

9.4.5 Phenotypic switching

C. albicans is a very adaptable microorganism with the ability to survive in diverse and distinct anatomic sites. Micromorphologic and physiologic properties of C. albicans are rapidly modified in response to dif- ferent growth conditions and environmental changes (Kennedy and Sandin 1988; Rams and Slots 1991; Soll 1992). Accordingly, pathogenicity may be increased after adaptation to the environment and phenotypic switching (Soll 2014).

Many studies including healthy individuals have demonstrated strain specialization for particular anatomic sites (Soll et al. 1991; Hellstein et al. 1993; Kam and Xu 2002). However, many studies have indi- cated the emergence of new, highly successful oral strains in particular geographic areas (Tamura et al. 2001). In two consecutive studies, Hannula et al.

(1997, 2001) revealed no difference in distribution of oral yeast species and of C. albicans phenotypes and genotypes between Finnish, US, and Turkish subjects obtained from the oral samples (periodontal pocket, oral mucosa, saliva). In an earlier study, Odds et al. (1983) also reported similar findings and found no significant differences among C. albicans phenotypes from different anatomic sources. However, in the same study they had found some differences among the phe- notypes of strains from the different geographic areas. From these and other studies emerged an interest in the phenotypic variability among strains and the devel- opmental capacity for phenotypic variability within strains, which includes the capacity to differentiate between the yeast and hyphal forms, and the capac- ity to switch frequently and reversibly between gen- eral phenotypes that can be distinguished by colony morphology (Soll et al. 1994). Other than the differ- ences in colony morphology, the general characteris- tics of switching in different strains were similar (Soll 1992; Soll et al. 1994) and included: (i) high- and low-frequency modes of spontaneous switching; (ii) a basic original smooth phenotype; (iii) reversibility and interconvertibility between phenotypes; (iv) a limited number of predominant phenotypes; and (v) stimula- tion by low doses of ultraviolet irradiation.

Switching is associated with changes in micromor- phology and physiologic properties as well as a number of putative virulence traits. It therefore seems reason- able to suggest that switching may provide C. albicans and related infectious yeasts with the diversity that is expected of such pervasive and successful pathogens. It has been proven that switching can provide an organ- ism with the capacity to invade diverse body locations, evade the immune system, and/or change antifungal resistance (Slutsky et al. 1985). Switching has been shown to occur not only in standard laboratory strains, but also in strains of C. albicans isolated from mouths of healthy patients. However, the fact that the genetic heterogeneity of strains increases with periodontal dis- ease (Song et al. 2005) and HIV infection (Pizzo et al. 2002, 2005) was clearly demonstrated in several stud- ies. One study evaluated the C. albicans phenotypes and genotypes from infected root canals reported broad spectrum of heterogeneity of the strains, which was quite similar to previous reports from other oral and nonoral sources. Furthermore, the data implied that unusual strains of C. albicans are not involved in root canal infections. However, the systemic condi- tions of these patients were not described in this study (Waltimo et al. 2001).

Fungi in Endodontic Infections 203

9.5 Presence and pathogenicity of yeasts in different dental tissues

9.5.1 Yeasts and dental caries

Dental plaque has a diverse microbial population (Marsh 2004). Streptococci and lactobacilli have been generally regarded as important microorganisms of dental plaque (Loesche 1986), while Candida is believed to be present temporarily as harmless sapro- phytic microorganisms. In addition to new microe- cologic concepts of dental plaque and caries research, comprehensive studies on yeasts and their by-products have led to a renewed interest in Candida species and their possible role in the etiopathogenesis of dental caries.

C. albicans is highly acidogenic (Samaranayake et al. 1983, 1986; Odds 1988), aciduric, and aci- dophilic (Odds 1988; Marchant et al. 2001), and may have a cariogenic potential (Nikawa et al. 2003). It demonstrates a high affinity for uncoated (Nikawa et al. 2003) and coated hydroxylapatite (Cannon et al. 1995b; Nikawa et al. 1998), specifically through elec- trostatic interactions, and dissolves hydroxylapatite to a greater extent (approximately 20-fold) when com- pared with S. mutans. In an ex vivo study (Charone et al. 2013), biofilms of C. albicans isolated from oral mucosa and dentin caries of an HIV-infected child were grown on enamel blocks for several time peri- ods and the change in microhardness of enamel was measured. C. albicans from dentin caries decreased enamel microhardness on day 3, while the one from oral mucosa on day 5. The authors have concluded that the control of fungi particularly in HIV-infected children to prevent oral candidiasis and dental caries is very important.

In addition to findings related to enamel, C. albi- cans also adheres to both denatured and intact collagen (Makihira et al. 2002a,b) and possesses collagenolytic activities (Kaminishi et al. 1986; Hagihara et al. 1988; Nishimura et al. 2002). Therefore, it is clear that the effect of Candida on dental hard tissues evolves in two ways. First, it dissolves the inorganic material of dental hard tissues with its acidogenic properties and removes calcium. Second, it attacks the exposed col- lagen and causes dissolution of the organic material with its collagenolytic enzymes. However, the degree to which Candida can contribute to the pathogenesis of caries in vivo remains to be determined.

In a scanning electron-microscopic study, Sen et al. (1997a) have shown that C. albicans was able to

Fig. 9.1 A colony of C. albicans consisting of yeast cells and hyphal extensions on untreated enamel surface. The extracellular material indicating dense cellular activity can be observed in the middle of the colony (original magnification 1000×). Courtesy of Bilge Hakan Sen, Kamran Safavi, and Larz Spangberg.

adhere to normal or EDTA/NaOCl-treated enamel and cementum, demonstrating close adaptation (Fig- ure 9.1). The surfaces were covered with separate but dense colonies. Different types of cell morphology including yeasts and hyphal structures were observed (Figures 9.2 and 9.3). Hyphae showed penetration into cracks and grew over the edges of the cavity in intimate contact. Yeast cells and hyphae were attached to the surfaces by strands of organic material (Figure 9.4). Taken together, with its acidogenic, collagenolytic,

Fig. 9.2 A colony of yeast cells on EDTA/NaOCl-treated cementum. The cells are in the stage of active budding (arrows) (original magnification 1500×). Courtesy of Bilge Hakan Sen, Kamran Safavi, and Larz Spangberg.

204 Endodontic Microbiology

Fig. 9.3 A dense colony of C. albicans with numerous yeast cells on hyphal structures on untreated cementum surface (original magnification 550×). Courtesy of Bilge Hakan Sen, Kamran Safavi, and Larz Spangberg.

adhesive, and plaque-forming properties, C. albicans may contribute to the pathogenesis of caries.

Candida spp. have been frequently isolated from dental plaque in different clinical occasions (Hodson and Craig 1972; Brown et al. 1978, 1979; Beighton and Lynch 1995; de Carvalho et al. 2006; Eliasson et al. 2006). In addition to its presence in dental plaque, Candida has been also isolated from several forms of dental caries. Primary root carious lesions contain a high number of yeasts (Lynch and Beighton 1994; Beighton and Lynch 1995). Marchant et al. (2001) and de Carvalho et al. (2006) have demonstrated that there is significant association between the presence of C. albicans and early childhood caries. Sziegoleit et al. (1999) and Hossain et al. (2003) have also demon- strated that carious dentine has a high concentration of

Candida spp., providing a significant ecologic niche for the dissemination of these yeasts. Moreover, Can- dida spp. have been found to be closely related with postirradiation caries, particularly depending on the xerostomia after radiation therapy (Brown et al. 1978).

Oral presence of fungi in immunocompetent patients may not demonstrate a clinical pathology in most of the instances. However, there are indeed partic- ular circumstances such as cancer or AIDS in which the immune system has been compromised. Damm et al. (1988) and Bunetel and Bonnaure-Mallet (1996) have reported that colonization of carious lesions by Can- dida may be responsible for development and recur- rence of oral candidiasis in patients with cancer. Jacob et al. (1998) observed that Candida was frequently present (77%) in dental caries of HIV-infected people,

Fig. 9.4 Two hyphal extensions showing close attachment to the enamel surface with secreted extracellular mucous material (arrows) (original magnification 5000×). Courtesy of Bilge Hakan Sen, Kamran Safavi, and Larz Spangberg.

Fungi in Endodontic Infections 205

and there was a significant association between clinical oral candidiasis and candidal colonization of carious dentin. Similarly, Domaneschi et al. (2011) demon- strated that Candida colonization in pediatric AIDS patients is directly associated with untreated dental caries in addition to other factors such as frequent use of antibiotics, sulfa drugs, and alteration in the oral mucosa.

9.5.2 Dentin colonization and infection by yeasts

Colonization and penetration of dentin by microorgan- isms is considered an important step for initiation and persistence of root canal infection. There are two main factors for invasion of dentin through dentinal tubules: colonizing species need to compete for space to adhere to the surface and sustain the infection, and for nutri- ents to survive. In addition, growth and survival in dentinal tubules can protect microorganisms from the effects of endodontic procedures such as instrumenta- tion, irrigation, and disinfection.

There are a few in vitro studies showing infection of dentin by Candida. Sen et al. (1997b) investigated the interaction of C. albicans with smear-free root canal walls and the growth patterns of this fungus in relation to radicular dentin in a nutritionally stressed medium. The diameter of dentinal tubules after smear layer removal was 2.0–4.5 μm. While the size of the yeast cells ranged from 2 to 5 μm, the width of grow- ing hyphae was 1.5–2.0 μm and the length extended to 100 μm. The canal walls were covered with yeast

Fig. 9.5 A colony of yeast cells and hyphal structures grown on EDTA/NaOCl-treated radicular dentin. Note the penetration of hyphae (arrows) into dentinal tubules (original magnification 1100×). Courtesy of Bilge Hakan Sen, Kamran Safavi, and Larz Spangberg.

cells and hyphal structures forming dense, but separate, colonies (Figure 9.5). Not only germ tubes, hyphae, and pseudohyphae, but also yeast cells showed evi- dence of penetrating into dentinal tubules (Figure 9.6) and budding in the tubules, representing growth poten- tial and active penetration. It was proposed that this contact-sensing (thigmotropism) ability of Candida supported dentinal invasion.

Fig. 9.6 Yeast cells migrating into the dentinal tubules. There are bud scars (arrows) on some of the cells (original magnification 5500×). Courtesy of Bilge Hakan Sen, Kamran Safavi, and Larz Spangberg.

206 Endodontic Microbiology

Fig. 9.7 A colony of yeast cells and hyphae at the base of a smear-free dentin cavity. Note that part of the dentin is coated by an extracellular material (original magnification 1500×). Courtesy of Bilge Hakan Sen, Kamran Safavi, and Larz Spangberg.

Contact sensing is a well-known property of neu- rons and other cells growing in close contact to sur- faces (Bourett et al. 1987; Clark et al. 1990). This ability is actually a habituation of organisms to grow on the surfaces of solid materials or tissues (Sherwood et al. 1992; Gow et al. 1994). With this invasive affin- ity to dentin, C. albicans has been considered as a dentinophilic microorganism. The emergence of bud- ding cells on previously formed pseudohyphae after 15 days in the nutritionally stressed medium with no addition of any type of sugars suggests that C. albi- cans can use dentin as a source of nutrition (Sen et al. 1997b).

In a parallel study from the same group (Sen et al. 1997a), colonization pattern of C. albicans was investigated in dentin cavities with or without smear layer. When there was no smear layer, a network of yeast cells and branching hyphae with bud clusters were present (Figure 9.7). However, no dense colony was observed at the base of the cavity or on the cavity walls, and dentin was still visible. Few hyphae demonstrated penetration into dentinal tubules. However, in the presence of smear layer, there was a dense mass of yeast cells and hyphae forming a thick biofilm layer at the base and the walls of the cavity (Figures 9.8 and 9.9a). Fungal cells and mycelia were

Fig. 9.8 A dense mass of yeast and hyphae at the base of a smeared dentinal cavity (original magnification 550×). Courtesy of Bilge Hakan Sen, Kamran Safavi, and Larz Spangberg.

Fungi in Endodontic Infections 207

(a) (b)

Fig. 9.9 (a) A dense colony of C. albicans particularly consisting of yeast cells on the smeared cavity wall (original magnification 750×). (b) Note that the yeast cells and hyphae are in close adaptation to the smeared surface (original magnification 1500×). Courtesy of Bilge Hakan Sen, Kamran Safavi, and Larz Spangberg.

in close contact with the smear layer and its particles (Figure 9.9).

In a follow-up study, Sen et al. (2003) developed a reproducible, quantitative model for microbial adhe- sion using a colorimetric method. After preparation of dentin disks, smear layer was either left intact or removed through EDTA/NaOCl irrigation. It was determined that presence of smear layer increased the adhesion of C. albicans to human dentin. Smear layer is composed of organic and inorganic material (Sen et al. 1995b). It has been previously observed that Candida has a specific affinity for dentinal collagen (Kaminishi et al. 1986; Hagihara et al. 1988) and type I collagen significantly increases adhesion of Candida (Makihira et al. 2002a,b). Furthermore, calcium ions control Candida morphogenesis (Holmes et al. 1991) and the adherence potential of C. albicans to various extracellular matrix proteins (Klotz et al. 1993). Therefore, they have hypothesized that the increase in adhesion of C. albicans to smeared dentin is caused by the presence of available resources of exposed collagen and calcium ions as a source of growth and adhesion. EDTA/NaOCl irrigation decreases the organic and the inorganic content of dentin. Hence, C. albicans loses its affinity to this poor substrate and shows less attachment to smear-free dentin. This find- ing is in accordance with the previous observational

scanning electron-microscopic (SEM) study (Sen et al. 1997a).

In an in vitro study by Waltimo et al. (2000a), penetration of C. albicans and Enterococcus fae- calis into dentinal tubules was comparatively inves- tigated through macroscopic and microscopic exam- ination. C. albicans growth was weak and observed in 6 of 12 test systems during the 30-day incubation. Both microorganisms were capable of penetrating the 2 mm thick dentin disks. However, histologic sections showed that continuous penetration of yeast cells and hyphae was limited to 60 μm in a few tubules and sin- gle yeast cells were found in low numbers throughout the dentin specimens. However, E. faecalis penetrated all dentin slices in a short period (1–5 days). While the infection was heavy in some of the tubules, the majority of the tubules were free of bacterial cells. The authors concluded that the difference in pene- tration capacity of both microorganisms was mainly because of the different cell sizes of the species.

Siqueira et al. (2002a) investigated the coloniza- tion pattern of five fungal species—C. albicans, C. glabrata, C. guillermondii, C. parapsilosis, and Sac- charomyces cerevisiae—by SEM. Regardless of the species, main growth form was single or budding yeast cells, but hyphal forms were not observed. While C. albicans colonized most of the specimens, the other

208 Endodontic Microbiology

fungal species showed sparce or no colonization on the radicular dentin. C. albicans demonstrated different patterns of penetration into dentinal tubules. However, the majority of the tubules remained free of fungal cells.

Infection pattern of C. albicans on radicular dentin was similar in these in vitro studies (Sen et al. 1997a,b; Waltimo et al. 2000a; Siqueira et al. 2002a). C. albi- cans demonstrated slight to dense colonization and less or no penetration. However, there are case reports or clinical studies presenting heavy yeast infection of carious and normal dentin (Kinirons 1983; Damm et al. 1988; Jacob et al. 1998). A thick biofilm layer of C. albicans presenting different growth forms was always observed on exposed dentin. Hyphal extensions were detected in almost all dentinal tubules penetrating toward the pulpal space. Yeast cells were also shown in root canals in situ (Eidelman et al. 1978; Sen et al. 1995a). The root canal walls were covered by dense masses of yeast cells and hyphal structures. In addi- tion, dentinal tubules were totally filled with hyphae (Eidelman et al. 1978). It is apparent that there is a dis- tinct difference between in vitro and in vivo infection patterns of C. albicans. The reason for less penetration and colonization in vitro may be the use of irrigation solutions to remove the smear layer during experimen- tal procedures. As stated previously, NaOCl and EDTA (or other acids) render dentin a deficient substrate, depleting available organic and inorganic content of dentin.

To test this hypothesis, the colonization patterns of C. albicans on chemically treated or untreated radicu- lar dentin were investigated by SEM (Turk et al. 2008). The colonization on untreated dentin surfaces was so dense that the yeast colonies could not be identified clearly. A dense mass of yeast cells forming a thick layer of biofilm was observed. However, the coloniza- tion was not extensive on treated dentin specimens and there were separate colonies. Few hyphal struc- tures penetrated into dentinal tubules. The untreated dentin was considered to present available nutrition for Candida because it did not receive any chemical treatment. As observed in this study, Candida has an ability to change its colonization pattern depending on the condition of dentin surfaces. This fact should be considered in the future in vitro studies.

Most of our current knowledge about the colo- nization pattern of Candida originates from research using chemically modified dentin, grown in nutrient- rich media under optimal conditions. Therefore,

extrapolation of the results from such studies (Sen et al. 1997a,b; Waltimo et al. 2000a; Siqueira et al. 2002a) to the in vivo situation may be misleading. Care should be taken when developing microbial models to eval- uate adherence, colonization, and penetration patterns of Candida on root canal dentin.

9.5.3 Yeast infection of periodontal tissues

The interaction between the microorganisms present in plaque and the host’s immunologic response is the cause of periodontal diseases (Kornman et al. 1997; Azuma 2006). Although the cause and effect relation- ship of specific pathogens to periodontal disease is not fully established, some particular bacterial species are reported to be risk factors in periodontitis (Darveau et al. 1997; Ezzo and Cutler 2003). In addition to these bacteria, yeasts have also been isolated in many cases, suggesting a possible role for these microorgan- isms in the pathogenesis of periodontitis (Slots et al. 1988; Rams et al. 1990; Listgarten et al. 1993; Dahlen and Wikstrom 1995; Reynaud et al. 2001). The inci- dence of yeasts in relation with periodontal diseases is reported to be as high as approximately 15–24% in sev- eral studies (Slots et al. 1988; Najzar-Fleger et al. 1992; Reynaud et al. 2001; Järvensiu et al. 2004). However, they are not found to exceed 10% of the total viable count (Dahlen and Wikstrom 1995).

Certain immunocompromising conditions such as HIV infection, cytotoxic treatment, and broad use of antibiotics may cause superinfection of periodontal structures with yeasts (Peterson et al. 1987; Rams et al. 1990; Odden et al. 1994). Presence of yeasts in subgingival areas is a frequent finding in HIV-related periodontitis (62% of the subjects; 55% of the sites) (Zambon et al. 1990). Chattin et al. (1999) compared the presence of microorganisms at the periodontal sites of HIV-positive or HIV-negative subjects. The average cell numbers of C. albicans were significantly higher in samples from the HIV-positive group.

Overgrowth of yeasts in periodontal tissues has been demonstrated after systemic antibiotic therapy. González et al. (1987) found yeast cells invading gin- gival connective tissue in juvenile (currently aggres- sive) periodontitis. Large numbers of yeast cells were observed particularly after treatment with spiramycin. They concluded that antibiotics might favor over- growth of yeasts. Another interesting finding in this study was that the researchers were unable to grow yeasts in Sabouraud agar (which is a specific medium

Fungi in Endodontic Infections 209

for fungi) although they demonstrated their presence by means of SEM. Rams et al. (1990) also reported that systemic doxycycline therapy caused more than 10-fold increase in subgingival numbers of Enterobac- ter aerogenes, Escherichia coli, staphylococci, and C. albicans.

Yeasts can be actively responsible in the pathogen- esis of the tissue breakdown in periodontal diseases. Odden et al. (1994) presented dense neutrophil infil- tration of gingival epithelium and numerous mitoses as a reaction to candidal invasion. Järvensiu et al. (2004) studied the extent of candidal penetration into gingival tissues by immunochemistry and periodic acid–Schiff (PAS) staining. They found that hyphal germination started in the gingival pocket and Candida was typi- cally present in the outer layers of the plaque, acting as a barrier between the host’s immune system and the inner layers of microbial biofilm. Hyphae were also present deep in the connective tissue of periodontium, indicating candidal penetration and attachment.

According to the findings of these studies, it seems reasonable to consider yeasts not only as a frequently occurring microorganism in the periodontal microbial flora, but also as a part of the periodontal disease process.

9.5.4 Yeasts in root canals

9.5.4.1 Primary root canal infections

The infection caused by microorganisms colonizing the necrotic pulpal tissues and root dentin is defined as primary root canal infection (Siqueira 2002). In gen- eral, primary infections are mixed and predominated by facultative or obligate anaerobic bacteria, depend- ing on the microenvironmental changes and stresses as discussed in Chapters 4 and 5.

As fungi were not isolated in initial microbial flora of most root canal infections (Haapasalo 1989; Sundqvist et al. 1989), they were not usually reported to be a common member of the microbial popu- lation isolated from primary endodontic infections (Sundqvist 1994). Even though the yeasts may be present in the original sample, they may not grow on culture plates because they have low colony-forming unit (CFU) numbers compared to bacteria (Peciuliene et al. 2001; Waltimo et al. 2004a). In addition, they may be frequently considered as a contaminant, par- ticularly from the air (Waltimo et al. 2004a). However, there are multiple reports demonstrating presence of

yeasts in infected root canals using either culturing, light and electron microscopy, or molecular techniques (Table 9.1).

The incidence of yeasts cultured from primary endodontic infections varies widely. When a selective media had not been used, the incidence was reported to be as low as 0.5–10%. When the studies using selective medium such as Sabouraud’s agar or broth are reevaluated, the occurrence of yeasts falls in the range of 1.9–61.5%. Najzar-Fleger et al. (1992) clearly demonstrated that the incidence was increased when Sabouraud’s dextrose agar was used for cultivation, instead of nonselective blood agar.

Even though Candida spp., particularly C. albicans, have been isolated in most of the studies (Jackson and Halder 1963; Najzar-Fleger et al. 1992; Baum- gartner et al. 2000; Lana et al. 2001; Egan et al. 2002), presence of other yeasts has also been reported. Saccharomyces cerevisiae was recovered from both the root canal and the patient’s blood samples taken during and after endodontic therapy (Debelian et al. 1995, 1997). Egan et al. (2002) investigated the rel- ative prevalence and diversity of yeasts in saliva and root canals of teeth associated with apical periodonti- tis from the same patients. In addition to C. albicans and C. sake, Rodotorula mucilaginosa was isolated from the root canals. They found that the presence of yeasts in root canals was significantly associated with their presence in saliva. Similar to Egan et al.’s (2002) study, Miranda et al. (2009) determined the diversity and frequency of yeasts from the dorsum of the tongue and root canals with necrotic pulp associated with pri- mary apical periodontitis. After isolation on Saboraud agar, yeasts were characterized by standard methods. Molecular techniques were used to confirm identifi- cation. Seventy-seven yeast colonies (45.8%) origi- nated from the tongue, whereas 38 colonies (22.6%) were isolated from the root canals. The most fre- quently identified species at both sites was C. albi- cans (90.4%). These two studies collected the mate- rial from patients with no significant medical history. However, a question usually arises about the frequency of yeasts in root canals of medically compromised patients. Therefore, the prevalence of Candida species in saliva and root canals of HIV+/AIDS patients in comparison to HIV– patients was evaluated by cul- turing (Chugal et al. 2007). Even though 37% of HIV– patients had Candida species in their saliva, no yeast cells were isolated from their root canals. Con- versely, Candida was found in saliva, tooth surface,

210 Endodontic Microbiology

Table 9.1 Literature relating to prevalence of yeasts isolated from root canals with primary endodontic infections

Authors Number of samples

(or teeth) Method/culture Prevalence of yeasts in root canals (%)

MacDonald et al. (1957) 46 Dextrose broth Thioglycolate broth

2.2

Leavitt et al. (1958) 154 Trypticase soy broth 10

76 Dextrose broth 5

Hobson (1959) 98 Nutrient broth Robertson’s broth

0.6

Jackson and Halder (1963) 214 Sabouraud’s broth 26

Wilson and Hall (1968) 263 Robertson’s meat broth Sabouraud’s broth

1.9

Goldman and Pearson (1969) 563 Trypticase soy broth Blood agar

0.5

Slack (1975) 560 Nutrient broth Robertson’s broth

5.2

Najzar-Fleger et al. (1992) 292 Sabouraud’s agar 55

Sen et al. (1995b) 10 SEM 40

Debelian et al. (1995) 26 Trypticase soy agar 3.8

Baumgartner et al. (2000) 24 PCR 20.8

Lana et al. (2001) 27 Sabouraud’s agar 7.4

Akdeniz et al. (2002) 13 Sabouraud’s broth 61.5

Egan et al. (2002) 35 Sabouraud’s agar 5.7

Siqueira et al. (2002a) 50 PCR 2

Siqueira et al. (2002c) 15 SEM 6.6

Ferrari et al. (2005) 25 Sabouraud’s agar 4

The rows shown in bold indicate the studies in which a selective medium has been used for culturing of yeasts.

and root canals of all HIV+/AIDS patients, indicat- ing that Candida species may have an important role in pathogenesis of endodontic infections in immuno- compromised patients. However, in a recent study, Brito et al. (2012) compared the microbiologic pro- file of root canal samples of HIV+ or HIV– patients by multiple-displacement amplification and checker- board DNA–DNA hybridization. C. albicans was the most predominant yeast in both groups. However, its prevalence was low in HIV+ individuals, in contrast to Chugal et al.’s (2007) study. This difference can be explained by the differences in identification methods or in the cohort studied.

Fungi have also been observed electron- microscopically in root canals associated with primary endodontic infections (Sen et al. 1995a; Siqueira et al. 2002b). Sen et al. (1995a) investigated

the topography of the microbial flora of infected root canals. They observed that the root canals were heavily infected by cocci and rods in six teeth (Figure 9.10). In the other four teeth, there were no bacteria, but yeast colonies were observed throughout the length of the root canals (Figures 9.11 and 9.12).

In addition to culturing and electron-microscopic methods, polymerase chain reaction (PCR) assay has been used for detection of yeasts in root canals. Baum- gartner et al. (2000) used PCR primers specific for the 18 S ribosomal RNA gene of C. albicans and stated that the PCR could detect very low concentrations of DNA of this yeast. The presence of C. albicans was detected in 5 of 24 (20.8%) samples. It was concluded that yeasts might be involved in root canal infections more often than previously believed. However, Siqueira et al. (2002c) used PCR along with species-specific primers

Fungi in Endodontic Infections 211

Fig. 9.10 Bacteria (arrows) in dentinal tubules approximately 100 μm from the root canal wall in the middle third of root (original magnification 2000×). Courtesy of Bilge Hakan Sen, Beyser Piskin, and Tijen Pamir.

Fig. 9.11 Yeast cells on the root canal wall in the middle third (original magnification 1500×). Courtesy of Bilge Hakan Sen, Beyser Piskin, and Tijen Pamir.

Fig. 9.12 Yeast cells on the root canal wall in the apical third. Note that the cells are attached to each other and the dentinal wall with numerous organic strands (original magnification 5000×). Courtesy of Bilge Hakan Sen, Beyser Piskin, and Tijen Pamir.

212 Endodontic Microbiology

and found fungi only in 1 of 50 specimens. The dif- ference may be because of the use of different primers with different sensitivity, and/or from the result of dif- ferences in geographic locations (Rôças et al. 2006; Machado de Oliveira et al. 2007). In a PCR-based study, Cogulu et al. (2008) found that prevalence of C. albicans was similar in both deciduous and perma- nent teeth, and less than 5%. In addition, in root canal- treated teeth with posttreatment apical periodontitis, species-specific PCR detected E. faecalis in 8 (47%) and C. albicans in 1 (6%) of 17 cases (Rôças et al. 2008).

There are case reports showing that pure cultures of C. albicans were isolated from primary endodontic infections associated with apical periodontitis (Eidel- man et al. 1978; Matusow 1981; Kinirons 1983; Damm et al. 1988).

9.5.4.2 Secondary root canal infections

This type of endodontic infection is caused by microor- ganisms that have not been in the root canal previ- ously, but have penetrated into the endodontic space during treatment, between appointments, or after the endodontic treatment (Siqueira 2002).

As stated by Waltimo et al. (2003b), yeasts may be either present in low numbers or not present at all in primary endodontic infections. During endodontic procedures, they may reach higher percentages in the total cultivable flora or new yeast species may pen- etrate into the root canal system. Lana et al. (2001) investigated the microbial status of intact root canals with necrotic pulps. C. tropicalis and S. cerevisiae were recovered from two root canals (7.4%) before endodontic procedures were initiated. After instru- mentation, irrigation with 2.5% NaOCl and disinfec- tion with calcium hydroxide, these yeasts were not present in the root canal; however, C. guilliermondii and C. parapsilosis were recovered in the second and third collections, respectively. It is very likely that the latter species gained access to the root canal because of poor isolation or cavity seal. Pinheiro et al. (2003) demonstrated a significant association between coro- nally unsealed teeth and Candida spp. Wilson and Hall (1968) reported the prevalence of yeasts in primary endodontic infections as 1.9%. In the subsequent sec- ond and third visits, this incidence was increased to 6.8%. When they examined the cases with positive yeast culture, they observed that either the tempo- rary restorations had defects or a very long period of

time (3–4 months) had elapsed between the two visits (four cases). Jackson and Halder (1963) determined the presence of yeasts as high as 26% at the initial visit of endodontic therapy. At the subsequent visits after using chloramphenicol as an intermediate dress- ing, they isolated yeasts from the teeth that had been negative at the initial culture. They concluded that use of an antibacterial agent might favor the overgrowth of yeasts in the root canal.

9.5.4.3 Persistent root canal infections

After gaining access into the root canal system, the microorganisms may survive against harsh conditions such as intracanal procedures, disinfection, and obtu- ration, and cause persistent infections. In addition to the establishment of presence of yeasts in primary and secondary infections in previous studies, there are con- siderable data that yeasts can also take part in the root canal microbiota of failed endodontic treatments.

The incidence of yeasts cultured from persistent endodontic infections is reported to be 2.9–22.2%. As stated previously, the use of a selective medium signifi- cantly affects the prevalence in root canals (Table 9.2). When a selective medium is used, their incidence is increased from 2.9–8.3% to 6.8–18%.

Yeasts are isolated either as pure cultures or together with other bacteria in endodontic therapy-resistant cases (Siren et al. 1997; Waltimo et al. 1997; Sundqvist et al. 1998; Peciuliene et al. 2001; Siqueira and Rôças 2004). Waltimo et al. (1997) isolated 48 fungal strains from 47 microbial samples, representing 7% of the 692 culture-positive samples. While C. albicans was the most common isolate, C. glabrata, C. guilliermondii, C. inconspicua, and Geotrichium candidum were also isolated. Fungi were demonstrated as pure cultures in six cases. Therefore it was proposed that they had a definite pathogenic role in developing apical periodon- titis. In most of the fungi-positive cases, facultative Gram-positive bacteria such as α- and nonhemolytic Streptococcus species were present. However, Gram- negative isolates were found randomly.

Peciuliene et al. (2001) reported the prevalence of C. albicans in root-filled teeth with chronic apical peri- odontitis to be as high as 18%, and this fungus was recovered from the root canals 50% with E. faecalis and 50% with other bacteria. However, yeasts consti- tuted only <1% of the total cultivable microbial flora. They have concluded that the ecology in the root canals that were inadequately filled may favor the particular

Fungi in Endodontic Infections 213

Table 9.2 Literature relating to prevalence of yeasts isolated from root canals with persistent endodontic infections

Authors

Number of culture-positive

samples (or teeth) Method/culture Prevalence of yeasts in root canals (%)

Nair et al. (1990) 9 LM and TEM 22.2 Siren et al. (1997) 70 TSBV agar 12.9 Waltimo et al. (1997) 692 TSBV agar 6.8 Sundqvist et al. (1998) 24 Thioglycolate 8.3 Hancock et al. (2001) 34 Agar 2.9 Peciuliene et al. (2001) 33 Sabouraud’s agar 18.2 Cheung and Ho (2001) 18 Sabouraud’s agar 11.1 Egan et al. (2002) 25 Sabouraud’s agar 16 Pinheiro et al. (2003) 51 Nonselective 3.9 Siqueira and Rôças (2004) 22 PCR 9

The rows shown in bold type indicate the studies in which a selective medium has been used for culturing of yeasts. LM, light microscopy; TEM, transmission electron microscopy; TSBV, tryptic soy-serum-bacitracin-vancomycin.

growth of E. faecalis and C. albicans. Adib et al. (2004) identified the cultivable microbial flora in root-filled teeth with persistent apical periodontitis and evident coronal microleakage. In addition to the presence of Gram-positive facultative anaerobes (75%), six strains of Candida were isolated from coronal dentin, root dentin, and root gutta-percha samples in three of eight subjects.

Apart from culture studies, the presence of fungi in failed root canal therapy has been shown using either microscopic or molecular techniques. Nair et al. (1990) analyzed the apical part of nine therapy-resistant cases with light and electron microscopes. Two specimens revealed yeast-like microorganisms in the root canal and apical foramen. There were numerous budding yeasts, indicating that they were in active prolifera- tion. They demonstrated fungi as a potential nonbacte- rial, microbial cause of nonhealed apical periodontitis. Siqueira and Rôças (2004) studied root canal samples taken from 22 root-filled teeth with persistent peri- radicular lesions by means of PCR. While E. faecalis was the most prevalent bacterial species (77%), C. albi- cans was isolated in 9% of the samples.

9.5.5 Extraradicular yeast infections

Generally speaking, bacteria are located within the root canals of teeth with apical periodontitis and can occasionally penetrate into the periapical tissues in clinical conditions such as acute apical periodontitis, acute apical abscess, periapical actinomycosis, and osteomyelitis (Nair 1997; Dahlen 2002). However,

many studies have shown that microorganisms can survive even in chronic apical pathologies of treated or untreated endodontic cases (Haapasalo et al. 1987; Tronstad et al. 1987, 1989; Iwu et al. 1990; Kiryu et al. 1994; Gatti et al. 2000). However, attention has been focused on bacteria in these studies and any information related to presence of yeasts in periapi- cal tissues has not been reported. In addition, it has been shown recently that C. albicans antigens can contribute to proinflammatory reactions and antimi- crobial responses by upregulating IL-6 and IL-10 and downregulating IL-12 and also by production of tumor necrosis factor α (TNF-α) and nitric oxide (NO) (Lima et al. 2015).

Nair et al. (1990) demonstrated electron- microscopically that yeasts were present in the apical part of root canals, but not extended to the periapical tissues. However, in a study investigating root surfaces of teeth with chronic apical periodontitis using SEM, Lomcali et al. (1996) reported that there was a multi- layered microbial plaque in most of the specimens and yeast cells were harbored in resorption lacunae.

Baumgartner et al. (2000) studied 19 samples of aseptic aspirates of cellulitis/abscesses of endodon- tic origin by means of PCR. They found that all samples were negative for the presence of C. albi- cans DNA. Waltimo et al. (2003a) investigated refrac- tory periapical granulomas for the presence of Can- dida spp. using three different methods. They were able to extract DNA from 68 of 103 paraffin- embedded samples. PCR products demonstrated pos- sible occurrence of Candida spp. in 18 of those 68

214 Endodontic Microbiology

DNA-positive samples. However, PAS staining and immunohistologic examination did not confirm this finding, and all PCR-positive samples were found to be negative for presence of Candida. Further sequencing of the PCR products revealed that the sequences were not typical for Candida spp. Therefore, they empha- sized both the importance of primer selection in molec- ular microbiologic studies and the use of sequencing to confirm the findings. Sunde et al. (2002) examined the periapical microbiota of 36 teeth with refractory apical periodontitis. Fifty-one percent of the bacte- rial strains were anaerobic and Gram-positive species had predominance (79.5%). In addition to bacterial species, they cultivated C. albicans in two patients. Even though the number of lesions with Candida was very small, this finding was in contradiction with the findings of Baumgartner et al. (2000) and Waltimo et al. (2003a).

Several case reports have also demonstrated the presence of yeasts in periapical tissues (Eidelman et al. 1978; Matusow 1981; McManners and Samaranayake 1990). Eidelman et al. (1978) showed that C. albicans was present in root canals and periapical granu- loma of a patient with chronic urticaria. Histologic examinations revealed that the granuloma exhibited invasive Candida infection. The complete cure of the patient was achieved only after the extraction of Candida-infected teeth and curettage of the granuloma.

Damm et al. (1988) presented a patient with carci- noma ex-pleomorphic adenoma. Radicular and cer- vical soft tissues of extracted teeth of this patient were examined histologically using PAS, Gomori methenamine silver, and Brown and Brenn stains. The microscopic analysis revealed that fibrous connective tissues contained mixed inflammatory cellular infil- trate with large areas of necrosis and abscess forma- tion. The tissues were invaded by numerous colonies of yeast cells together with pseudohyphae, which were morphologically consistent with C. albicans.

Debelian et al. (1995, 1997) reported the presence of S. cerevisiae in the root canal and blood sample of a patient undergoing endodontic therapy. With the aid of phenotyping and genotyping tests, it was found that the root canal and blood isolates were identical. It was concluded that the root canal was the source of the blood isolate and fungemia occurred uninten- tionally during endodontic therapy. It was interesting to note that the patient had been an alcoholic for 20 years and alcoholism could be a predisposing factor

for yeast infections (Bardwell et al. 1986; Trowbridge et al. 1999).

As seen in these cases, extraradicular presence of yeasts seems to be associated mostly with immuno- compromised state of the patients.

9.6 Antifungal activity of endodontic irrigating solutions and disinfectants

During the 1950s and 1960s, local use of antibiotics had been extensively used in endodontic treatment. Most of the antibiotic pastes contained an antifun- gal component, particularly nystatin or sodium capry- late. It is clearly evident that there used to be a clin- ical approach to antifungal treatment of infected root canals. Since the 1970s, the popularity of local use of antibiotics in endodontics decreased mainly because of the high risks of producing resistant microorgan- isms and host sensitization. Since then, antifungal con- siderations in endodontic therapy have received little attention.

If there is a possible yeast infection in the root canal, use of common antibacterial solutions or disin- fectants, which may have limited antifungal activities, may favor the overgrowth of yeasts between the visits (Sen et al. 1999). As stated previously, single Can- dida spp. may cause persistent root canal infections in vivo (Waltimo et al. 1997). Therefore, in addition to antibacterial effectiveness, it is desirable that root canal irrigating solutions or disinfectants should have antifungal capacity as well.

9.6.1 Sodium hypochlorite

Sodium hypochlorite (NaOCl) is perhaps the most widely used irrigant in endodontics today. It has a broad spectrum of antimicrobial activity against bac- teria, yeasts, and viruses (Mentz 1982). Its particular antifungal properties have been investigated in sev- eral studies with different methodologies (Ayhan et al. 1999; Waltimo et al. 1999a; Ferguson et al. 2002; Rad- cliffe et al. 2004).

Ferguson et al. (2002) reported that NaOCl was very effective against C. albicans having a minimumal inhibitory concentration (MIC) of <10 μg/mL. Differ- ent concentrations of NaOCl ranging 0.5–5% showed complete antifungal activity in a range from 10 s to 30 min (Smith and Wayman 1986; Harrison et al. 1990; Radcliffe et al. 2004; Vianna et al. 2004). According

Fungi in Endodontic Infections 215

to Radcliffe et al. (2004), over 9 million CFU were decreased to below the limit of detection even with 0.5% NaOCl within a 10-s contact time. Waltimo et al. (1999a) used filter paper method to determine the susceptibilities of seven strains of C. albicans to endodontic disinfectants. Among a group that included iodine potassium-iodide (IKI), chlorhexidine acetate, and calcium hydroxide, NaOCl was the most effective disinfectant. Both 5 and 0.5% concentrations showed complete killing in 30 s. However, 0.05 and 0.005% concentrations were not effective even after 24 h.

Sen et al. (1999) investigated the antifungal effects of 0.12% chlorhexidine (CHX), 1 and 5% NaOCl on C. albicans biofilms grown on the radicular dentin walls and the effect of smear layer on antifungal capac- ity of these solutions. In the presence of smear layer, antifungal activity was not observed in 1, 5, and 30 min groups of any solution. Only 1-h treatment groups for all solutions were effective. When the smear layer was absent, 5% NaOCl alone started to exert its antifun- gal properties after 30 min. It is apparent that both smear layer and biofilms of C. albicans delayed or stopped the antifungal capacity of NaOCl and CHX and it took more time to reach the complete anti- fungal effect. This finding was different from that of other studies, which used direct exposure tests. Even though the direct exposure test is a practical labora- tory method, the clinical relevancy of the methodology should be taken into consideration with the knowl- edge of complex root canal anatomy, polymicrobial root canal infections, and biofilm formation (Orstavik and Haapasalo 1990; Spratt et al. 2001; Clegg et al. 2006). When embedded in a biofilm, microorganisms become resistant to antimicrobial agents that are active against the planktonic form of the same organisms in suspension (Evans and Holmes 1987). During irriga- tion of root canals with microbial biofilms, the top layer of microorganisms will be directly affected by a relatively high concentration of the irrigating or disin- fecting solution. However, the extracellular matrix of the biofilm will interfere with the solution’s ability to affect the deeper layers, which may contain still viable microorganisms. Endodontic instrumentation may dis- rupt the biofilm and expose its embedded microor- ganisms to irrigating or disinfecting solutions. How- ever, it is also well-known that endodontic files cannot touch all root canal walls (Wu et al. 2002, 2003), and microorganisms may still remain in the root canal and dentinal tubules after instrumentation and irrigation (Moodnik et al. 1976; Nair et al. 2005).

Clegg et al. (2006) developed a model to study the effect of biofilm in root canal disinfection. Polymicro- bial biofilms were grown on apical dentin and the effect of different irrigating solutions was evaluated. It was concluded that 6% NaOCl was the only irrigant capa- ble of both disrupting the biofilm physically and ren- dering bacteria nonviable. However, Ruff et al. (2006) evaluated antifungal efficacy of several irrigants as a final rinse after developing biofilms of C. albicans in root canals. On 1 min application, 6% NaOCl could not destroy all yeast cells, but lowered the growth potential considerably from 105 to 102 CFUs.

9.6.2 Ethylene diamine tetraacetic acid

EDTA has been mainly used to increase the efficacy of root canal instrumentation (Fraser 1974; Hülsmann et al. 2002; Lim et al. 2003). It reacts with calcium ions in dentin and forms soluble calcium chelates (Fraser 1974). It is also highly effective in removing smear layer from root canal walls (Sen et al. 1995b; Tora- binejad et al. 2002). In addition, EDTA does not only remove smear layer, but also removes the microor- ganisms entrapped in the smear layer (Bystrom and Sundqvist 1985; Yoshida et al. 1995). However, EDTA itself has not been considered as a particular antimicro- bial agent and has no effect on Gram-positive species (Heling and Chandler 1998). Orstavik and Haapasalo (1990) demonstrated that EDTA did not have any disinfecting action, but rather increased antibacterial effects of other agents by opening the dentinal tubules. However, some antibacterial activity of EDTA has been reported against streptococci and staphylococci (Masillamoni et al. 1981; Root et al. 1988). According to Haapasalo et al. (2005), EDTA extracts surface pro- teins of microorganisms by interacting with the metal- lic ions from the cell membrane and leads to microbial death only if the microorganisms are exposed to EDTA for a long time. EDTA alone or in combination with other antimicrobial agents is used for disruption and eradication of biofilm layers of several microorgan- isms such as Pseudomonas aeruginosa, Staphylococ- cus aureus, Staphylococcus epidermidis, and C. albi- cans in catheters or polycarbonate chips (Kite et al. 2004; Percival et al. 2005; Banin et al. 2006). EDTA has been found to be effective even on microorgan- isms cultivated on these artificial surfaces that contain no divalent ions. Its activity may be more evident on biofilms grown on organo-inorganic surfaces such as dentin, particularly affecting the substrate as well.

216 Endodontic Microbiology

From an endodontic point of view, antifungal prop- erties of EDTA were first evaluated by Sen et al. (2000) in an agar diffusion study. They observed that EDTA had the highest antifungal activity among a variety of common antifungal drugs and endodontic irrigat- ing solutions including nystatin, ketoconazole, NaOCl, and CHX. In addition, it was demonstrated that the root canal isolate of C. albicans was more susceptible to all disinfectants when compared with the oral cavity isolate. This potential antifungal activity of EDTA is considered interesting because it does not affect pro- tein and DNA synthesis of C. albicans (Gil et al. 1994). However, there may be two particular ways for EDTA to show its antifungal activity: first, anticolonization; second, antigrowth effects. According to Odds (1988), there is a direct relation between adherence and colo- nization capacity of C. albicans. The presence of cal- cium ions in the medium or environment has a critical role in the control of morphogenesis of C. albicans (Holmes et al. 1991) and its adherence capacity to var- ious extracellular matrix proteins (Klotz et al. 1993). Hence, EDTA may prevent binding of C. albicans to proteins by chelating calcium ions. In addition to anti- colonization effect, EDTA may also reduce the growth potential of C. albicans. By removing calcium ions from the cell walls, it causes collapses in the cell wall and inhibits the enzyme reactions (Pugh and Cawson 1980).

The antifungal effect of EDTA has been confirmed by other studies (Grawehr et al. 2003; Ates et al. 2005). The effects of different calcium-chelating or calcium- binding agents on C. albicans were evaluated by deter- mining MIC and minimum fungicidal concentrations (MFC) of the solutions (Ates et al. 2005). Ketocona- zole and EDTA showed the highest antifungal and fungicidal activities followed by titanium tetrafluoride. EGTA and sodium fluoride demonstrated weak activ- ity against C. albicans. Even though EGTA is con- sidered as a specific calcium ion chelator, it did not demonstrate an antifungal activity similar to that of EDTA. As calcium-binding capacities of EDTA and EGTA are similar (Ueno et al. 1982), high antifun- gal activity of EDTA may be related to chelating not only calcium ions, but also other divalent ions such as magnesium, manganese, and zinc. These divalent ions are also considered important for the growth and morphogenesis of C. albicans (Bedell and Soll 1979; Holmes et al. 1991; Sohnle et al. 2001). In addition to the chelating activity, EDTA inhibits a pericellu- lar metalloenzyme of C. albicans, which presents a

cell-associated collagenolytic property (Nishimura et al. 2002).

In contrast to the abovementioned studies presenting clear antifungal activity of EDTA, Ruff et al. (2006) demonstrated in a root infection model that a final rinse with 1 mL of 17% EDTA at a contact time of 1 min was not sufficient to remove C. albicans from the root canals. It is probable that EDTA interacts rapidly with root canal dentin through chelation mechanisms, reaches a saturation point with excessive calcium ions, and becomes ineffective against C. albicans. Accord- ingly, Grawehr et al. (2003) have reported that micro- bial growth inhibition of EDTA is strongly reduced when it is preincubated with dentin powder. This may be the explanation for the weak antibacterial effect of EDTA in infected dentin tubules (Orstavik and Haa- pasalo 1990; Heling and Chandler 1998). However, this problem may be solved in a clinical situation by increasing the volume and contact time of EDTA used for irrigating root canals.

Because of its limited antibacterial effectiveness, EDTA may not be recommended as a routine work- ing solution during endodontic treatment by itself. However, because of its strong antifungal and biofilm- disrupting properties, copious irrigation with EDTA may be advised particularly in persistent root canal infections or in root canals of medically compromised patients who are prone to oral candidosis.

9.6.3 Chlorhexidine

CHX is one of the most commonly used biocides in antiseptic products in general medicine and dentistry. It has a wide antimicrobial spectrum and is effective against both Gram-positive and Gram-negative bac- teria as well as yeasts. It permeates the cell wall or outer membrane and attacks the cytoplasm of bac- teria or inner membrane or the yeast plasma mem- brane (McDonnell and Russell 1999). When Candida is exposed to CHX, both macroscopic and micro- scopic profound effects on the viability and structural integrity of Candida are observed (MacNeill et al. 1997). Nucleoproteins coagulate with inhibition bud- ding and cell wall changes with possible escape of cytoplasmic components through the cell membrane (Bobichon and Bouchet 1987). Candida grown in the presence of CHX is more susceptible to spheroplas- ting, indicating further that the antiseptic affects the cell surface composition of Candida (McCourtie et al. 1986).

Fungi in Endodontic Infections 217

CHX has long-term antimicrobial properties because of its unique ability to bind to organic and inor- ganic dental tissues (Rolla et al. 1970; Parsons et al. 1980). It also inhibits the initial adherence of yeasts and other microorganisms efficiently, and perhaps further accumulation and biofilm formation (Waltimo et al. 2004b). Although it has strong antimicrobial properties with relatively low toxicity (Chang et al. 2001; Tanomaru Filho et al. 2002), its activity is mainly pH dependent and is considerably reduced by organic materials (Russell and Day 1993).

Use of CHX as an irrigating solution in endodontics has been suggested by Delany et al. (1982). Later, its gel form has been proposed as an intracanal dressing (Siqueira and de Uzeda 1997; Gomes et al. 2003). Even though the literature about the antibacterial effect of CHX is abundant, the scientific exploration of its antifungal effects in endodontics has been growing at a slower pace in recent years.

Ferguson et al. (2002) reported that MIC of CHX gluconate needed to inhibit the growth of C. albicans was <0.63 μg/mL. In a broth dilution test, both liquid and gel formulations of 2% CHX gluconate eliminated C. albicans in 15 s (Vianna et al. 2004). Similarly, Gomes et al. (2006) demonstrated that 2% CHX glu- conate gel eliminated C. albicans in 15 s in a direct con- tact test. Waltimo et al. (1999a) evaluated the in vitro susceptibility of seven C. albicans strains to four dis- infectants and their combinations, using filter paper method. They found that 0.5% CHX acetate killed all yeast cells within 5 min. Tenfold dilution of this solu- tion became effective within 1 h and there was a slight variation in susceptibility of the strains to this dilution. It was also stated in this study that combinations of the disinfectants were equally or less effective than the more effective component. Sena et al. (2006) evaluated the effect of 2% CHX in liquid and gel forms against C. albicans biofilms grown on cellulose nitrate mem- branes. The contact time required to achieve negative cultures for CHX ranged between 30 s and 30 min, depending on the formulation type and mechanical agitation. The liquid form and mechanical agitation lowered the effective contact time.

It is apparent that CHX is very effective against C. albicans in direct contact tests or after grow- ing biofilms on artificial membranes. However, these methods may not be clinically relevant. Considering the fact that organic matter, which is abundant in the root canal environment, reduces the activity of CHX, it may be expected that antimicrobial performance

of CHX will be affected considerably. Haapasalo et al. (2000) demonstrated that the effect of CHX was reduced by the presence of dentin. Similarly, Portenier et al. (2002) showed that CHX was strongly inhibited by organic content of dentin as well as heat-killed cells of E. faecalis and C. albicans. However, dentin pre- treated with EDTA showed only slight inhibition in this study. Loss of antimicrobial activity of CHX by bovine serum albumin had also been reported previ- ously (Portenier et al. 2001).

These inhibition studies may help to explain rela- tively poor performance of CHX in the root canal. Sen et al. (1999) reported that antifungal activity of 0.12% started in 1 h regardless the presence or absence of smear layer. Menezes et al. (2004) infected the root canals with C. albicans for 7 days and observed that 2% CHX irrigation following instrumentation could not prevent further growth of C. albicans. In another tooth infection model, antifungal activity of 2% CHX gel was evaluated on biofilms of C. albicans in root canals (Ercan et al. 2006). It took 7 days for com- plete inhibition of C. albicans growth. In contrast to these studies, it was demonstrated that a final rinse of 2% CHX with 1 min application reached a neg- ative culture in the root canals having biofilms of C. albicans. However, it should be strongly empha- sized that no attempt was made in this study to neutral- ize CHX left in the root canals, to reduce the carry-over effect.

There is substantial evidence that CHX has a good potential of antifungal effects in general. However, its activity may be limited in the root canals by the pres- ence of high amounts of organic structures. In addi- tion, CHX does not have any tissue-dissolving activ- ity (Okino et al. 2004), cannot remove smear layer (Yamashita et al. 2003), and cannot disrupt biofilms (Clegg et al. 2006). In this regard, use of CHX can be modified with the aid of other irrigating solutions. After completion biomechanical instrumentation with NaOCl as a working solution, smear layer is removed with sequential use of EDTA and NaOCl. Hence, the amount of organic structures present in the root canal and dentin walls is greatly reduced and CHX solution is used as a final rinse or gel form as a dressing. This may create an optimal environment for CHX to present its potent antimicrobial properties and bind to dentin to release back in a longer period. Antimicrobial action of several medicaments on the external root surfaces were investigated in an in vitro study by Gomes et al. (2009). Interestingly, 2% CHX gel diffused well into

218 Endodontic Microbiology

the dentin in presence or absence of cementum and showed good antifungal and antibacterial effects.

9.6.4 MTAD

Biopure MTAD (Tulsa Dentsply, Tulsa, OK) is a mix- ture of tetracycline isomer (doxycycline), an acid (cit- ric acid), and a detergent (Tween 80). Torabinejad et al. (2003a,b) have demonstrated that Biopure MTAD is an effective solution to remove smear layer without causing any erosion in dentinal tubules. MTAD was found to be very effective in eradicating E. faecalis (Shabahang and Torabinejad 2003; Shabahang et al. 2003; Torabinejad et al. 2003c). However, its activity was lower than EDTA, NaOCl, and their combinations (Dunavant et al. 2006; Baumgartner et al. 2007).

The antifungal effect of MTAD is not clear and has been studied in only one study so far. In a tooth model, it was demonstrated that a final rinse of 5 mL of Biop- ure MTAD at a contact time of 5 min was not effective against biofilm of C. albicans (Ruff et al. 2006). Doxy- cycline, which is the active antimicrobial ingredient in Biopure MTAD, demonstrates considerable antimi- crobial activity against a wide spectrum of Gram- positive and Gram-negative bacteria, but not against fungi (Liu et al. 2002). It has been previously reported that a well-recognized potential disadvantage of tetra- cycline administration is the induction of opportunistic infections such as candidiasis (Rams et al. 1990). In addition, MacNeill et al. (1997) observed that a solu- tion of tetracycline hydrochloride at a concentration of 3.0 mg/mL caused a heavy and constant uniform growth of C. albicans during the study period of 10 days. The other component of MTAD which is citric acid also does not have any antifungal activity (Smith and Wayman 1986). With the current knowledge of poor antifungal properties of both tetracycline and cit- ric acid, it is not surprising to find out that Biopure MTAD is not effective against C. albicans. From the clinical point of view, it may be concluded that MTAD should be carefully used in persistent root canal infec- tions that may have yeasts already in the microflora or in the root canals of the patients who already have oral candidosis.

9.6.5 Calcium hydroxide

Calcium hydroxide (CH) has been routinely used as an interappointment medicament in endodontics for many years. In cases of endodontic infections, a CH

dressing for at least 1 week is advocated for use in root canals. However, it has limited effectiveness in elimi- nating bacteria from human root canal when assessed by culture techniques (Peters et al. 2002; Zerella et al. 2005; Sathorn et al. 2007). After isolation of yeasts particularly from root canals with secondary or per- sistent infections, there has been some interest in the antifungal capacity of calcium hydroxide preparations.

Waltimo et al. (1999b) demonstrated that saturated aqueous solutions of CH were ineffective against clin- ically important Candida spp. Even though they used a direct contact test, it required 16 h to kill 99.9% of the CFUs of C. albicans strains. When compared with E. faecalis, all Candida spp. showed either equally high or higher resistance to aqueous CH solution. In a parallel study from the same group (Waltimo et al. 1999a), they saturated irrigating solutions with CH powder and observed that these solutions were always more effective than CH alone. Similarly, Ferguson et al. (2002) reported ineffectiveness of saturated CH solution against C. albicans. Because C. albicans is able to survive in a wide range of pH, alkalinity of CH solutions may not have any effect on this organism. In addition, saturated CH solution readily presents cal- cium ions necessary for growth and morphogenesis of Candida (Holmes et al. 1991; Klotz et al. 1993). How- ever, it has been reported that antifungal effectiveness of CH is increased when used as a paste (Ferguson et al. 2002). Siqueira et al. (2003) demonstrated that CH–glycerin paste started to disinfect the dentin spec- imens infected with C. albicans in 2 days and reached complete disinfection in 7 days.

Efforts have been made to increase antifungal prop- erties of CH paste. In this regard, mixing it with other disinfectant solutions, particularly with CHX, has been proposed. However, the outcomes of this approach are somewhat contradictory. While Siqueira et al. (2003) demonstrated that CH–CHX combination was ineffec- tive in disinfecting C. albicans-infected dentin speci- mens even after 7 days, Haenni et al. (2003) did not observe any additive antimicrobial effect of CHX, IKI, and NaOCl when mixed with CH powder. In contrast to these findings, Ercan et al. (2006) and Gomes et al. (2006) reported that CH mixed with 2% CHX solu- tion or gel considerably improved antimicrobial effec- tiveness of CH in comparison to CH–sterile water combination. Zerella et al. (2005) studied potential interactions of CH with CHX in a preliminary study before the main clinical research. They found that there was a significant loss (>99%) of CHX when mixed

Fungi in Endodontic Infections 219

with CH. However, despite this potential loss of CHX, the antimicrobial effectiveness of this mixture was as effective as CHX alone on E. faecalis. Therefore, it was considered that the combined effect of CH and CHX might have a clinical value. In the clinical study, CH– CHX mixture more effectively eliminated microorgan- isms from the root canal in comparison to CH–sterile water combination. However, this difference was not statistically significant because of the low number of subjects in the study. Clinical performance of CH– CHX combination seems to be promising; however, further in vitro studies and larger clinical studies are needed to elucidate whether CHX has an additive effect when mixed with CH.

9.6.6 Other antifungal measures

9.6.6.1 Effect of endodontic procedures

Biomechanical preparation of root canals cannot com- pletely eliminate the bacteria from the root canals. The same concept may also be true for the presence of yeasts in various types of root canal infections.

Lana et al. (2001) analyzed the root canals with pulp necrosis microbiologically before and after endodontic procedures. At the first collection, yeasts (C. tropicalis, S. cerevisiae) were recovered from two root canals having intact pulp chambers. In the second collection taken after endodontic instrumentation, irrigation, and disinfection of 1 week, C. guilliermondii was isolated in one canal. After sealing the root canals without any dressing for another week, C. parapsilosis was present in another root canal.

In another clinical study (Ferrari et al. 2005), 25 single-rooted teeth with pulp necrosis, intact pulp chamber, and periradicular lesions were used to detect enterococci, enteric bacteria, and yeasts before (first sampling) and after (second sampling) endodontic instrumentation. While 0.5% NaOCl was the work- ing solution, 10 mL EDTA was used for final irriga- tion. After sealing with temporary restoration for 1 week, a third sample was obtained. At the first micro- bial sample, there was only one root canal infected by yeasts (C. albicans). After biomechanical instrumen- tation, yeasts were no longer detected. However, two root canals (C. glabrata and C. magnoliae) contained yeast cells after 7 days without intracanal dressing.

In an ex vivo study (Menezes et al. 2004), the teeth were contaminated with C. albicans. After biomechan- ical preparation up to size 50 K file was accomplished

with sterile saline, the root canals were irrigated with 2.5% NaOCl or 2% CHX. Following the first sampling, the root canals were filled with Sabouraud’s broth and were sealed with temporary cement for 1 week. Then, a second microbial sampling was performed. While the root canals were negative for C. albicans at the first sampling, the yeast cells were present at the sec- ond sampling. The results showed that C. albicans was able to recolonize the root canals after instrumentation and irrigation with 2.5% NaOCl or 2% CHX.

In contrast to the result of the abovementioned stud- ies, Peciuliene et al. (2001) did not recover yeasts after endodontic procedures, including instrumenta- tion and irrigation with 2.5% NaOCl and 17% EDTA. Because of low numbers of Candida-positive cases, they stated that it was too early to draw a conclusion that yeasts were very sensitive to endodontic biome- chanical instrumentation. In a recent study (Nakamura et al. 2013), the effect of instrumentation technique (manual and rotary) and type of irrigating solutions (saline; 1 or 5.25% NaOCl; 1%NaOCl with 15% cit- ric acid) were investigated in root canals infected with E. faecalis and C. albicans. Regardless of the instru- mentation technique and type of the irrigating solu- tion, more than 98% of the microbial population was removed. While saline and 1% NaOCl were less effec- tive than the other groups, there was no particular dif- ference between manual or rotary instrumentation.

It is a fact that yeasts can be present in a wide range of percentages in various types of endodontic infec- tions. There are two possible ways for their persis- tence in the root canal even after endodontic proce- dures. First, they may be very low in numbers at the beginning, but survive against endodontic treatment modalities and show considerable growth afterward. Second, new species may gain access to the root canal as a result of poor sealing and ineffective disinfectants. It is a requirement henceforth that both irrigating solu- tions and disinfectants should have a good antifungal capacity in addition to their antibacterial properties, because the microbial ecology of the root canals con- tains both bacterial and fungal species.

9.6.6.2 Antifungal agents

It has been emphasized in recent reports that the oral cavity may act as a reservoir of resistant yeast iso- lates and cause persistent yeast infections (Berthold et al. 1994; Kuriyama et al. 2005). Considering the fact that Candida species are highly resistant to calcium

220 Endodontic Microbiology

hydroxide (Waltimo et al. 1999a,b) and that the other endodontic disinfectants may be inactivated by the presence of organic structures such as dentin, serum albumin, and dead microbial cells (Haapasalo et al. 2000; Portenier et al. 2001, 2002), possible presence of yeasts in the root canals becomes a more pronounced issue. Therefore, use of specific antifungal agents may be considered after routine endodontic procedures in cases of particular yeast infections of root canals. How- ever, there is limited information about the effects of classic antifungal agents against yeasts isolated from root canals.

Sen et al. (2000) compared antifungal effects of endodontic irrigating solutions or disinfectants with classic antifungal agents, nystatin, and ketoconazole in an agar diffusion study. They found that both anti- fungal agents were superior to all solutions or disin- fectants, except EDTA. In a subsequent study (Ates et al. 2005) determining MIC or MFC, ketoconazole was the most effective agent against three strains of C. albicans. While the root canal isolate was the most sensitive strain in the first study, there was no dif- ference among the same strains in a follow-up study. It was concluded that the type of antimicrobial sus- ceptibility test might affect the results of the studies when evaluating the antifungal capacity of the agents or resistance of the strains.

Waltimo et al. (2000b) determined MIC values of amphotericin B, 5-fluorocytosine, and three azole- based agents (fluconazole, miconazole, and clotrima- zole) against 70 C. albicans strains isolated from either persistent cases of apical periodontitis or marginal periodontitis. All isolates were susceptible to ampho- tericin B and 5-fluorocytosine with an MIC below 1 μg/mL, and there was no difference between endodontic and periodontal isolates. However, their susceptibility to the three azoles varied. Fluconazole was mostly effective on endodontic isolates. Two peri- odontal and one endodontic strains presented azole cross-resistance. According to Waltimo et al. (2004a), fungal antibiotics should be used only for the treatment of acute endodontic cases after a substantial microbi- ologic diagnosis is made.

9.7 Conclusions

During standard endodontic treatment procedures, most of the bacteria may be removed from the root canals, but fungi can survive. Then, they can

demonstrate an overgrowth because of their oppor- tunistic character. They can also gain access to the root canals during or after root canal therapy.

The incidence of yeasts in primary, secondary, or persistent endodontic infections has been reported to be in the range of 0.5–61.5%. However, it should be kept in mind that all of these studies have been accom- plished in systemically healthy dental subjects. Con- sidering that the presence of yeasts in oral and dental tissues of immunocompromised patients is relatively higher than that of the healthy population, it may be proposed that their incidence will also be higher in the root canals of these patients. At this point, we should continue searching for new remedies not only against bacteria, but also against fungi in endodontics.

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Chapter 10 Severe Head and Neck Infections Jaime S. Brahim and Robert A. Ord

10.1 Introduction 10.2 Etiology and epidemiology 10.3 Microbiology 10.4 Anatomy and pathogenesis of spread

10.4.1 Submandibular space 10.4.2 Sublingual space 10.4.3 Submental space 10.4.4 Buccal space 10.4.5 Lateral pharyngeal space 10.4.6 Masticator space 10.4.7 Canine space

10.5 Diagnosis 10.5.1 History 10.5.2 Systemic examination 10.5.3 Head and neck examination 10.5.4 Laboratory investigations 10.5.5 Imaging

10.6 Airway management 10.7 Medical and surgical management 10.8 References

10.1 Introduction

This chapter discusses the microbiology, diagnosis, and management of severe, life-threatening, aggres- sive odontogenic infections of the head and neck. Minor intraoral soft tissue infections and bony infec- tions, for example, osteomyelitis and bisphosphonate- related osteonecrosis of the jaw are not addressed. Dra- matic changes have occurred in the management of infections since the early seventeenth century when teeth were the fifth or sixth most common cause of death. The discovery of penicillin in 1928 and its sub- sequent introduction to clinical practice in 1940 led to a decrease in mortality from 54% to 4% in patients with Ludwig’s angina.

There is no accurate estimate of the frequency of deep neck space infections worldwide, but they are still associated with high rates of morbidity and mortality. Early recognition by the oral health care professional of potentially severe infections and prompt management can prevent progression of the

infection, with the later need for hospitalization and life-saving surgery. If not recognized and treated at an early stage, these infections may progress to cause airway obstruction, necrotizing infections with septic shock, or fatal outcomes from complications such as brain abscess, cavernous sinus thrombosis, or mediastinitis. Although medical advances in imaging, antibiotics, and critical care have increased our ability to treat infectious diseases, the outcome remains a bal- ance between the virulence of the infectious agent and the immune response of the host. The development of bacterial strains resistant to multiple antibiotics (MRSA, VRE) “super bugs,” the proliferation of bacteria causing necrotizing infections “flesh eating bacteria” (Deans 1994), and the increasing elderly population with multiple comorbidities and compro- mised immune systems (AIDS, diabetes, renal failure, chemotherapy) have more than kept pace with these medical advances. Today, severe aggressive odonto- genic infections remain an important cause of mor- bidity, mortality, and utilization of hospital resources.

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The cost of treatment for odontogenic infections in the USA is staggering. According to Eisler et al. (2013), between 2001 and 2010 the total national cost of inpatient care approached $200 million annually. Kim et al. (2012) reported that in 2007 a total of 302 507 emergency department visits were attributed to facial cellulitis in the USA, with a total cost of $241 541 694.

10.2 Etiology and epidemiology

Although odontogenic sources, for example, periapical infections, pericoronitis, and periodontal abscesses, account for most of the deep neck infections (DNI), they may also be secondary to facial fractures, pene- trating neck wounds, skin lesions, for example, furun- cles, folliculitis, infected sebaceous cysts, and sali- vary gland infections or sinusitis. In addition, we must beware of malignancy presenting as an infection. In Wang et al.’s (2005) series, 157 of 250 maxillofacial infections (63%) were of odontogenic origin. Bakir et al. (2012), in a series of 173 cases, report odonto- genic origin as the primary cause in 48.6%, periton- sillar in 19.7% and tuberculosis in 6.9%. Potter et al. (2002) found 69% of deep space infections to be of odontogenic origin and 11% peritonsillar (Riggio et al. 2007). In subcategorizing odontogenic infections, 65% were secondary to caries, 22% pericoronitis, and 22% were periodontal (some patients had more than one source), with 68% of cases originating in the mandibu- lar third molars, followed by the mandibular bicuspids and molars (Flynn et al. 2006a,b). Wang et al.’s (2005) study also confirmed previous study findings that most patients were adults, older than 18 years, and although 15% of patients were under 12 years old, severe odon- togenic infections were rare in teenagers (2%). Peters et al. (1996) reviewed 128 patients with only odonto- genic maxillofacial infections and found their average age to be 35.8 years, with a slight female predomi- nance (54%). However, other authors have described more male patients (62%) and also a high incidence of ethnic minorities: African American 54%, Hispanic 22% (Flynn et al. 2006a,b). In children, Dodson et al. (1989, 1991) showed that most had maxillary buccal infections, in contrast to adults who had more infec- tions related to the mandible.

Many publications have looked at these patients for comorbid conditions that may have immunosuppres- sive potential, with conflicting results reported con- cerning HIV and diabetes. Liu et al. (2013) studied a

total of 9888 patients with HIV matched with 49 440 randomly selected subjects; the log rank test indicated that patients with HIV had a significantly higher 8- year incidence rate of DNI than the control group. Zheng et al. (2012) reported a retrospective study of 191 diabetic and nondiabetic patients, and concluded that diabetic patients had more fascial spaces involved in the infections, required more incisions for drainage, had longer hospital stay, and developed more compli- cations. There is a wide range reported in the literature for the association of a compromised immune system with severe deep space infections, from 8% (Flynn et al. 2006a,b), 24% (Peters et al. 1996), up to 64.5% (Wang et al. 2005). In this last series, Wang et al. found that all 26 of their patients under the age of 18 years were healthy; however, of the 131 patients over 18 years of age, 64.2% were compromised: 8 with dia- betes, 35 with alcohol addiction, 34 with intravenous drug abuse, and 6 with HIV infection. However, this may reflect the patient population of the study which was set in a large urban public hospital, rather than a potential specific predilection for developing these serious infections.

In necrotizing infections, such as necrotizing fasci- itis, underlying medical compromise with diabetes, steroid or chemotherapy treatment, leukemia, renal failure, or HIV is very frequent and may, at least in part, account for the increased mortality rate of 20–73% (Lin et al. 2001). In one large series of head and neck infection patients with necrotizing fasciitis, 89.4% had an underlying medical condition which included 72.3% with diabetes, 19.1% with acute or chronic renal failure, and 12% with underlying malignancy (Lin et al. 2001). A comprehensive review article of necrotizing fasciitis concluded that 70% of adult cases have an underlying immune compromising disease (McGurk 2003). Odontogenic infection is less frequent (10.6%) as an etiology (Lin et al. 2001), as most cases result from trauma or surgery.

10.3 Microbiology

In most odontogenic infections there is a polymi- crobial mixed aerobic and anaerobic infection with intraoral bacteria in 70% of the cases. Pure aerobic infections are much less common comprising approxi- mately 5%, and pure anaerobic infections comprise the remaining 25%. Cultures show mixed infections with

Severe Head and Neck Infections 233

Gram-positive cocci and Gram-negative rods predom- inating. Wang et al. (2005) reported that in their 250 cases, obtaining cultures and sensitivity reports did not appear to be clinically helpful as it failed to lead to any antibiotic or treatment change. Other authors recom- mend cultures to guide antibiotic selection for exten- sive or rapidly spreading infections, in immunocom- promised patients, recurrent infections, where there is no response to antibiotics, nosocomial infections, or necrotizing and gas-producing infections as opposed to purely empiric therapy (Jones and Cadelaria 2000). Lastly, some researchers have described the use of molecular microbiologic methods. As noted in Chap- ter 5, this methodology has been shown to detect uncul- tivable species of bacteria (Riggio et al. 2007). The clinical applicability of these types of diagnostics is questionable because the phenotypic properties of the microorganisms and their responses to antibiotics can- not be detected with certainty. Therefore, at this time it does not appear as though molecular microbiology can change the initial management of these types of cases. However, there may be some benefit in assist- ing with the selection of a narrow-spectrum antibi- otic after surgical therapy has been implemented. In Flynn et al.’s (2006b) prospective study, a total of 45 different species of bacteria were isolated, both aero- bic and anaerobic, in their cultures of severe odonto- genic infections. The most frequent pathogens isolated were Prevotella spp., Streptococcus viridans (which includes the Streptococcus milleri group), and Pep- tostreptococcus spp. (some species are now classified under Parvimonas spp.). These findings agree with other publications since 1998 (Sakamoto et al. 1998; Kuriyama et al. 2000; Stephanopoulus and Kolokotro- nis 2004).

In Flynn et al.’s study, a positive culture for Pep- tostreptococcus spp. was a negative predictor for abscess formation, but positively associated with a cellulitis. Unless patients were allergic or had a necro- tizing infection, all were started on intravenous peni- cillin G, and subsequently 19% of isolated species were found to be penicillin-resistant, and one or more of these resistant bacteria were found in 54% of cases. In addition, clindamycin-resistant strains were found in 17% of cases. All six patients who failed penicillin treatment were found to have one or more penicillin- resistant strains, and six out of ten (60%) patients with resistant bacteria were treatment failures. However, the authors point out that the identification of penicillin- resistant organisms was often delayed up to 2 weeks,

at which time clinical decisions had already been made (Flynn et al. 2006a).

Huang et al. (2005) analyzed a cohort of 56 patients with diabetes and 129 patients without diabetes pre- senting with DNI. They found the expected bacte- rial cultures in the nondiabetic group, with S. viri- dans (43.7%), followed by Peptostreptococcus spp. and Klebsiella pneumoniae; however, the predominant pathogen in the diabetic group was K. pneumoniae (56.1%) followed by S. viridans, and this difference was highly significant (p = 0.0004).

Because of the very predictable polymicrobial mixed infections, most patients are treated empiri- cally on hospital admission with regimens that usu- ally include a penicillin, clindamycin, and/or metron- idazole. Reported antibiotic regimes are penicillin + metronidazole, or clindamycin in 60% and 20% of 157 patients, respectively (Wang et al. 2005), and penicillin G with or without oxacillin in 54% or clindamycin in 33% of patients (Flynn et al. 2006a), or cefotaxime + metronidazole + corticosteroids in one trial of antibi- otics without surgical drainage (Mayor et al. 2001).

In necrotizing fasciitis, most infections particularly those of the limbs, perineum, and abdomen have been attributed to group A streptococci, which appears to have an increasing incidence (Kaul et al. 1997). How- ever, although the classic disease is seen with group A streptococci alone or in combination with Staphylo- coccus aureus, this disease may also be polymicrobial with prominent anaerobic species present (Brook and Frazier 1995). In Lin et al.’s (2001) paper, K. pneu- moniae was the most common organism followed by S. aureus and Streptococcus spp. Because of the severe systemic compromise and high mortality rate, triple antibiotic therapy is usually mandated. Regimens rec- ommended include a penicillin, aminoglycoside, and antianaerobe (Lin et al. 2001), or gentamycin, metron- idazole, and clindamycin (Flynn et al. 2006a).

10.4 Anatomy and pathogenesis of spread

Essentially, there are three patterns of spread seen in severe odontogenic infections, although a mix- ture of these types is frequently seen. These are abscess formation, cellulitis, and necrotizing fasci- itis. Once the infection has spread beyond the apex of the involved tooth into the alveolar process of the maxilla or mandible, the bacteria can propagate and spread through the medullary spaces until the cortical

234 Endodontic Microbiology

Fig. 10.1 Pathways of spread of odontogenic infections.

plate of the jaw is reached and eventually perforated. Infection then involves the primary spaces (vestibular/ palatal, canine, buccal, submandibular/submental, sublingual), secondary spaces (masseteric, pterygo- mandibular, temporal, lateral pharyngeal, retropha- ryngeal, danger space, prevertebral), or distant sites (mediastinum, brain) (Figure 10.1).

An abscess is a localized collection of pus classically caused by Staphylococcus species which produce the coagulase enzyme, which “walls off” infections. Cel- lulitis is an infection that spreads widely through the tissues, not usually producing much pus but permeat- ing tissue spaces, and is classically caused by strep- tococci that produce collagenase and hyaluronidase to break down the ground substance of connective tissue and promote spread. In necrotizing fasciitis, there is a rapid progressive liquefaction of subcutaneous fat and fascia with thrombosis of the subdermal veins. The skin becomes reddened and then necrotic as its blood supply is lost. Although the underlying muscle is usu- ally preserved, myositis can occur as can gas formation (“gas gangrene”). Extensive underlying skin necrosis may be present with comparatively little in the way of clinical signs.

The patterns of spread of the cellulitic infections will determine symptoms and signs and also whether the airway is liable to be compromised. Pus and cel- lulitic fluid will usually follow the path of least resis- tance and track along fascial planes in the neck and face. The direction of spread for odontogenic infec- tions is therefore determined by the anatomic rela- tionships of the tooth root to the jawbone and the muscle insertions into the jaw. The majority of severe odontogenic infections are related to the mandibular third molars, followed by the other molars and pre- molars. It should also be appreciated by the reader that most of these spaces or potential spaces are inter- connected so that many of these infections involve multiple spaces. Additionally, fascial layers extending

along the retropharyngeal–prevertebral and carotid– jugular sheath may allow infection to spread into the mediastinum and thorax, far from the head and neck.

Once pulp necrosis has occurred, periapical infec- tion is located within the medullary bone of the jaw. This infection will eventually perforate the cortical plate to gain access to the soft tissue spaces. In the mandibular molar region, the roots tend to be situated lingually and lingual perforation is more common. If the apex of the tooth is inferior to the attachment of the mylohyoid muscle and to the mylohyoid ridge (which is usually the case), pus can directly enter the sub- mandibular space. If the apex is superior to the mylohy- oid attachment then it will enter the sublingual space. Both these spaces are connected posteriorly at the free edge of the mylohyoid muscle. If the pus perforates buccally in the mandibular molar region and it is infe- rior to the buccinator muscle attachment, it can involve the buccal space. If the apex of the tooth is supe- rior to the buccinator insertion, the pus will present intraorally as a vestibular abscess. (This can easily be drained intraorally and is not discussed further.) Severe pericoronitis and periapical infection from mandibular third molars may track posteriorly into the masticator and lateral pharyngeal spaces. In the maxilla, buccal perforation from posterior teeth may enter the buccal space and from the canine to the canine space, which can lead to orbital or intracranial involvement. Infec- tions perforating the palatal side of the alveolus, for example, from lateral incisors or the palatal roots of molars, are usually well confined by the thick bound down palatal mucosa of the hard palate and simply require intraoral drainage.

In order to understand the presentation and man- agement of these infections, some knowledge of the anatomy of these spaces is essential. A brief outline of their anatomy and intercommunications is given next.

Severe Head and Neck Infections 235

Fig. 10.2 Anatomic boundaries of the submandibular space. Source: Hohl et al. (1983).

10.4.1 Submandibular space

Several reports have identified the submandibular space the most frequent site of infection. Poeschl et al. (2010), in a study of 206 patients with the diagnosis of an odontogenic head and neck deep space infec- tion, reported 34.1% in the submandibular space (Fig- ure 10.2).

The submandibular space lies inferior to the mandible and contains the submandibular gland, lymph nodes, the facial artery, and the anterior facial vein. The mandibular branch of the facial nerve lies superiorly and the lingual and hypoglossal nerve poste- riorly and deeply. The medial–deep boundary is delin- eated by the mylohyoid muscle with contributions from the hypoglossal and styloglossus, while laterally and superficially are skin and platysma. Anteriorly is the anterior belly of the digastric muscle although the submandibular space communicates freely anteriorly with the submental space. Posteriorly are the posterior belly of digastric and the stylohyoid muscles. Supe- riorly is the lower border of the mandible and the pterygomasseteric sling and inferiorly the hyoid bone. The space communicates with the submental anteri- orly, the sublingual space posterosuperiorly, and the lateral pharyngeal posteriorly.

10.4.2 Sublingual space

The sublingual space contains the sublingual gland, Wharton’s duct, and the lingual nerve. Medially it is bounded by the genioglossus, geniohyoid, and sty- loglossus muscles, and laterally by the lingual plate

of the mandible. Posteriorly it connects with the lat- eral pharyngeal space. Superiorly is the mucosa of the floor of mouth and inferiorly the mylohyoid muscle (Figure 10.3).

10.4.3 Submental space

The submental space lies between the anterior bel- lies of the digastric muscles, superiorly bounded by the mylohyoid muscle, and inferiorly bounded by the platysma (Figure 10.4).

Ludwig’s angina (Figure 10.5a,b), first described by William Frederick von Ludwig in 1836, is important to highlight here. This is a bilateral infection of the submandibular, sublingual, and submental spaces; the term angina means “to constrict.” Ironically, Ludwig died at the age of 75 of an acute infection of the neck. Some feel he succumbed to the very disease he is famous for describing.

10.4.4 Buccal space

The buccal space contains fat, branches of the facial nerve, the parotid duct, and the facial artery and vein. Medially lies the buccinator muscle and laterally skin. Anteriorly is the oral commissure with zygomaticus major and depressor anguli oris muscle, and the infraorbital space, while posteriorly is the pterygo- mandibular raphe. Superiorly lies the zygomatic arch and inferiorly the mandible. Infections can track to the submandibular and masticator spaces (Fig- ure 10.6).

236 Endodontic Microbiology

Fig. 10.3 Anatomic boundaries of the sublingual space. Source: Hohl et al. (1983).

10.4.5 Lateral pharyngeal space

This is a pyramid-shaped space with its base superiorly which can be divided into an anterior and posterior compartment by the styloid muscles. It contains fat, lymph nodes, the carotid artery, and jugular vein as well as cranial nerves IX–XII. The medial boundary is the superior and middle constrictor muscles and laterally are the medial pterygoid and parotid glands. The base of the skull is superior and the hyoid bone inferior. Anteriorly lies the pterygomandibular raphe, but the space communicates freely with the

submandibular and sublingual spaces, while posteri- orly it communicates directly with the retropharyngeal space, which runs inferiorly to C6–T4 region where its fascia fuses with the alar fascia. If pus perforates the alar fascia, it enters the prevertebral (danger) space and can track along the whole length of the spinal column (Figures 10.7 and 10.8).

10.4.6 Masticator space

This space is composed of those spaces formed by the insertions of the masticatory muscles to the vertical

Fig. 10.4 Anatomic boundaries of the submental space. Source: Hohl et al. (1983).

Severe Head and Neck Infections 237

(a)

(b)

(c)

Fig. 10.5 (a) Anatomic boundaries of the Ludwig’s angina (Source: Hohl et al. 1983); (b) clinical example of Ludwig’s angina.

ramus of the mandible, and it is subdivided into the masseteric, pterygomandibular, and temporal spaces.

10.4.6.1 Masseteric space

This small potential space is bounded medially by the buccal plate of the vertical ramus of the mandible and laterally by the masseter muscle. Superiorly it can

pass into the infratemporal space, but inferiorly it is restricted by the attachment of the pterygomasseteric sling to the mandible (Figure 10.9a).

10.4.6.2 Pterygomandibular space

This space is bounded medially by the medial ptery- goid muscle and laterally by the lingual surface of the

238 Endodontic Microbiology

Fig. 10.6 Anatomic boundaries of the buccal space. Source: Hohl et al. (1983).

(a) (b)

Fig. 10.7 (a) Anatomic boundaries of the lateral pharyngeal space; (b) anatomic boundaries of the retropharyngeal space. Source: Hohl et al. (1983).

vertical ramus of the mandible. Superiorly lies the lat- eral pterygoid muscle and inferior the pterygomasse- teric sling. Anteriorly are the pterygomandibular raphe and the parotid gland, respectively (Figure 10.9b).

10.4.6.3 Temporal space

This is divided into the superficial and deep spaces. In the deep space the pus lies between the temporal sur- face of the skull medially and the temporalis muscle

p e rc

e n ta

g e

De ep

te m

po ra

O rb

ita l

M as

se te

ric

Ca ni ne

fo ss

Su bm

en ta

Pt er

yg om

an di bu

la r

Bu cc

Su bm

an di bu

la r

35 30 25 20 15 10 5 0

Fig. 10.8 Spaces involved as percentages of all cases. Source: Adapted from Poeschl et al. 2010 [151–156]. Reproduced with permission of Elsevier.

Severe Head and Neck Infections 239

(a) (b)

Fig. 10.9 (a) Anatomic boundaries of the submasseteric space; (b) anatomic boundaries of the pterygomandibular space; (c) anatomic boundaries of the superficial and deep temporal space. Source: Hohl et al. (1983).

240 Endodontic Microbiology

Fig. 10.10 Anatomic boundaries of the canine space. Source: Hohl et al. (1983).

laterally, while in the superficial space the temporalis muscle is medial and the thick temporalis fascia lateral. These spaces communicate with the rest of the masti- cator spaces inferiorly via the infratemporal space and the attachment of the temporalis tendon to the coronoid process (Figure 10.9c,d).

10.4.7 Canine space

This space contains terminal branches of the facial artery and vein (angular vein). Medially lies the nasal bone and laterally the buccal space. Anterior is the facial skin and posterior the maxilla, while inferiorly are the levator labii superiori, levator labii, and alaeque nasi muscles. The orbital septum is the superior bound- ary (Figure 10.10).

Lastly, an infectious process worth brief discus- sion for the sake of thoroughness is NOMA, from the Greek “to devour.” Otherwise known as cancrum oris, NOMA is associated with a virulent organism, Fusobacterium necrophorum (Paster et al. 2002). This infectious process, some feel, is an extensive form of acute necrotizing ulcerative gingivitis (ANUG). It typi- cally begins with necrosis of the periodontium and then extends into the soft tissue of the face, hence its pre- tense “to devour” (Figure 10.11). The common denom- inator in NOMA is malnutrition and immunosuppres- sion; in African NOMA there is also an association with AIDS, malaria, measles, and anemia. NOMA is rarely seen in western countries because of their low malnutrition rates; however, it may develop in severely malnourished immune-compromised patients, such as those undergoing chemotherapy or transplanta- tion, and, as previously mentioned, the AIDS pop- ulation. NOMA is initially managed by nutritional

Fig. 10.11 Example of cancrum oris in an HIV patient.

supplementation, fluid resuscitation, blood transfu- sion, and soft tissue debridement.

Reconstruction for these patients can be surgically challenging, especially if the functional units of the face are involved. Many reconstructive surgeons will follow the principles of the “reconstructive ladder”; which is as follows:

� Healing by secondary intention; � Skin grafting; � Local flaps; � Regional flaps; � Free tissue transfer.

10.5 Diagnosis

10.5.1 History

The diagnosis for patients with severe aggressive odontogenic infections will usually be evident but

Severe Head and Neck Infections 241

commences with a history and physical examination. Important areas to cover in the history include precipi- tating factors (toothache or trauma), and specific symp- toms that may alert the clinician to the fact that that the patient has impending airway obstruction. Symp- toms that are very concerning are dysphagia and/or odynophagia, or difficulty in and/or pain with swallow- ing, or speaking with a muffled or “hot potato” voice quality. Patients with these symptoms represent true surgical emergencies and can quickly progress to a life- threatening situation. A complaint of inability to fully open the mouth (trismus) is suggestive of pus causing spasm of the masticatory muscles. The examination is directed toward an overall systemic assessment of the patient and a good head and neck examination.

10.5.2 Systemic examination

Initial examination may reveal a patient sitting with his/her head held forward, drooling, and using his/her accessory muscles of respiration. This is the worst case scenario, with the patient leaning forward to pro- tect his/her airway and unable to control his/her own saliva because of tongue edema. It can be predicted that even attempting to lie the patient flat to intubate him/her can precipitate complete obstruction. Fortu- nately, the majority of patients do not present at such an advanced stage. Fever and tachycardia may be marked especially in children, and this may exacerbate dehy- dration in patients with inability to swallow. These systemic manifestations of acute infection may desta- bilize preexisting diseases (e.g., diabetes) and cause hyperglycemia and ketoacidosis. In necrotizing fasci- itis, severe systemic effects may be seen; more than 50% have hypotension and 10–30% display one or more of the following: acute renal failure, coagulopa- thy, abnormal liver function, acute respiratory distress syndrome, abnormal liver function, or hemolytic ane- mia (McGurk 2003).

10.5.3 Head and neck examination

The classic signs of acute inflammation, swelling, red- ness, pain, and loss of function are usually present in patients with abscess or cellulitis but not always in those with necrotizing fasciitis, which can have gross necrosis with little in the way of obvious inflamma- tory signs. In an abscess, the swelling is localized and painful and fluctuant to the touch. The overly- ing skin may be shiny or reddened and in advanced cases becomes thinned prior to spontaneous drainage

Fig. 10.12 Clinical picture of a facial swelling and abscess of submandibular space.

of pus (Figure 10.12). In cellulitis, the swelling is dif- fuse, firm, and brawny to palpation. The overlying skin may be red and pitting edema can occur. In necro- tizing fasciitis, the skin is diffusely reddened over a wide area, and with progression becomes white and then black and necrotic. Blisters or bullae may occur and the skin is tender to palpation. Crepitus due to gas-forming microorganisms may be elicited, such as Clostridium perfringens, Clostridium novyi, and Kelb- siella pneumoniae.

In abscess or cellulitis, the swelling may be obvious on initial visual examination, for example, swelling of the cheek in buccal space infections or of the submandibular region in submandibular space infections. Considerable swelling may occur in the lateral pharyngeal–masticator space, which can only be detected on intraoral examination when the bulging of the pharyngeal wall, soft palate, and deviation of the uvula will be noted. However, in infections around the masticatory muscles severe trismus may occur, which will prevent a good intraoral examination and be a problem for airway access. Some space infections, such as the masseteric space, have trismus as the most prominent sign. Lastly, because odontogenic infec- tions can be life-threatening as a result of airway loss, the practitioner must thoroughly evaluate swelling on the floor of mouth and tongue, and oropharynx (Figure 10.12).

10.5.4 Laboratory investigations

Blood tests will usually show a leukocytosis with increased percentage of granulocytes and a left shift

242 Endodontic Microbiology

(i.e., an increase in immature cells as the body attempts to produce more granulocytes). In these infections, the presence of a “normal” white cell count is clinically abnormal and usually indicates a deficient immune response. Blood glucose and electrolytes will be essen- tial for diabetic patients and for suspected necrotizing fasciitis to diagnose underlying systemic problems.

10.5.5 Imaging

Two-dimensional radiographs such as a panoramic view are useful in identifying an odontogenic etiology. Soft tissue lateral views of the neck have been used to visualize increased prevertebral soft tissue thickening in retropharyngeal abscess (Haug et al. 1991). A CT scan is now the imaging modality most commonly used to assess severe neck infections (Figure 10.13) Lazor et al. (1994) reviewed 38 patients with retro- or parapharyngeal abscesses and found that intraop- erative findings correlated with CT scan in 76.3% of cases, with a false positive rate of 13.2% and a false negative rate of 10.5%. Miller et al. (1999) compared clinical examination with contrast-enhanced CT in a blinded prospective trial of 35 patients to predict the presence of a drainable purulent infection in suspected deep neck abscess. Twenty-two patients had purulent drainable collections. Clinical examination was 63% accurate with sensitivity of 55% and specificity of 73%, whereas CT was 77% accurate with sensitivity of 95% and specificity of 53%. If the two modalities were combined, accuracy was 89%, sensitivity 95%, and specificity 80%. The authors concluded that both clinical examination and contrast-enhanced CT were critical components in diagnosing these infections. Recently, Smith et al. (2006) attempted to use Hounsfield units from their CT scan images to predict the presence of pus or cellulitis and found this was unreliable. Because of their negative exploration rate of 25%, these authors emphasized the importance of clinical decision-making for surgical drainage. Although the contrast-enhanced CT appears to be the accepted “gold standard” in imaging, ultra- sound has also been advocated in children to assess abscess formation (Yeow et al. 2001; Duque et al. 2006).

In other studies, MRI was considered superior to CT in the initial evaluation of neck infections. Munoz et al. (2001) evaluated 47 patients with DNI: all patients underwent CT and MRI. The results demonstrated that MRI was superior to CT in regard to lesion conspicuity,

number of anatomic spaces involved, extension, and source. These findings suggest that MRI may be used as the first and perhaps the only modality to initially evaluate patients with neck infections when clinically feasible. At the University of Maryland, CT scan with contrast is the gold standard for deep space infection imaging; the difficulties with MRI are the cost, time, and availability.

CT scanning has many advantages for the clinician. It shows all the spaces of the head and neck to demon- strate involvement of areas that were not clinically suspected, as well as visualizing spaces that cannot be directly examined in patients with severe trismus, such as the brain and mediastinum; when extensive spread and complications need to be ruled out, clini- cal assessment is paramount. Patients with impending airway obstruction should not be delayed from having an airway secured in the operating room to wait for a CT scan, nor should the surgeon forget that placing these patients in a supine position for scanning places them at risk for airway obstruction. The CT scanner is often in a remote area of the hospital and not ideally suited to deal with airway emergencies. Despite the valuable information that can be gained from imaging, the clinician has to balance the benefits against the risk in any case where the airway is an issue.

10.6 Airway management

It is essential to secure the airway in patients with severe DNI. This is frequently a challenging task because of generalized fascial swelling with edema of the tongue, pharynx, and larynx, often accompanied by severe trismus. When the cords can be visualized then standard oral endotracheal intubation is a safe way to proceed, especially with the use of a Glidescope. How- ever, in the presence of edema, inappropriate manip- ulation of the airway can cause further swelling and bleeding, requiring emergent surgical intervention. In addition, a retropharyngeal or peritonsillar collection of pus may be ruptured by injudicious use of the laryn- goscope with aspiration of purulent material and sub- sequent pulmonary or mediastinal complications. In most severe infections, standard oral intubation is not possible so the choice of airway management will be either a fiber optic intubation or a tracheotomy. In Potter et al.’s (2002) review of the literature, they found that in cases managed by otolaryngologists tra- cheotomy was the treatment of choice and in those

Severe Head and Neck Infections 243

(a)

(b)

(c)

Fig. 10.13 (a) Panorex showing gross decay of molar teeth; (b) CT scan of right submandibular, submasseteric, pterygomandibular, and lateral pharyngeal abscess formation; (c) clinical photo of patient.

managed primarily by oral and maxillofacial surgeons fiber optic intubation was preferred.

Ovassapian et al. (2005) reported 26 patients with DNI and 17 with Ludwig’s angina who underwent awake fiber optic intubation. Three patients were intu- bated in the sitting position, 2 in Fowler’s position,

and 21 supine with head 10–15◦ raised. Twenty-five of the 26 intubations were successful. Postsurgery, seven patients were kept intubated and five under- went tracheotomy. Twelve patients remembered part of the procedure (Ovassapian et al. 2005). In Flynn et al.’s (2006a,b) series, 49% underwent fiber optic

244 Endodontic Microbiology

(a) (b)

Fig. 10.14 (a) Glidescope. (b) Fiberoptic intubation.

intubation, 43% direct laryngoscopic intubation, and 8% no intubation; only one patient (3%) had a tra- cheotomy and this was the patient who required reoper- ation. Obviously, awake fiber optic intubation requires a skilled anesthesiologist and a surgeon on hand should intubation prove unsuccessful. The major advantage with intubation is avoiding the complications asso- ciated with tracheotomy; however, the disadvantages are that the airway is not secured and there is an ele- ment of discomfort for the patient, at times requiring mechanical ventilation. In this setting it is often diffi- cult to assess when the patient is suitable for extuba- tion despite the presence of a cuff leak and ventilatory weaning parameters being met. Reintubation can be hazardous in these patients.

Tracheotomy requires skill and experience as swelling of the neck may obscure the usual landmarks. In the series by Potter et al. (2002), 34 patients under- went tracheotomy and 51 endotracheal intubation. One patient in the tracheotomy group had a cerebrovascular accident from rupture of a carotid aneurysm during the tracheotomy, and two patients in the intubation group died: one from an unplanned extubation and the other from laryngeal edema postextubation with inability to reintubate. Patients with tracheotomy had a shorter hospital stay, significantly less time in the intensive care unit, and 60% less hospital cost (Potter et al. 2002). It was felt that tracheotomy provided better

utilization of critical care resources. In true airway emergencies, cricothyroidotomy is probably a quicker and safer choice for the nonspecialist surgeon to secure an airway, but again swelling and edema may make palpation of the cricoid and thyroid cartilages difficult. In these cases, the use of a lumbar needle on a syringe to identify the cricothyroid ligament by aspirating for air may be very helpful (Figure 10.14).

10.7 Medical and surgical management

Obviously, initial therapy for these patients will be directed to their overall systemic condition. In the case of necrotizing fasciitis, fluid resuscitation for sep- tic shock and control of hyperglycemic ketosis may be required prior to surgical intervention. Diabetic patients who are uncontrolled should also be stabi- lized as much as possible prior to surgery. The tradi- tional management of these infections is reflected in the statement from the article by Wang et al. (2005) that “early dental extractions, incision and drainage, coupled with intravenous antibiotic therapy is the most effective treatment.” Most surgeons would feel that pus is most effectively treated by surgical drainage (Fig- ure 10.15). Even in the case of cellulitis that is causing systemic signs and involving important fascial spaces, aggressive incision and drainage will help earlier

Severe Head and Neck Infections 245

Fig. 10.15 Incision and drainage of left submandibular abscess.

resolution (Flynn 1991). This may be due to decom- pression of the space or changing the microenviron- ment to promote a more aerobic background. However, it is currently debated in the literature whether there is a place for nonsurgical management or radiologically guided aspiration, and for which types of patients it may be indicated (Box 10.1).

Box 10.1 Principles of surgery

� Establish airway � Removal of the source � Dependent drainage � Culture � Drain placement � Antibiotics

It would appear from the literature that peritonsil- lar and parapharyngeal abscesses, especially those in children, can possibly be treated medically with intra- venous antibiotics alone. Sichel et al. (2002) undertook a prospective study on patients with infection limited to the parapharyngeal space. Twelve patients presented with this diagnosis but five were excluded as other spaces were involved. Seven patients without systemic signs of shock were treated with intravenous amoxi- cillin/clavulanic acid for 9–14 days and all were cured without need for drainage. Six of the seven cases were of pediatric patients (Sichel et al. 2002). In another prospective study of 34 patients with parapharyngeal

abscesses (confirmed by contrast-enhanced CT scan), all of whom received antibiotics, 15 patients were sur- gically drained and 19 cases had antibiotics alone or needle aspiration. Length of hospital stay was 8.2 days in the medical group and 11.6 days in the surgical group. One patient in the medical group developed mediastinitis. The conclusion was that parapharyngeal infections may localize and be effectively treated with- out drainage (Oh et al. 2007). However, in a review of 205 children with lateral neck infections, it was found that the clinical diagnosis corresponded with the radiologic findings in only 73.6% of cases and ultrasound with the surgical findings in only 65.2%. It was also found that clinical assessment of lateral neck infections was poor because of underestimating suppuration, although the diagnosis of an abscess clin- ically correlated highly with surgical findings (Court- ney et al. 2007). Therefore, diagnosis and selection of patients for conservative treatment or drainage is fraught with inaccuracy.

In peritonsillar abscesses, catheter or needle drainage is the method of choice and ancillary steroids may reduce morbidity (Herzon and Martin 2006). Image-guided aspiration using either CT or ultrasound has been applied to DNI. Poe et al. (1996) reported the use of CT-guided aspiration in a small series of 4 cases without complications. In 15 cases of unilocular neck abscess, 13 were successfully treated with nee- dle or catheter aspiration, 2 of whom required reaspira- tion. Two patients failed and required surgical drainage (Yeow et al. 2001). In a prospective controlled study of 14 patients with well-defined and/or unilocular abscesses, all were successfully treated by ultrasound- guided drainage, and eight patients had an indwelling catheter (Chang et al. 2005). There are a few trials that examine this method for space infections from purely odontogenic sources. One report described that the surgeon undertook aspiration drainage under ultra- sound guidance of 11 patients with a submasseteric space infection. Eight of 11 (73%) required no surgical drainage. Clinically, when mouth opening improved to 1.5 cm or more following aspiration drainage, cure was more likely (Al-Belasy 2005).

However, in most severe DNI from odontogenic causes, abscesses are not well defined and may occupy more than one space, and cellulitic processes are poorly confined with impending airway problems. In these circumstances, aggressive drainage using a large incision to allow adequate exploration of all spaces and breakdown of loculations with digital examination

246 Endodontic Microbiology

is preferred. Prior to drainage, needle aspiration may be performed to obtain a pus sample for culture and sensitivity in patients for whom this information may be necessary, for example, immunocompromised patients. The principles of drainage include placing incisions in noninvolved skin in a site that allows dependent drainage and avoids important anatomic structures, for example, facial nerve branches. Blunt dissection by opening hemostats or finger dissection will break down loculi and allow wide exploration of all spaces. Latex or rubber drains are placed to keep the wound open for drainage and to allow irrigation. In cases of cellulitis, a large incision to open the spaces widely and exploration of adjacent spaces with the placement of multiple drains is essential although cel- lulitic edema fluid is usually seen with little frank pus. Postdrainage there will initially be increased edema, and endotracheal tubes should not be removed when airway compromise is an issue. Wide drainage of these spaces will reduce the chance of missing loculations or involved spaces and the need for subsequent reop- eration for patients whose fever and swelling are not resolving. An alternative approach with small inci- sions for Ludwig’s angina in 113 patients has been published. In 62 there was extension to the parapharyn- geal space and 32 had retropharyngeal involvement. In this series, 33 patients had major complications such as mediastinitis, sepsis, or death, but the authors con- cluded that drainage using small incisions was safe and effective in Ludwig’s angina (Bross-Soriano et al. 2004) (Figure 10.16).

In necrotizing fasciitis, aggressive surgical debride- ment is essential and delay is associated with an increased death rate, so initial fluid resuscitation and stabilization should be rapid and not unduly delay surgery. The area of reddened skin is usually delin- eated with a surgical marking pen so that postsurgical progression can be followed. The skin is incised and usually no bleeding is observed due to blood ves- sel thrombosis. The underlying fascia is necrotic and “dishwater” pus is classic, although foul-smelling pus and gas may be obtained. The skin and fas- cia are excised radically until bleeding skin edges are observed. The underlying muscle is usually unin- volved; however, if it is involved, it is also vigorously debrided. The wound is packed open and irrigated with hydrogen peroxide and saline, with frequent dressing changes. Further visits to the operating room are done daily as the disease declares itself and more necrotic skin is excised. The surgeon will usually have mul- tiple operating room sessions before the disease is stabilized. It is usually recommended waiting 7–10 days until healthy granulation tissue is present prior to reconstruction. If available, hyperbaric oxygen may be helpful in these cases, but aggressive surgical man- agement is undoubtedly the primary treatment.

Reconstruction is not considered until after the wounds are stabilized with signs of a healthy granula- tion tissue bed. In addition, these patients are typically debilitated as a result of sepsis, poor nutritional status, and diminished immunologic response. Sequencing reconstructive strategies for patients with aggressive

(a) (b)

Fig. 10.16 (a) Aspiration of right submandibular space abscess being performed under sterile conditions to avoid contaminant organisms; (b) incision and drainage of right submandibular space abscess.

Severe Head and Neck Infections 247

head and neck infections (i.e., necrotizing fasciitis) can be a monumental challenge. These wounds can have extensive tissue loss following aggressive debridement, and therefore may require composite (e.g., skin, muscle, bone) tissue replacement. The chal- lenges of reconstruction for these aggressive head and neck infections include compromised airway support, impaired sensory and motor control, facial deformity, inability to control secretions, and impaired speech. The goals of reconstruction are similar to those in oncologic head and neck surgery, which are restoration of function and form. In order to achieve these goals, the surgeon must understand the impact of the recon- struction ladder on the surgical and prosthetic phases of reconstruction. The stages (“rungs”) of the ladder include primary closure, followed by skin grafts, local flaps, distant flaps, and finally free flaps (Figure 10.17).

A true comprehension of the architectural hard and soft tissue defects helps the surgeon in developing a strategic approach to these difficult and challenging reconstructions. The differences between a graft and flap are based on the donor tissues’ blood supply; a graft depends on the recipient bed’s blood supply and lacks its own. A flap is independent of the recipient bed’s blood supply and carries its own vascular sup- ply. There are different types of flaps available for uti- lization by the surgeon. These depend on the size and complexity of the defect and the underlying comor- bidities of the patient. A local flap transposes healthy tissue based on a random blood supply to the defect (Figure 10.18c); the regional flap rotates distant tis- sue based on an axial blood supply (blood vessel) to the defect site, and the free flap transfers distant tissue based on an axial blood supply by the donor vessels

(a)

(c)

(b)

Fig. 10.17 (a) Alar defect of infected wound; (b) wound thoroughly debrided and a bilobed local flap marked; (c) rotation and closure of local flap.

248 Endodontic Microbiology

(a) (b)

(c)

(d)

Fig. 10.18 (a) Upper lip debridement after a necrotizing infection; (b) example of a radial forearm free flap with the skin paddle based over the radial artery; (c) example of the raised flap prior to inset; (d) 1 month after upper lip reconstruction.

which are intentionally transected and then anasto- mosed to vessels in the recipient bed to restore blood flow (Figure 10.18a–d). The advantage of free tissue transfer it offers is a unique combination of vascu- larized tissue (bone, muscle, skin) in a single stage. The initial sequencing of reconstruction is focused on restoring oral lining, followed by soft tissue coverage of the face and then skeletal support. Prior to imple- menting any of the reconstruction options available, it is important the patient is medically stabilized; also, all the necrotic and infected tissues must be debrided with no signs of progression. As some of these cases

can result in a large surface area of skin loss, they are reconstructed in a manner that is analogous to a burn patient, with multiple skin-grafting procedures. How- ever, other patients may have composite tissue loss and are suitable candidates for free tissue transfer. There is a population of patients that may not be suitable candidates for a free flap surgery because of medi- cal comorbidities, and are more suitable for regional flaps. Because each option has its own inherent advan- tages and disadvantages, the surgeon can employ a combination in order to achieve the best reconstruction possible. Some of these cases can be extraordinarily

Severe Head and Neck Infections 249

complex, and require well thought-out planning and sequencing of treatment.

10.8 References

Al-Belasy FA. 2005. Ultrasound-guided drainage of submas- seteric space abscesses. J Oral Maxillofac Surg 63(1): 36– 41.

Bakir S, Hali Tanriverdi M, et al. 2012 Deep neck space infections: a retrospective review of 173 cases. Am J Oto- laryngol 33: 56–63.

Brook I, Frazier EH. 1995. Clinical and microbiological fea- tures of necrotizing fasciitis. J Clin Microbial 33: 2382– 2387.

Bross-Soriano D, Arrieta-Gomez JR, Prado-Valleros HS, Schimelmitz-Idi J, Jorba-Basave S. 2004. Management of Ludwing’s angina with small neck incisions 18 years expe- rience. Otolaryngol Head Neck Surg 130(6): 712–717.

Chang KP, Chen YL, Hao SP, Chen SM. 2005. Ultrasound- guided closed drainage for abscesses of the head and neck. Otolaryngol Head Neck Surg 132(1): 119–124.

Courtney MJ, Miteff A, Mahadevan M. 2007. Management of pediatric lateral neck infections: does the adage “… never let the sun go down on undrained pus …” hold true? Int J Pediatr Otorhinolaryngol 71(1): 95–100.

Deans M. 1994. Flesh eating bug scare. Lancet 343: 1418. Dodson TB, Barton JA, Kaban LB. 1991. Predictors of out-

come in children hospitalized with maxillofacial infections a linear logistic model. J Oral Maxillofac Surg 49: 838– 842.

Dodson TB, Perrot DH, Kaban LB. 1989. Pediatric max- illofacial infections: a retrospective study of 113 patients. J Oral Maxillofac Surg 47: 327–330.

Duque CS, Guerra L, Roy S. 2006. Use of intraoperative ultrasound for localizing difficult parapharyngeal space abscesses in children. Int J Pediatr Otolaryngol 71(3): 375–378.

Eisler L, Wearda K, Romatoski K, Odland RM. 2013. Mor- bidity and cost of odontogenic infections. Otolaryngol Head Neck Surg 149(1): 84–88.

Flynn TR. 1991. Odontogenic infections. Oral Maxillofac Clin North Am 3: 311–329.

Flynn TR, Shanti RB, Hayes C. 2006a. Severe odontogenic infections, Part 2: Prospective outcomes study. J Oral Maxillofac Surg 64: 1104–1113.

Flynn TR, Shanti RB, Levi MH, Adams AK, Kraut RA, Trieger N. 2006b. Severe odontogenic infections, Part 1: Prospective report. J Oral Maxillofacial Surg 64: 1093– 1103.

Haug RH, Wible RT, Lieberman J. 1991. Measurement stan- dards for the prevertebral region in the lateral soft-tissue radiographs of the neck. J Maxillofac Surg 49: 1149– 1156.

Herzon FS, Martin AD. 2006. Medical and surgical treat- ment of peritonsillar, retropharyngeal, and parapharyngeal abscesses. Curr Infect Dis Rep 8(3): 196–202.

Hohl TH, Whitacre RJ, Hooley JR, Williams BL. 1983. Diag- nosis and Treatment of Odontogenic Infections. Seattle, WA: Stoma Press.

Huang T-T, Tseng F-Y, Liu T-C, Hsu C-J, Chen Y-S. 2005. Deep neck infections in diabetic patients: Comparison of the picture and outcomes with nondiabetic patients. Oto- laryngol Head Neck Surg 132: 943–947.

Jones JL, Cadelaria LM. 2000. Head and neck infections. In: Fonseca R (ed.), Oral and Maxillofacial Infections in Oral and Maxillofacial Surgery, p. 81. WB Saunders.

Kaul R, Mcgeer A, Low DE, Green K, Schwartz AE, Simar AE. 1997. Population based surveillance for group A strep- tococcal necrotizing fasciitis. Am J Med 103: 18–24.

Kim MK, Allareddy V, Nalliah RP, Kim JE, Allareddy V. 2012. Burden of facial cellulitis: estimates from the Nationwide Emergency Department Sample. Oral Surg Oral Med Oral Pathol Oral Radio Endod 114: 312– 317.

Kuriyama T, Karasawa T, Nakagawa K, Saiki Y, Yamamoto E, Nakamura S. 2000. Bacteriologic features and antimi- crobial susceptibility in isolates from odontogenic infec- tions. Oral Surg Oral Med Oral Path Oral Radiol Endod 90: 600–608.

Lazor JB, Cunningham MJ, Eavey RD, Weber AL. 1994. Comparison of computed tomography and surgical find- ings in deep neck infections. Otolaryngol Head Neck Surg 111: 746–750.

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Chapter 11 Endodontic Infections and Pain Anibal Diogenes and Ken M. Hargreaves

11.1 Introduction 11.2 Biology of the pain system 11.3 Central sensitization 11.4 Persistent pain following endodontic

therapy

11.5 Mechanisms of pain due to endodontic infections

11.6 Clinical strategies for treating pain due to endodontic infections

11.7 References

11.1 Introduction

Odontogenic pain is a prevalent condition. Epidemi- ologic studies indicate that dental pain is reported by about 12–14% of the population, which translates to tens of millions of patients in the USA alone (Locker and Grushka 1987; Lipton et al. 1993). As odontalgia is often directly related to microbial infection and result- ing tissue inflammation, this indicates that one of the most common forms of acute pain is actually a conse- quence of endodontic infection. Research conducted in the last several decades has greatly increased our understanding of the pain system and it is now widely appreciated to be a highly dynamic sensory system that rapidly alters its response properties due to con- ditions such as inflammation resulting from bacterial infection. Increased knowledge of the pain system and its properties contributes to improved clinical skills for diagnosis and treatment of odontogenic pain. In this chapter, we review the pain system and its interaction with microbial and inflammatory factors and use this knowledge base to make evidence-based recommen- dations for managing endodontic-related pain.

Although many patients may view pain from a binary perspective (“it hurts” or “it doesn’t hurt”), it is important to realize that this sensory system undergoes

substantial dynamic alterations following tissue injury. This neuronal plasticity is manifested in three major pain symptoms: allodynia, hyperalgesia, and sponta- neous pain.

Allodynia is defined as a reduction in pain thresh- old where normally nonpainful stimuli can now elicit pain. Mechanical allodynia is a reduction in mechan- ical pain thresholds and classically is evaluated by percussing a tooth with a mirror handle. Studies of nearly 1000 patients indicate that mechanical allody- nia can occur in 57% of patients with a diagnosis of irreversible pulpitis (Owatz et al. 2007), indicating that this is an early feature of pulpal inflammation. New technologies for diagnosing mechanical allody- nia have been introduced, which provide an actual measure of mechanical allodynia in painful teeth (i.e., in newtons of force) rather than the outcome of the percussion test (Khan et al. 2007). In this study, teeth diagnosed with symptomatic irreversible pulpitis dis- played a 77% reduction in the mechanical allodynia threshold (maximum tolerable bite force). This reduc- tion was largely blocked by local anesthesia, indi- cating that it was, at least in part, due to peripheral sensitization. Interestingly, the same study detected a reduction in the mechanical allodynia threshold in healthy contralateral teeth, suggesting that a “central

251

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sensitization” (discussed later) was also established in cases of symptomatic irreversible pulpitis. Thus, pul- pal inflammation is not isolated to the pulpal tissues, but extends to the surrounding apical tissues causing sensitization of periradicular nociceptors and mechan- ical allodynia. The robust activation of pulpal and peri- radicular nociceptors in this presentation may lead to the development of central sensitization.

Hyperalgesia is defined as an increase in the per- ceived magnitude of a noxious stimulus; in other words, a stimulus that is painful when applied to healthy tissue is perceived as being much more painful under conditions of hyperalgesia. This is classically evaluated by the response to cold stimulation of teeth, where the response becomes much more exaggerated for a diseased tooth than a healthy tooth.

Spontaneous pain is a prevalent presentation in cases of symptomatic irreversible pulpitis. Possible factors leading to this presentation include continued tissue damage by invading microorganisms, and both periph- eral and central sensitization. Sensitized nociceptors may display spontaneous depolarization, discharges to subthreshold stimuli (allodynia) and increased dis- charges to suprathreshold stimuli (hyperalgesia). Thus, allodynia, hyperalgesia, and spontaneous pain are not mutually exclusive, instead they are often seen as coex- isting symptoms in cases of pulpal and periradicular inflammation due to infections.

11.2 Biology of the pain system

Historically, many patients harbor a negative asso- ciation between dentistry and pain. Moreover, clini- cians often face the challenge of managing pain and suffering that occurs in their patients. It should be appreciated that the oral cavity is exquisitely inner- vated by pain-sensing afferent neurons (nociceptors). Indeed, most studies indicate that all physiologic stim- uli applied to dental pulp results in a sensation of pain. Moreover, there is a large area of the cerebral cortex solely dedicated to interpreting sensory inputs from the orofacial structures (Penfield and Ramussen 1950).

Pain is a complex sensation with three major pro- cesses: detection, processing, and perception. First, detection is initiated by activation, depolarization, and conduction of action potentials by nociceptor neurons. Second, processing occurs when the action potentials reach the trigeminal nucleus where considerable infor- mation processing and modulation occurs. Third, these

action potentials are relayed to the thalamus and ulti- mately to the cerebral cortex where they are perceived as pain, with both affective (e.g., suffering) and sensory intensity components. Increased understanding of the pain system provides considerable clinical insight into strategies for adequate treatment. In this chapter, we address the pain system and its relation to odontogenic infections.

Dental pulp and periradicular tissues are innervated primarily by sensory neurons originating from the trigeminal ganglia where their cell bodies reside. Each neuronal cell body has one axon that divides into a peripheral projection, where it terminates into free nerve endings innervating a target tissue (e.g., den- tal pulp) and a central projection to the trigeminal brainstem nuclei complex, where it synapses with sec- ond order or projection neurons. These projection neu- rons ascend through the contralateral trigeminothala- mic tract and synapse with higher order neurons in the thalamus with final neuronal projections into the sen- sory cerebral cortex (Penfield and Ramussen 1950). Although the dental structure is one of the most heav- ily innervated tissues in the body with pain-sensing neurons (nociceptors), not all neurons in the dental pulp and periodontal ligament are nociceptors. There are postganglionic sympathetic fibers from the cervical sympathetic ganglia and other afferent low threshold (A-β) trigeminal fibers responsible for detecting non- noxious tactile information (Hargreaves and Goodis 2002).

Nociceptive afferent nerve fibers in dental pulp and periodontal ligament (PDL) are normally composed of C-fibers (25–50%) and A-δ (approximately 25%) (Byers 1984; Mengel et al. 1992, 1993). Therefore, the dental pulp and PDL are mainly innervated by noci- ceptors and to a lesser extent by A-β and sympathetic fibers. The C-fibers and A-δ fibers innervating the den- tal pulp and PDL are typically sensitive to thermal, mechanical, and chemical stimuli (polymodal noci- ceptors). There are significant differences between C- fiber and A-δ nociceptors regarding anatomy (neuronal morphology and distribution) and conduction velocity.

Electrophysiologic studies measuring the conduc- tion velocity of sensory neurons form the basis of widely used nomenclature of these fibers. The C-fibers have a slower conduction velocity (<2 m/s) than A-δ fibers (≥2 m/s conduction velocity). Anatomically, C-fibers are unmyelinated and originate from small neurons and, in contrast, A-δ fibers are lightly myeli- nated and originate from small to medium diameter

Endodontic Infections and Pain 253

cell bodies in one of the three divisions of the trigem- inal ganglion. Myelination provides axonal insulation allowing action potentials to propagate faster in A-δ fibers. Although trigeminal neurons project their cen- tral terminals to different brainstem nuclei (the nuclei interpolaris, oralis, and caudalis), much of the noci- ceptive input is thought to be located in the outer layers of the nucleus caudalis (Dubner and Bennett 1983; Hargreaves and Milam 2001; Sessle 2005). In an elegant study, mustard oil was used as a dental pulp irritant in rodents. Central sensitization was ver- ified by the increase in the mechanoreceptive field in the mice orofacial region and a decrease in mechani- cal activation threshold (mechanical allodynia) (Park et al. 2006). The detection of significant long-lasting central sensitization following noxious stimulation of the dental pulp suggests that this robust activation of primary afferent neurons in the dental pulp is capa- ble of causing central changes leading to allodynia in areas other that the tooth itself. Importantly, in this study, microinjections of a synaptic inhibitor into the nucleus caudalis completely abolished central sensiti- zation, whereas injections into the other nuclei (oralis and interpolaris) had no effect. Thus, the dental pulp is richly innervated by nociceptors that project and con- nect to second order neurons in the nucleus caudalis where neuronal plasticity and central sensitization may take place (discussed below).

In the dental pulp, the greatest density of inner- vation is found in the coronal aspect at the level of the pulpal horns (Byers 1984). At these regions, nociceptors are presented as intertwined ramified free nerve endings forming a plexus structure (Luthman et al. 1992). In general, nociceptive innervation den- sity decreases in the apical direction. A-δ fibers are known to traverse the odontoblastic layer, lose their myelination, and project approximately 0.1–0.2 mm far into the dentinal tubules, whereas C-fiber innerva- tion is found deeper in the dental pulp. The innerva- tion of the dentinal tubules by A-δ nociceptive fibers agrees qualitatively with the sharp and transient pain usually originated when the dentin is stimulated (e.g., dentinal sensitivity). According to the hydrodynamic theory, this pain is the result of fluid moving in the dentinal tubules due to thermal (cold drinks or 1,1,1,2 tetraflouroethane [Endo Ice] application during pulp vitality examination), drilling (during cavity prepara- tion), or hypertonic (sweets) stimuli (Brannstrom and Johnson 1978). This theory has been substantiated by several studies showing that the movement of fluid

in the tubules generates discharges in nociceptors and that closing of the dentinal tubules confers resistance to those noxious dentinal stimuli (Ahlquist et al. 1994; Andrew and Matthews 2000; Charoenlarp et al. 2007).

Besides the movement of fluid through the dentinal tubules and subsequent activation of mechanosensitive fibers, transdentinal cooling or heating could poten- tially activate C-fibers located deeper in the dental pulp. In order for this to happen in uninjured tissue, the temperature gradient needs to diffuse from the min- eralized tissues and reach C-fibers at a noxious levels sufficient for activating these high threshold fibers. Indeed, in an experimental setting, heating of the tooth generated a sharp pain that subsided (consistent with A-δ fiber activation) followed by a pain characterized by a slow, burning pain (consistent with C-fiber acti- vation) (Jyvasjarvi and Kniffki 1987). Although C- fiber nociceptors likely encode this deep pulpal ther- mal nociception, perhaps their most important role is detection of inflammatory pain.

There is growing recognition that odontoblasts are likely involved in nociception (Allard et al. 2006; Magloire et al. 2010). These highly specialized cells have functions that extend far beyond their best role as dentin secreting cells. They have been found to express a complex array of receptors and inflam- matory mediators primarily found in cells of the innate immune response. Importantly, odontoblasts also express ionotropic channels primarily found in nociceptors such as transient receptor potential chan- nels (discussed later) and voltage gated sodium chan- nels. In addition, these odontoblast are selectively acti- vated by agonist known to activate nociceptors (e.g., capsaicin, a TRPV1 agonist; Okamura et al. 2005) and are capable of demonstrating membrane depolar- ization and action potentials similar to those seen in nociceptors (Ichikawa et al. 2012). However, odonto- blasts do not share direct cellular communication with neurons and a synaptic communication between these two cell types has never been demonstrated. Therefore, despite increased gained knowledge in odontoblast physiology, their direct role in nociception remains elusive.

Transient receptor potential (TRP) ionotropic chan- nels are an important class of cation permeable chan- nels expressed in many cell types including sensory neurons (Tominaga 2007). These channels are known to be activated by physical (e.g., changes in temper- ature), chemical (e.g., lipids), and mechanical stim- uli (e.g., cellular stretch due to osmotic changes)

254 Endodontic Microbiology

(Tominaga 2007). Several members of this ionotropic family have been directly implicated in nociception when expressed in sensory neurons (Julius 2013). The transient receptor potential vanilloid -1 (TRPV1) has been the focus of much research because it is expressed, within the trigeminal, exclusively in a subclass of nociceptors, and is required for the development of inflammatory hyperalgesia (Caterina et al. 2000). This thermosensitive channel is activated by noxious heat (temperature >43◦C) through the recently discovered release of endogenous oxidized metabolites of linoleic acid. In addition, TRPV1 is also activated by low pH (<5.5), capsaicin (the pun- gent ingredient of peppers), and other lipids (Ikeda et al. 1997; Caterina et al. 1999; Morgan et al. 2005). TRPV1 is widely expressed in human dental pulp noci- ceptors and its activation leads to robust neuronal acti- vation and the release of vasoactive peptides (Fehren- bacher et al. 2009). Further, there is a substantial body of evidence demonstrating that TRPV1 integrates the signaling of several inflammatory mediators (Bhave et al. 2003; Diogenes et al. 2006), including many known to be involved in pulpitis (Hargreaves et al. 1994; Narhi et al. 1994; Huang et al. 2006; Jankowski and Koerber 2010).

A myriad of inflammatory mediators are released upon tissue damage. These inflammatory mediators are usually released in a cascade that comprises an “inflammatory soup”(Julius and Basbaum 2001; Cunha et al. 2005). Nociceptors express an array of receptors for inflammatory mediators such as the B2 receptor for bradykinin, TrkA receptor for nerve growth factor (NGF), prostaglandin receptors and oth- ers (Julius and Basbaum 2001). Whenever the con- centration of any given mediator is sufficient to bind and activate its receptors in the nociceptors, intracel- lular signaling pathways are triggered. Many intra- cellular signaling pathways are known to sensitize or activate nociceptors. For example, local elevation of prostaglandin E2 activates protein kinase A lead- ing to nociceptor sensitization, while increased tis- sue amounts of bradykinin activates phospholipase C and protein kinase C, leading to nociceptor activa- tion (i.e., depolarization) (Nicol et al. 1992; Cesare et al. 1999). Some of these intracellular signaling path- ways are involved in phosphorylating receptors such as TRPV1 resulting in a decreased activation threshold (manifested as allodynia) or an augmented response to a suprathreshold stimulus (contributing to hyperal- gesia). For example, bradykinin and prolactin trigger

phosphorylation of TRPV1 reducing its temperature of activation from 43◦C to temperatures as low as 36◦C (Cesare et al. 1999; Diogenes et al. 2006). Thus, allodynia can be produced by inflammatory media- tors that sensitize nociceptors. This reduced activation threshold could explain how minor, innocuous trans- dentinal heating of inflamed pulp might still activate TRPV1 leading to pain perception (Ahlberg 1978). More importantly, the lowering of the temperature of activation of TRPV1 to less than 37◦C may lead to continuous activation of this channels contributing to spontaneous pain. Interestingly, the dull, throbbing pain sensation typically associated with activation of C nociceptors is often reported by patients with irre- versible pulpitis. In many of these patients, cooling the tooth (i.e., drinking ice water) appears to promote a transient alleviation of the symptoms. It can be hypoth- esized that this alleviation could be due to the cooling of the pulp to temperatures below the threshold for activation of the sensitized TRPV1 receptor.

11.3 Central sensitization

The central circuitry at the level of nucleus caudalis is far from being a simple static “connector” that relays the original burst of action potential from the peripheral neuron to the cerebral cortex. Instead, there is significant processing of the nociceptive signal at this level. The process known as central sensitization occurs when the primary afferent signal is amplified at this central terminal. Central sensitization represents a major component in inflammatory hyperalgesia and allodynia (Woolf 1996). It is triggered by an increase in nociceptive peripheral afferent barrage (e.g., aug- mented responses due to inflammation or nerve injury) (McMahon et al. 1993; Woolf 1996; Urban and Geb- hart 1999). This constant primary afferent barrage evokes the release of neuropeptides (e.g., substance P) and neurotransmitters (e.g., glutamate) from cen- tral terminals of these neurons (Ren and Dubner 1999; Urban and Gebhart 1999).

Glutamate activation of the glutamate N-methyl d- aspartate (NMDA) receptor is an important mech- anism in central sensitization. Interventions at this receptor in the central terminals induce either reduc- tion or enhancement of central sensitization gen- erated by repeated C-fiber discharge. For example, an NMDA antagonist reduces central sensitization, whereas brain-derived neurotrophic factor (BDNF)

Endodontic Infections and Pain 255

facilitates NMDA activation, thereby enhancing cen- tral sensitization (Woolf and Thompson 1991; Kerr et al. 1999).

Clinically, central sensitization can be observed in patients complaining of persistent pain even after the removal of inflamed peripheral tissue with endodon- tic and restorative procedures or in allodynia felt in teeth and tissue surrounding an offending tooth. Stud- ies have shown that the presence of preoperative pain increases the risk of postoperative pain (Torabinejad et al. 1994; Polycarpou et al. 2005). Furthermore, pre- emptive analgesia during surgical procedures is asso- ciated with reduced postoperative pain in many situ- ations (Woolf and Chong 1993; Reuben 2007). This suggests that patients who seek endodontic assistance at the first sign of symptoms, and who receive treat- ment under adequate profound anesthesia, have better postoperative prognosis because of the decreased like- lihood of developing central sensitization.

11.4 Persistent pain following endodontic therapy

There have been significant advances in endodontics in the last couple of decades with the development of microscopy, advanced imaging (i.e., cone beam com- puter tomography), nickel-titanium rotary instruments, ultrasonic and sonic instruments, and negative pres- sure irrigation, among others. Despite these signifi- cant technical advances, pain may persist following adequate therapy, with a reported frequency of 5.3% (Nixdorf et al. 2010a). Although an endodontic treat- ment may be deemed adequate when evaluating peri- apical radiographs, pain may persist for longer than 6 months.

The evaluation of periapical radiographs has been traditionally used as an outcome measure of endodontic success (i.e., healing of apical periodon- titis). Despite efforts to establish standardized cri- teria such as the periapical index (Ørstavik et al. 1986), the two-dimensional radiographic interpreta- tion is afflicted with poor inter-examiner and intra- examiner agreement (Goldman et al. 1972). The use of advanced imaging with cone-beam computer tomography (CBCT) has been shown to significantly increase the detection of apical radiolucencies (Abella et al. 2014; Patel et al. 2015). Therefore, persis- tent postendodontic pain of odontogenic origin may be associated with symptomatic apical periodontitis

not detected with conventional radiographic imaging methods. From this perspective, persistent pain may result from the failure to completely eliminate the orig- inal etiologic factors (i.e., microorganisms and their antigens), instead of an undiagnosed nonodontogenic pain condition. In a study using the National Den- tal Practice-based Research Network (PBRN), radi- olucencies were detected in 100% of the patients with persistent odontogenic pain using CBCT, whereas they were detected only in 57% when periapical radio- graphs were used (Nixdorf et al. 2015). While non- odontogenic pain conditions must be considered in the differential diagnosis of persistent postendodontic pain, these findings indicate that certain cases of post- treatment pain may result from persistent symptomatic apical periodontitis.

There are many factors that predispose patients to persistent post-treatment apical periodontitis. These include host factors such as systemic diseases that modulate the immune system (e.g., diabetes mellitus), virulence of the endodontic microflora, presence of complex root canal anatomy, genetic factors such as single nucleotide polymorphisms (SNPs), and epige- netic factors (Campos et al. 2013; Chan et al. 2013). SNPs have been shown to be associated with persis- tent apical periodontitis. A single nucleotide substitu- tion on the interleukin-1 beta (IL-1β) gene has been shown to increase the odds ratio for persistent api- cal periodontitis from about 6- to 12-fold for patients with either heterozygous or homozygous mutations, respectively (Morsani et al. 2011). However, another study found no correlation of IL-1β polymorphisms with persistent apical periodontitis following adequate root canal therapy. This discrepancy may be brought about, at least in part, by inadequate sample sizes for the detection of low frequency SNPs, or inadequate control of additional variables that influence the occur- rence of apical periodontitis (Aminoshariae and Kullid 2015). Importantly, the inflammatory mediators tumor necrosis factor α (TNF-α) and IL-1β are known to be directly involved with the development and main- tenance of apical lesions detected as radiolucencies (Tani-Ishi et al. 1995). In addition, both cytokines are also known to sensitize nociceptors, being impor- tant players in inflammatory thermal hyperalgesia and mechanical allodynia (Ferreira et al. 1988; Cunha et al. 2005). Therefore, genetic mutations (i.e., SNPs) that result in a gain of function for these inflammatory mediators have the potential to increase osteoclastic activation, lesion formation, and the sensitization of

256 Endodontic Microbiology

periradicular nociceptors. Thus, these SNPs could pre- dispose patients to apical periodontitis following root canal therapy with the clinical presentation of radiolu- cencies and pain upon function or percussion (mechan- ical allodynia).

The frequency of nonodontogenic pain lasting longer than 6 months following root canal therapy has been found to be about 3% of cases (Nixdorf et al. 2010b). This suggests that, at least in part, some teeth may have been misdiagnosed as being the source of the pain and have been erroneously endodontically treated. Another study found that approximately half of the cases with diagnosis of nonodontogenic pain following root canal therapy were related to referred pain from temporomandibular disorders (Nixdorf et al. 2015). Importantly, the majority (63%) of the patients reporting persistent pain following root canal therapy also reported a history of chronic pain elsewhere in the body. This is an important observation because there is growing recognition that patients with a history of chronic pain such as temporomandibular disorders have higher levels of psychosocial symptoms, affective distress, somatic awareness, and pain catastrophizing (Fillingim et al. 2011). Indeed, new research suggests that many chronic pain disorders reflect a centralized pain state that amplifies pain perception far beyond levels perceived by patients without chronic pain dis- orders (Tracey et al. 2009; Williams and Clauw 2009; Woolf 2011). Simply put, patients with a history of chronic pain are predisposed to persistent pain follow- ing root canal therapy. These findings are in agreement with the finding that moderate to severe preoperative pain and presence of chronic pain are predictors of postoperative pain (Polycarpou et al. 2005). It is note- worthy that nonodontogenic pain following root canal therapy is a relatively rare occurrence in patients with nonchronic pain. Therefore, infections remain the pre- dominant etiology of endodontic pain.

11.5 Mechanisms of pain due to endodontic infections

Odontogenic infection is one of the most prevalent infectious diseases that affects humans. When bacte- ria gain access to the pulp, a significant inflammatory response is initiated. Bacteria can induce tissue injury and inflammation through three general pathways. First, bacteria can release tissue modifying agents such as enzymes capable of degrading host tissue elements

(e.g., metalloproteinase and proteases) (Sedgley et al. 2005; Reynaud af Geijersstam et al. 2007). Second, bacteria can stimulate the innate immune system by activation of pattern recognition receptors such as the toll-like receptors (TLR) (O’Neill 2004; Wadachi and Hargreaves 2006). Third, chronic bacterial infections can lead to activation of specific or adaptive immune response (Baumgartner and Falkler 1991).

These three pathways could lead to nociceptor acti- vation by either indirect or direct mechanisms (Fig- ure 11.1). First, it is known that endodontic infections are polymicrobial with a predominance of strict anaer- obic and facultative aerobic bacteria (Fabricius et al. 1982). Many of these bacterial species (e.g., black- pigmented bacteria) have strong proteolytic activity and have been directly correlated with severity of symptoms (Hashioka et al. 1992; Haapasalo 1993; Siqueira et al. 2001, 2004; Gomes et al. 2005). Inter- estingly, some of the proteases released by these bac- terial species have been found to directly activate noci- ceptors through proteinase activated receptors (PARs). Trypsin and trypsin-like enzymes released by Pre- votella spp. and Porphyromonas spp., for example, are direct activators of the PAR-2 receptor in trigeminal nociceptors (Patwardhan et al. 2006). Another mech- anism by which bacteria could activate nociceptors upon direct tissue damage is the release of adeno- sine triphosphate (ATP) from damaged resident cells in the invaded tissues. The released ATP in the extra- cellular milieu is readily available to bind to the P2X purinoceptor 3(P2×3) ligand-gated ion channel (Chen et al. 1995; Alavi et al. 2001; Renton et al. 2003). This direct activation by either proteases or free ATP released from damaged cells leads to nociceptor acti- vation, pain, and the subsequent release of vasoactive neuropeptides from the free nerve endings in the pulp and periradicular tissues (Figure 11.1).

The recognition of microbial pathogen-associated molecular pattern (PAMPs) represents the very first step in host defense. The detection of this invading microorganisms depends on TLRs and other pattern recognition receptors (PRRs) (Janssens and Beyaert 2003). The TLR family of receptors represents an family of proteins capable of detecting a broad range of microbial substances (Table 11.1). These recep- tors recognize specific and highly conserved nonself molecular microbial signatures. The binding of these molecules to TLRs triggers cellular molecular events that trigger the upregulation and release of inflam- matory mediators (e.g., IL-1 and TNF-α) involved in

Endodontic Infections and Pain 257

Fig. 11.1 Direct and indirect mechanisms by which bacteria can activate pulpal or periradicular nociceptors. CGRP, calcitonin gene-related peptide; LPS, lipopolysaccharide; LTA, lipoteichoic acid; PAR, proteinase activated receptor; SP, substance P; TLR, toll-like receptor.

the initiation of the inflammatory response (Kirschn- ing and Bauer 2001; Takeda and Akira 2001). Thus, these specialized receptors represent the gateway of the innate immune response against invading microorgan- isms. It is noteworthy that in the dental pulp the expres- sion of TLRs is not only restricted to cells classically associated with the innate immune response (e.g., den- dritic cells, macrophages, and neutrophils), but TLRs have also been found to be expressed in odontoblasts (Jiang et al. 2006), fibroblasts (Staquet et al. 2008), and in nociceptors (Wadachi and Hargreaves 2006).

Thus, nociceptors in the dental pulp are particularly equipped to directly sense bacteria (Wadachi and Har- greaves 2006).

The TLR4 receptor detects lipopolysaccharides (LPS) derived from the surface of Gram-negative bac- teria (Poltorak et al. 1998; Qi and Shelhamer 2005). As illustrated in Figure 11.2, the activation of TLR4 is greatly enhanced by the participation of other membrane-anchored receptors that lack an intracellu- lar domain: myeloid differentiation-2 receptor (MD2) and cluster differentiation-14 receptor (CD14). These

258 Endodontic Microbiology

Table 11.1 The toll-like receptor (TLR) family: an innate immune system capable of detecting microbial substances

TLR Foreign substance

TLR-1/2 Bacterial lipopeptides TLR-2 Bacterial lipopeptides, lipoteichoic acid

from Gram-positive bacteria, zymosan from yeast

TLR-3 Double-stranded RNA from viruses TLR-4 Endotoxin (lipopolysaccharide) from

Gram-negative bacteria TLR-5 Flagellin TLR-7/8 Uridine-rich single-stranded RNA from

viruses TLR-9 CpG DNA TLR-10 Unknown TLR-11 Uropathogenic bacteria TLR-12/13 Unknown

Source: Wickelgren (2006).

two accessory proteins bind to LPS but are unable to trigger any intracellular signaling because they lack an intracellular domain. However, the MD2 receptor binds to LPS and to an extracellular domain in the TLR4 receptor itself. The formation of a TLR4/MD2 dimer and its LPS binding is essential for LPS sig- naling in immune cells. This interaction is believed to promote TLR4 activation by LPS and stabilize the molecular conformational change of the activated LPS/TLR4/MD2 complex (Fitzgerald et al. 2004). However, the CD14 receptor has the capacity to bind to multiple LPS molecules, increasing the concentration of the ligand that is then presented to TLR4. Therefore,

O-specific Polysaccharide

Lipid A

TIRAP TRAM

TRIFFMyD88

M D

-2

C D

-1 4

TLR-4

Core Polysaccharide

Fig. 11.2 The structure of lipopolysaccharides (LPS) and the pattern recognition receptor, TLR4, which detects LPS and its associated intracellular signaling molecules.

the formation of a LPS /CD14/TLR4/MD2 complex has been shown to evoke greater cellular responses than a LPS/TLR4/MD2 interaction alone. It is impor- tant to note that soluble LPS in the extracellular space is often bound to a LPS-binding protein (LPSB), which serves as a carrier protein allowing better interaction of LPS with any of the members of the MD2/CD14/TLR4 complex. Thus, there are several extracellular proteins that serve to greatly amplify the ability of host cells to detect the presence of LPS.

LPS at very low concentrations promotes maximal activation of immune cells, particularly macrophages and neutrophils. In these cells, LPS evokes a NFκB- dependent gene upregulation and release of cytokines such as TNF-α and IL-1α (Hahn et al. 2000), and chemotactic agents such as leukotriene B4 (Doyle and O’Neill 2006). Upon the release of these cytokines, the inflammatory reaction amplifies rapidly as infiltrating immune cells start releasing more inflammatory medi- ators which act in concert to promote the cardinal signs of inflammation: edema and pain.

Nociceptors express the receptors for numerous inflammatory mediators and activation of these recep- tors leads to the sensitization of these pain-sensing fibers (Byers et al. 1990b; Fried and Risling 1991; Goodis et al. 2000; Chang et al. 2006). The concen- tration of inflammatory mediators such as bradykinin, IL-1α and NGF have been found to be dramatically increased in inflamed dental pulp compared to asymp- tomatic control dental pulp (Nakanishi et al. 1995; Shi- mauchi et al. 1997; Wheeler et al. 1998; Lepinski et al. 2000). Some of these mediators such as bradykinin and prostaglandins evoke an acute sensitization of noci- ceptors by triggering local intracellular events (e.g., phosphorylation of channels).

Other mediators such as NGF bind to their receptors and are retrogradely transported from the periphery to the cell body located in the trigeminal ganglion. In the cell body, NGF triggers the transcription of genes for ionotropic channels (e.g., transient receptor potential A1, TRPA1) and G-protein coupled recep- tors (GPCRs) that are anterogradely transported back to the periphery where they will be involved in an exac- erbated nociceptive signaling (Obata et al. 2005; Dio- genes 2007a). Also, NGF promotes neuronal sprout- ing at the site of injury, increasing the local density of nociceptors (Byers et al. 1990a). This process requires hours to days to occur and has long-lasting effects. In fact, peripherally administered NGF is known to gener- ate hyperalgesia and allodynia that lasts days to months

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in both humans and experimental animals (McMahon 1996; Mendell et al. 1999). Thus, bacterial infections are capable of causing significant neuronal plasticity via the release of neurotrophins such as NGF that could result in long-lasting changes in nociceptor phenotype and function.

The activation of a peptidergic subset of nocicep- tors is often accompanied by a vasodilation reaction (Simone et al. 1989). This occurs because these neu- rons release vasoactive neuropeptides such as cal- citonin gene-related peptide (CGRP) and substance P when depolarized (Figure 11.1). Certainly, the enhanced activation of these nociceptors in inflam- matory conditions leads to neuropeptide release. For example, elevated levels of substance P have been found in the pulp of patients with irreversible pul- pitis (Bowles et al. 2003). CGRP and substance P pro- mote vasodilation and plasma extravasation, respec- tively, thus participating in the inflammatory process. The mechanism for neuronal-generated inflammation is called neurogenic inflammation and is often inter- twined with classic inflammatory processes due to a positive feedback loop. Therefore, it is hard to determine which event physiologically started first. Although inflammation and neurogenic inflamma- tion have different etiologies, they are often present together as part of an overall amplifying inflammatory response (Figure 11.1).

It has been suggested that odontoblasts have a monitoring and functional role in pulpal inflamma- tion because they represent the first biologic bar- rier, between mineralized tissues and the dental pulp, encountered by pathogens. Odontoblasts are highly specialized cells that express a “molecular repertoire” far greater than that needed for simple dentin produc- tion and deposition. Human odontoblasts express both TLR4 and TLR2 capable of recognizing cell wall com- ponents from Gram-negative and Gram-positive bac- teria, respectively (Veerayutthwilai et al. 2007). The activation of TLR2 and TLR4 in human odontoblasts results in the upregulation and release of soluble fac- tors such as TNF-α and IL-1β (Veerayutthwilai et al. 2007). Therefore, bacteria invading the pulp are most likely first detected by odontoblasts, in addition to sen- sory neurons within the dentinal tubules, via activa- tion of TLR4 and TLR2 receptors, with subsequent paracrine communication to nearby cells, including immune cells. Together, these cells then release inflam- matory mediators leading to the indirect sensitization of nociceptors by bacteria.

The sensitization of nociceptors in infections is classically believed to be caused by the release of inflammatory mediators from bacteria-activated immune cells. However, Wadachi and Hargreaves (2006) demonstrated, for the first time, that trigemi- nal nociceptors in the human dental pulp express both TLR4 and CD14 (Figure 11.3). This novel finding raises the possibility that sensory neurons are able to “sense” the presence of Gram-negative bacteria.

It had been demonstrated that LPS derived from the Porphyronomas gingivalis, which is a species known to be involved in odontogenic infections, and Escherichia coli directly sensitizes TRPV1 responses in cultured rat trigeminal neurons (Diogenes et al. 2007b; Ferraz et al. 2007). Exposure of trigeminal neurons in culture to LPS acutely sensitized nocicep- tors, significantly increasing the release of the neu- ropeptide CGRP when neurons were stimulated with capsaicin. These data have exciting clinical signifi- cance, because deeper carious lesions and primary periapical lesions are predominantly associated with Gram-negative bacteria. Moreover, elevated LPS con- centrations in infected root canal systems have been positively correlated with painful presentations of peri- radicular periodontitis (Jacinto et al. 2003).

The direct sensitization of nociceptors by LPS may explain why some immunocompromised patients present with devastatingly painful infections (Epstein 1988; Navarro et al. 1998). In these patients, there are decreased levels of inflammatory mediators, which normally would result in attenuated inflammatory pain. However, bacteria grow unchecked in these patients, elevating local LPS levels with the potential to sensi- tize nociceptors in the infected area. Another important clinical consideration is substantial LPS levels can be still be measured in root canals after complete chemo- mechanical debridement (Martinho and Gomes 2008; Endo et al. 2012). A study revealed that LPS penetrates up to 300 μm into root canal dentinal tubules (Horiba et al. 1990). This high penetration and dentin affinity may explain the persistence of this highly immuno- genic molecule following chemomechanical prepara- tion (Martinho and Gomes 2008). Therefore, tapered root canal preparations with greater apical diameter provide better circumferential removal of contami- nated dentin decreasing intracanal levels of LPS (Mar- inho et al. 2012). Although complete removal of LPS from infected root canals is not achievable, its resid- ual antigenicity can be completely neutralized with the use of calcium hydroxide as an intracanal medicament

260 Endodontic Microbiology

Fig. 11.3 Evaluation of the expression patterns of TLR4 and CD14 in human trigeminal sensory neurons. White arrows depict examples of neurons expressing both markers for each row of three images, and yellow arrows depict examples of neurons that express one but not both markers. Human trigeminal neurons were evaluated for colocalization of TLR4 (panel A), CD14 (panel J), with a marker for the capsaicin-sensitive subclass of nociceptors (TRPV1, panels B,C for TLR-4 and panels K,L for CD14. Source: Wadachi and Hargreaves (2006). Reproduced with permission of International and American Associations for Dental Research.

(Nelson-Filho et al. 2002; Guo et al. 2014). This is believed to be largely due to the alkaline hydrolysis of the “lipid A” moiety of the bacterial LPS (Safavi and Nichols 1993). Therefore, LPS from dead bacteria needs to be adequately removed or neutralized because there is the potential for direct activation and sensitiza- tion of nociceptors. Further research is needed to see if residual LPS–endotoxin levels underlie the cause of flareups and persistent disease following root canal procedures.

There is a growing body of evidence to suggest that LPS is not the only activator (ligand) of TLR4, because this receptor has been shown to be also activated by endogenous ligands derived from damaged surround- ing local tissue. These ligands include fibronectin- A (Okamura et al. 2001; Al-ofi et al. 2014), heat shock protein 60 (HSP60) (Ohashi et al. 2000), HSP70 (Lipsker et al. 2002; Gong et al. 2009), and tenascin-C (Midwood et al. 2009). Importantly, these tissue fac- tors are expressed in the dental pulp, and have levels increased upon injury (Martinez et al. 2000; Ohshima et al. 2000; Eberhard et al. 2005; Piva et al. 2006; Sens et al. 1997). Thus, injury to the dental pulp by

microbial infections followed by the release of tis- sue factors could lead to the activation of TLR4 in a process independent from LPS. The recent discov- ery of these endogenous ligands represents a novel mechanism of innate response in sterile injuries such as certain dental trauma and aseptic iatrogenic pulp exposures. However, in endodontic infections, these endogenous ligands and LPS are likely to be coac- tivators on TLR4 amplifying its crucial effect in the initiation and maintenance of a robust inflammatory response.

11.6 Clinical strategies for treating pain due to endodontic infections

The management of the patient with pain resulting from an odontogenic infection involves both pharma- cologic and non-pharmacologic therapies. Appropriate pharmacotherapy includes drugs that reduce pain per- ception or microbial infection. Pain perception can be reduced by the administration of local anesthetics or the use of analgesics. It is well recognized by clinicians

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that patients with odontogenic pain often experience considerably reduced efficacy of local anesthetics, par- ticularly in cases involving painful mandibular teeth. It has been speculated that this might be caused, in part, by direct effects of prostaglandins on sensitizing neuronal voltage-gated sodium channels via activation of protein kinase A and subsequent protein phospho- rylation (Hargreaves and Keiser 2002). This hypothe- sis suggests that preoperative administration of nons- teroidal anti-inflammatory drugs (NSAIDs) in patients who can tolerate this drug class might actually increase the efficacy of local anesthetics. Interestingly, two clin- ical trials have recently reported that oral analgesics do tend to increase the success of local anesthetics in odontogenic pain patients (Modaresi et al. 2006; Ianiro et al. 2007). In addition, several randomized clinical trials have indicated that the efficacy of anes- thetizing painful mandibular teeth can be increased about twofold (from ∼40% to ∼85% of patients) by combining conventional inferior alveolar nerve block injection with intraosseous injection of local anesthet- ics (Nusstein et al. 1998; Parente et al. 1998; Nusstein et al. 2003; Bigby et al. 2006). However, it should be noted that none of these clinical trials evaluated the efficacy of intraosseous anesthetics in cases of pain resulting from pulpal necrosis with periradicu- lar involvement. Indeed, at least some investigators have expressed concern about the efficacy and poten- tial for side effects when intraosseous anesthetics are used in cases involving pulpal necrosis (Reader and Nusstein 2002). Thus, additional clinical trials in this area appear warranted.

Oral analgesics have been shown to be useful in treating odontogenic pain. In general, the NSAIDs have been shown to have efficacy for reducing odonto- genic pain (Doroschak et al. 1999; Holstein et al. 2002; Keiser and Hargreaves 2002). Surveys of endodon- tists reveal that most clinicians would use NSAIDs for pain scenarios, with ibuprofen 600 mg being the most common (Mickel et al. 2006). Although NSAIDs are available in formulations combined with a narcotic drug, there are large and dose-related increases in the potential for adverse effects (Dionne 1999). Instead, there is increasing evidence that NSAIDs can be com- bined with acetaminophen to provide greater analgesia for treating odontogenic pain (Menhinick et al. 2004). This important clinical finding is bolstered by recent reports that acetaminophen is actually a pro-drug that is converted in the brain to a drug with considerable activity for enhancing the endogenous cannabinoid

analgesic system (Hogestatt et al. 2005; Bertolini et al. 2006; Ottani et al. 2006). Thus, important evidence from randomized controlled trials and from bench-top research indicates that moderate to severe pain often can be managed by combinations of an NSAID with acetaminophen.

Pharmacotherapy is also used to reduce microogan- isms in infected root canal systems. Several drug classes have been used for disruption or elimination of microorganisms in the infected root canal systems or associated tissues. Common endodontic pathogens in primary endodontic infections include members of Gram-negative anaerobic species. Accordingly, oral antibiotics including the penicillins and clindamycin comprise appropriate and popular forms of antibiotics used by endodontists (Yingling et al. 2002; Baum- gartner and Xia 2003). Although such surveys indi- cate appropriate usage in the majority of clinical sce- narios, it should be noted that they also reveal that some clinicians prescribe these drugs up to 80 times per week. In general, antibiotics are not indicated for cases of irreversible pulpitis, where pain of inflamma- tory origin is dominant (Nagle et al. 2000; Keenan et al. 2006). In addition, most (Walton and Fouad 1992; Walton and Chiappinelli 1993; Fouad et al. 1996; Henry et al. 2001; Pickenpaugh et al. 2001), but not all (Torabinejad et al. 1994) placebo-controlled clin- ical trials indicate that antibiotics have no effect for reducing odontogenic pain. Moreover, a comprehen- sive evidence-based review of the literature came to the same conclusion about the lack of efficacy of antibi- otics for relieving pain of odontogenic origin (Fouad 2002). Instead of systemic antibiotics, chemomechan- ical debridement procedures appear to provide effec- tive postoperative pain control because even placebo- treated patients report an ∼80% reduction in pain within 48 hours of endodontic instrumentation pro- cedures (Torabinejad et al. 1994).

The term “chemomechanical debridement” is of value because it reflects the notion that bulk removal of infected tissue combined with direct application of antimicrobial agents into infected root canal systems provides a useful strategy for achieving both short- term (pain) and long-term (healing) endodontic out- comes. Although mechanical debridement using rotary NiTi file systems can reduce bacterial load, most of the treated root canal systems still retain cultivable microorganisms when instrumentation is performed in the presence of sterile saline (Shuping et al. 2000). Instead, antimicrobial agents must be provided. Local

262 Endodontic Microbiology

antimicrobial therapy generally consists of delivery of compounds such as NaOCl, 2% chlorhexidine, cal- cium hydroxide, MTAD (Biopure®), or other agents into infected root canal systems (Gilad et al. 1999; Shuping et al. 2000; Torabinejad et al. 2003; Waltimo et al. 2005; Zerella et al. 2005; Hoelscher et al. 2006; Cook et al. 2007). However, despite the well-reported disinfecting action of these intracanal agents, they do not seem to improve postoperative pain (Torabinejad et al. 2005; Ehrmann et al. 2007). It can be hypothe- sized that this may be caused, at least in part, by the inability of these intracanal disinfectants to neutral- ize bacterial by-products (e.g., LPS) that have diffused into the periradicular tissues. In addition, it should be noted that ultrasonic delivery of NaOCl may improve local tissue debridement (Gutarts et al. 2005; Burleson et al. 2007), and the use of calcium hydroxide has been shown to neutralize LPS (Safavi and Nichols 1993). Further research is warranted to evaluate disinfection protocols that eliminate residual bacterial by-products.

Taken together, there is no one magic bullet for treating pain associated with odontogenic infections. Instead, local anesthetics, analgesics, and effective chemomechanical debridement provide an effective multidisciplinary strategy for the control of pain due to microbial infection.

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Chapter 12 Systemic Antibiotics in Endodontic Infections Ashraf F. Fouad

12.1 Introduction 12.2 General principles of antibiotic

prescribing 12.3 Efficacy of antibiotics 12.4 Classification and mode of action

of antibiotics 12.4.1 Cell wall and cell membrane

disruption 12.4.2 Interference with protein

production 12.4.3 Interference with DNA

structure and replication

12.5 Host factors 12.6 Antibiotic effectiveness and bacterial

resistance in endodontics 12.7 Antibiotic toxicities, allergies, and

superinfections 12.8 Role of antibiotics in clinical

management of endodontic infections 12.9 Indications for prophylactic antibiotic

therapy 12.10 References

12.1 Introduction

As noted throughout this textbook, endodontic dis- ease is fundamentally microbial in origin. The pulp is a connective tissue that is housed within the hardest tissues in the body, and is sterile under normal condi- tions. The pulp and periapical tissues react to advanc- ing microbial infiltration by initiating an inflammatory response, the function of which is to stop the advance of the microorganisms involved and eliminate their pathogenicity. Therefore, it is logical to conclude that systemic antibiotics may enhance the ability of the tissues to defend themselves and eliminate the dele- terious effects of these microorganisms. This chapter presents the available information on the appropriate systemic antibiotics for the microorganisms involved in endodontic infections, their mechanisms of action, and their efficacy in various presentations of the dis- ease process.

Antibiotic literally means “against life.” Antibiotics are synthetic or naturally occurring chemicals that inhibit the growth or kill microbes. The modern con- cept of specific antimicrobials to treat infections was introduced by Paul Ehrlich in 1906. In the 1930s, the use of sulfonamides pioneered the modern era of antibiotic treatment of infections. However, the so- called golden age of antibiotic therapy began with the use of penicillin in 1941. Although Fleming discovered the antimicrobial properties of the mold penicillium in 1929, it was not until Florey and Chain mass produced penicillin that it became available in the quantities needed for widespread clinical use. Since then, over 100 antibiotics have been used therapeutically with dramatic clinical efficacy in treating infectious dis- ease. However, because of widespread and often indis- criminate use of antibiotics, numerous microbes have developed resistance to previously effective antibi- otics. In addition, many of the antibiotics have toxic

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side effects, which has led to the discontinuation of several otherwise effective antibiotics.

The efficacy of antibiotics is based on the principle of selective toxicity for the infectious organisms but not for the host cells. Many antibiotics in use do have toxic side effects for patients. The amount of toxicity is related to the antibiotic’s mechanism of activity. The basic mechanisms of activity for antibiotics involve blocking a function necessary to the microbe or inter- fering with its structure, with or without affecting host cells. Penicillin is a prime example of an antibiotic that has selective toxicity for bacteria but not eukary- otic cells or viruses. Penicillin inhibits cell wall pro- duction in bacteria but has no effect on mammalian cells, fungi, viruses, or protozoa. The efficacy of many antibiotics is based on varying degrees of selective toxicity between mammalian cells and microbes. For example, an antibiotic that inhibits protein synthesis may have a profound effect on bacteria but mini- mal effect on mammalian protein synthesis. In this chapter, the discussion is restricted to that of systemi- cally administered antibiotics. A discussion of locally applied antibiotics is presented in Chapters 13 and 14.

12.2 General principles of antibiotic prescribing

Antibiotic prescribing for endodontic infections is intended to interfere with microbial cell structure and function, in order to reduce the rate of bacterial growth and production of toxins and destructive enzymes. This limits the expansion of infections and enhances the host’s own immunologic response. There are certain rules involved in prescription of antibiotics that maxi- mize their efficacy and enhance the patient’s recovery.

� The local source of the infection must be eliminated, such as by debridement and disinfection of the root canal system responsible for the infection, extraction of the offending tooth, and/or incision for drainage of a swelling. The site of the bacterial proliferation, such as the necrotic pulp space, a soft tissue abscess, or an infected fascial space, is devoid of a vascular supply. Therefore, systemic antibiotics do not reach these areas in sufficient concentration for therapeutic action, and should not be relied on without adequate elimination of the source of infection.

� The route of administration of an antibiotic must assure rapid access of the drug in a therapeutic dose to the site of the infection. For progressive infections

that extend to fascial planes, intravenous antibiotics are used for rapid absorption and dissemination. For oral administration, it is essential to assure that the antibiotic is administered on an empty stomach and that certain food products that interfere with its action are not concomitantly consumed. For exam- ple, tetracyclines can bind to calcium in dairy prod- ucts, and so it is necessary to alert the patient to this interaction.

� The antibiotic dosage should be high enough to ensure that it reaches the site of the infection in suffi- cient therapeutic concentration. With aggressive and rapidly progressive infections the initial dose could be doubled (loading dose) to allow rapid access with high concentration.

� The antibiotic dosage should be maintained at thera- peutic levels for a long enough period to assure effec- tiveness. There are insufficient data on the optimal length of an antibiotic course. However, this is gen- erally related to the degree of infection, the recovery of the patient, and the microorganisms involved. A typical course of antibiotics for an endodontic infec- tion ranges 5–8 days.

� The therapeutic index is a measure of toxic level of the drug divided by the minimum inhibitory concen- tration (MIC). Antibiotics with a high therapeutic index, such as penicillin and amoxicillin, allow the use of high doses for rapid dissemination and action.

� While the great majority of antibiotic prescribing is based on empirical recommendations, it is essential that the antibiotic selected is specific for the microor- ganisms that are targeted. This specificity can only be determined by culture and sensitivity testing. The latter is time consuming, and so is only carried out when the infection is not responding to the antibiotic treatment and local debridement, or if the infection is severe enough that empirically prescribed antibiotics need to be confirmed and reinforced after testing.

12.3 Efficacy of antibiotics

Success in management of acute infections relies on surgical drainage and debridement of the source of infection, together with the selection of the appro- priate antibiotic for control of the spread of infec- tion. Although antibiotics reduce the number of viable bacteria, ultimate healing is dependent on the host’s innate and specific immune responses. Endodontic infections are polymicrobial and should be treated as such. As discussed in Chapters 4–6, endodontic

Systemic Antibiotics in Endodontic Infections 271

microflora are primarily composed of strict anaerobic Gram-negative and Gram-positive bacteria, as well as facultative anaerobic Gram-positive bacteria. Wide- spectrum bactericidal agents are generally preferred over bacteriostatic agents in acute infections for sev- eral reasons:

1. They provide a decrease in number of bacteria by causing cell death, and so are bactericidal.

2. They produce a more rapid clinical effect than bac- teriostatic agents.

3. They bind to the bacteria and produce an effect even after falling below a MIC. An example of this are the β-lactam antibiotics (penicillins), which can cause delayed lysis of bacterial cells even after their tissue levels have fallen below the MIC.

12.4 Classification and mode of action of antibiotics

In this section we limit our discussion to those classes of antibiotics that have been shown to be most effi- cacious in dealing with orofacial and endodontic infections.

12.4.1 Cell wall and cell membrane disruption

12.4.1.1 𝛃-Lactam antibiotics

This group is composed of penicillins, cephalosporins, carbapenems, and monobactams. Penicillins owe their

antimicrobial activity to their ability to bind to a vari- ety of membrane-bound proteins, collectively referred to as penicillin-binding proteins (PBPs) (Spratt 1980). Gram-positive bacteria are the most sensitive to peni- cillin, primarily because of the importance of a transpeptidase that is also a PBP. This enzyme has a crucial role in cell wall synthesis, which is particu- larly significant in Gram-positive bacteria because of their extremely thick cell walls (50–100 molecules vs 1–2 molecules in Gram-negative bacteria). As shown in Figure 12.1, a new subunit of N-acetylmuramic acid (NAMA) and N-acetylglucosamine (NAGA) disaccha- ride with an attached peptide side chain are linked to an existing peptidoglycan polymer. This occurs by a glycine (G) covalent bridge from one peptide side chain to the other by enzymatic action of PBP. In the presence of a β-lactam, it binds the PBP and prevents it from cross-linking the two peptide side chains (Hauser 2013).

Although this process is thought to be an integral part of the bactericidal action of penicillin, it is still not clear how the inhibition of transpeptidation ulti- mately leads to cell death. The lysis of bacterial cells that result from penicillin action appears to require par- ticipation of autolytic enzymes (autolysins or murein hydrolases). The transpeptidase enzyme is just one of many PBPs and the interaction of penicillins with some of these other PBPs appears to contribute to the antimicrobial action of penicillins (Figure 12.2).

Because of the nature of penicillin’s actions, it is apparent that it is most effective when the pathogens are in their log phase of growth (i.e., when rapid cell division is occurring that requires considerable cell

Fig. 12.1 Penicillin structure and action. The penicillin β-lactam ring is seen to bind the penicillin-binding protein (PBP) and interfere with the peptidoglycan assembly process during bacterial cell wall formation. G, glycine; NAGA, N-acetylglucosamine; NAMA, N-acetyl-muramic acid. Data re-drawn from Hauser 2013.

272 Endodontic Microbiology

Fig. 12.2 The cross-linking step during cell wall synthesis in Staphylococcus aureus. Transpeptidase cleaves the terminal D-alanine from a pentapeptide chain, which allows the next D-alanine to bind to the terminal glycine from an adjacent peptidoglycan strand. This cross-links the two peptidoglycan strands to provide stability to the cell wall. Penicillins and cephalosporins are structurally similar to the two terminal D-alanines and serve as substrates for transpeptidase. Therefore these agents will compete with the pepidoglycan D-alanines for this enzyme and thus limit the cross-linking within the cell walls.

wall synthesis). Penicillins lose considerable efficacy in long-established infections where the pathogens are in the lag growth phase or in situations where a bacte- riostatic antibiotic has been used prior to the penicillin (Yagiela et al. 1998).

Of the penicillins commercially available, penicillin V (the phenoxymethyl derivative of penicillin G) is the mainstay for treating endodontic infections. However, amoxicillin offers advantages in certain situations over penicillin V. Amoxicillin is an aminopenicillin that was developed as an orally effective analogue of ampicillin. Although it is typically considered a “broader spectrum” antibiotic than penicillin V, this expanded spectrum mainly refers to activity against organisms not routinely associated with endodontic infections, such as Haemophilus influenzae, and Escherichia coli (Johnson 1999). This is why the advantage of amoxicillin has classically been for immunocompromised or hospitalized patients, who may be at risk for secondary infections. Both penicillin V and amoxicillin are susceptible to inactivation by β-lactamase. To counteract this problem, penicillins have been compounded with agents that will inhibit the actions of β-lactamase. The most convenient product for use in dentistry is the orally effective form, which is a combination of amoxicillin with clavulanate (Augmentin).

Penicillins can be given orally or parenterally, via intramuscular or intravenous administration. Penicillin G, ampicillin, oxacillin, ampicillin-sublactam and

piperacillin-tazobactam, and ticarcillin-clavulanate are all given parenterally. However, penicillin V, amox- icillin, and amoxicillin-clavulanate are common oral penicillins (Hauser 2013).

There have been reports of reduced effectiveness of oral contraceptives in women taking amoxicillin, ampicillin, or penicillin. Although the association is weak, it is recommended to advise women taking these medications to use alternative measures of birth control (Walker and Shaddox 2009).

Cephalosporins also contain the β-lactam ring, and act with the same general mechanism as penicillins. This explains the cross-allergy that patients who are allergic to penicillins have to cephalosporins (Ganda 2013). Five generations of cephalosporins have now been produced. These drugs have a broader spec- trum of activity than penicillins. The fourth generation drug cefepime and the fifth generation ceftaroline can resist β-lactamases and can even be effective against methicillin-resistant Staphylococcus aureus (MRSA). They can also cross the blood–brain barrier (Hauser 2013).

12.4.1.2 Glycopeptides

These drugs, which include vancomycin, have a large molecular weight and cannot penetrate the porins of Gram-negative bacteria, and have poor gastrointestinal absorption. They are very effective against Gram- positive bacteria such as streptococci, staphylococci,

Systemic Antibiotics in Endodontic Infections 273

clostridia (including Clostridium difficile), and ente- rococci. Vancomycin-resistant enterococci (such as Enterococcus faecium and to a lesser degree Entero- coccus faecalis) were discovered in the late 1980s, and represent a major public health problem, particularly in nosocomial infections.

It is worth noting here that antibiotics such as nys- tatin and amphotericin B used to treat fungal infections affect cell membranes. Damage to the cell membrane leads to changes in membrane permeability and inhibi- tion of growth or death of the cell. Because of similar- ities between mammalian and fungal cell membranes, these antibiotics can cause cell damage to mammalian cells.

12.4.2 Interference with protein production

Second line drugs for treating endodontic infections can be found among those agents that owe their antimi- crobial activity to an ability to inhibit microbial pro- tein synthesis. Because of the importance of pro- teins as receptors, enzymes, transport channels, and so on, these agents can interfere with routine func- tions within the microorganism. Generally speaking, these agents are considered bacteriostatic; however, at higher concentrations and with specific organisms

they can be bactericidal (Johnson 1999). To understand the mechanisms of action of these agents, it is worth- while to review briefly the process of bacterial protein synthesis.

Bacterial protein synthesis proceeds in three stages: initiation, elongation, and termination (Kalant and Roschlau 1998; Hardman et al. 2005). Initiation involves the formation of the 70S ribosomal subunit. To start the process, mRNA must bind to a 30S riboso- mal subunit. Once this union has been established, the appropriate tRNA, with its accompanying amino acid, can bind to the mRNA and this complex now can bind to a 50S ribosomal subunit to complete the formation of the 70S ribosome. Once the tRNA is bound to the “A” (acyl) site on the ribosome, elongation of the pep- tide chain can occur. This entails the translocation of the tRNA-aa to the “P” (peptidyl) site where the amino acid is added to the growing peptide chain. The final step is termination, which is initiated by a termination codon on the mRNA and causes the elongation process to cease and the peptide chain to be released from the ribosome (Figure 12.3). Although protein synthesis in mammalian cells proceeds in an almost identical man- ner as described previously, we are fortunate that the mammalian ribosome is an 80S molecule, which is not easily split into subunits. Therefore, the antibiotics that

Fig. 12.3 The three steps in protein synthesis. (a) Frame 1 represents the initiation step. In this example a small section of a strand of mRNA is depicted with three codons for three different amino acids (aa1, aa2, aa3). The anticodon for the tRNA carrying aa1 binds to the first codon to initiate protein synthesis. The tetracycline family of antibiotics can inhibit this first step in protein synthesis. (b) Frame 2 represents the elongation step. In this step the adjacent codon has been bound by the complementary tRNA which allows transpeptidation to occur (i.e., aa1 is freed from its tRNA and bound to the next aa). When this occurs the “donating” tRNA is released from the mRNA. (c) Frame 3 represents the translocation step. In this step, the ribosome repositions on the mRNA so that the tRNA carrying the growing peptide chain now occupies the correct location on the ribosome to pass the peptide chain on to the next amino acid. In this illustration, once the tRNA carrying aa3 attaches to the mRNA, the stage will be set for a repeat of the activity portrayed in Frame 2. Macrolide and lincosamide antibiotics will interfere with this third step in protein synthesis.

274 Endodontic Microbiology

inhibit protein synthesis in microbes have little influ- ence on this process in mammals. Most of the adverse side effects associated with the use of these antibiotics can be traced back to overgrowth of nonsusceptible bacteria or direct cellular actions not related to protein synthesis.

The three families of antibiotics used in endodon- tics that inhibit protein synthesis are lincosamides, macrolides, and tetracyclines. Of these, the most useful are the lincosamides, and specifically clindamycin.

12.4.2.1 Clindamycin

Clindamycin is a member of the lincosamide group of antibiotics, which has good effectiveness against Gram-positive and anaerobic Gram-negative bacteria. It functions by the inhibition of protein synthesis, via action on the 30S ribosomal subunit. In oral infections, clindamycin acts as a good replacement of β-lactam antibiotics, for patients who have allergic reactions to β-lactams.

Clindamycin is generally known to have good soft tissue and bone penetration. It is a bacteriostatic agent when prescribed at the customary dose of 150–300 mg, but is thought to be bactericidal at the higher dose of 600 mg.

12.4.2.2 Macrolides

Macrolides are a relatively narrow spectrum group of antibiotics, and the main drugs are erythromycin, clar- ithromycin, and azithromycin. Their function involves binding the 50S subunit of the bacterial ribosome, thereby blocking peptide formation (Hauser 2013).

Macrolides are effective against Gram-positive cocci and bacilli and, to a lesser extent, Gram-negative cocci. Azithromycin expands the spectrum of activity to more Gram-negative bacteria.

Erythromycin and clarithromycin suppress the cytochrome 4503A4 enzyme in the liver and gut. This enzyme metabolizes many other drugs or converts them to the active metabolite. In its absence, these drugs become toxic and/or ineffective. This is why these macrolides have many drug–drug interactions. In fact, erythromycin is no longer recommended for clinical use in dentistry as it is also associated with bacterial resistance and moderate to severe gastroin- testinal side effects (Ganda 2013).

Azithromycin is safe in this regard as it does not affect the 3A4 enzyme. However, recent studies have

suggested that a 5-day course of azithromycin may place the patient at a higher risk for arrhythmia and other forms of cardiovascular disease. Therefore, short duration regimens for this drug are recommended (Ganda 2013).

12.4.2.3 Tetracyclines and glycylcyclines

Tetracycline was discovered in the 1950s, and is still being used as it is an effective broad-spectrum antibi- otic. Doxycycline and minocycline are newer mem- bers of the tetracycline family that are also in common use. These antibiotics act by binding with the 30S subunit of the ribosome, in a way that interferes with its binding with tRNA, and thus prevent protein syn- thesis. They are effective against Gram-positive and Gram-negative bacteria, spirochetes, Chlamydia spp., and Mycoplasma spp. (Hauser 2013).

Tigecycline is a member of the glycylcycline group of antibiotics, which is closely related to tetracyclines. Its structure prevents its recognition by many bacterial efflux pumps and makes it insensitive to modification of the 30S subunit that confer resistance to tetracy- clines. Therefore it is an effective antibiotic against many bacteria that are resistant to tetracyclines (Hauser 2013).

Side effects of tetracyclines include hypersensitiv- ity, phototoxicity, and bluish black skin pigmentation by minocycline. Dentin discoloration by topical application of all tetracyclines or tigecycline are com- mon, especially minocycline, which causes severe dentin discoloration. Tetracycline are bacteriostatic antibiotics.

12.4.2.4 Aminoglycosides

Common members of this group include gentamicin, amikacin, kanamycin, neomycin, and streptomycin. They bind 30S subunit in the bacterial ribosome and cause mismatch between mRNA and tRNA, thus inter- fering with protein synthesis. They have a very nar- row spectrum of action, and are mainly active against aerobic Gram-negative and Gram-positive bacteria. Aminoglycosides have high incidence of nephrotox- icity and ototoxicity, which limit their use in dentistry.

12.4.2.5 Rifamycins

Rifampin and its analogs are effective antibiotics against mycobacteria and staphylococci. They inhibit

Systemic Antibiotics in Endodontic Infections 275

bacterial RNA polymerase, thus interfering with for- mation of mRNA. These drugs are usually used in combination with other drugs. They are also consid- ered effective against bacterial biofilms. However, they interfere with the cytochrome P450 system and thus inhibit metabolism of other medications.

12.4.3 Interference with DNA structure and replication

12.4.3.1 Metronidazole

Metronidazole is an older antibiotic that has a nar- row spectrum of activity limited to obligate anaero- bic bacteria. In this regard it is active against both Gram-positive and Gram-negative obligate anaerobic bacteria, including C. difficile. It has a small molecu- lar structure that can easily diffuse into bacterial cells and, through a process of electron transfer, is reduced to release oxygen radicals, which disrupt the DNA of anaerobic bacteria (Hauser 2013).

Because of its limited spectrum, metronidazole is used to complement other antibiotics, such as β- lactams, in the treatment of oral infections, which tend to be a mixture of anaerobic and facultative organisms. The addition of metronidazole is made if the infec- tion does not appear to be responding to the initial antibiotic, and is suspected to be mostly comprised of anaerobic bacteria. It is also one of the antibiotics that can be used for the treatment of pseudomembranous colitis.

12.4.3.2 Quinolones

Common quinolones, like ciprofloxacin, levofloxacin, moxifloxacin, and ofloxacin, are potent antibiotics against aerobic Gram-positive and Gram-negative bac- teria. With the exception of ofloxacin (which is an oral agent), they can be administered orally or parenter- ally. They act by inhibiting two enzymes that regulate DNA supercoiling, thus interfering with protein syn- thesis and leading to cell death (Hauser 2013).

12.4.3.3 Sulfa drugs

Sulfa drugs are the oldest antibiotics, dating back to the 1930s. They act by preventing the formation of folic acid, which is a key factor in DNA synthesis. Sulfa drugs, including agents such as trimethoprim– sulfamethoxazole, are active against many aerobic

Gram-positive and Gram-negative bacteria; however, they suffer from increased resistance by bacteria, pri- marily because of the length of time that they have been in use.

12.5 Host factors

The host immune response, anatomic spread of dis- ease, liver and renal function, and allergies are some of the factors that influence the type, dosing, and effec- tiveness of antibiotics. Age and adiposity are also clearly important factors, and, like with any drug, pre- scription charts should be consulted to provide optimal prescription for the patient’s age and weight profile. In recent years it has been revealed that the host’s microbiome in the oral cavity, gut, skin, and other sites has unique features, which are modulated by environ- mental, geographic, genomic, and dietary factors. The microbiome carries a number of antibiotic resistance factors that are significantly influenced by these fac- tors. For example, it was shown that Europeans who travel to Asia or Africa acquire antibiotic resistance to a variety of antibiotics in their gut microflora, with- out actually consuming any antibiotics (Bengtsson- Palme et al. 2015). In addition, previous exposure to antibiotics clearly affects the prevalence of antibiotic resistance (Willmann et al. 2015). Thus, the preva- lence of antibiotic resistance in oral infections can vary widely from patient to patient, and future molec- ular approaches to identifying antibiotic resistance in endodontic infections should optimally be personal- ized to the individual infection being managed.

12.6 Antibiotic effectiveness and bacterial resistance in endodontics

Among the dozens of antibiotics available for the prac- titioner, the selection of the right agent for the non- allergic patient primarily depends on specificity and effectiveness of the antibiotic for the type of infec- tion that the patient has. As endodontic infections are polymicrobial, and the actual virulent microor- ganism in each infection cannot be easily identified, agents with a relatively wide spectrum against Gram- positive and Gram-negative facultative and anaero- bic organisms are preferred. Numerous studies have been performed to identify the antibiotic sensitiv- ity of endodontic microorganisms. In these studies,

276 Endodontic Microbiology

specimens are obtained from different sources, such as the necrotic pulp or the periapical abscess. The identi- fication of antibiotic sensitivity necessitate culturing of the bacteria, and exposing them to the various antibi- otics to determine the MIC and minimum bactericidal concentration (MBC). This means that for the multi- tude of endodontic bacteria that cannot be grown in the laboratory, or that require specific growth condi- tions not widely available, this type of analysis cannot be performed. Therefore, in recent years studies have been published that use molecular methods to identify antibiotic resistance genes within these specimens, in an attempt to define antibiotic resistance in a speci- men regardless of bacterial growth. Tables 12.1 and 12.2 summarize the findings of important examples of both types of these studies.

Culture and sensitivity studies generally show that β-lactam antibiotics, particularly those that contain the inactivator of β-lactamase, are very effective for endodontic infections. Metronidazole is a very effec- tive antibiotic for Gram-negative anaerobic bacteria, and is frequently used to supplement the primary antibiotic if the infection is not responding to ther- apy after 1–2 days. However, it cannot be used as the primary antibiotic for endodontic infections. There is ample data to show that for the patient who is allergic to penicillin, clindamycin is the antibiotic of choice. However, there are not enough data at this time to support the primary use of macrolides, tetracyclines, or quinolones for primary endodontic infections.

There are two main limitations to molecular studies that use amplification techniques to identify antibi- otic resistance genes: (i) the presence of an antibiotic resistance gene indicates the potential for resistance, but not necessarily that this resistance is expressed at a level to render the flora resistant, and (ii) there are hun- dreds of known antibiotic resistance genes at this time, and effective and affordable methods to screen a spec- imen for the important genes present are not currently available for clinical use. Some common oral bacteria and the β-lactamases that they produce are shown in Table 12.3. There are numerous molecular techniques that are currently used for rapid identification of bac- teria (and viruses) in diseases in which the etiologic agent is a specific species or strain of bacteria. How- ever, the clinical use of these molecular techniques in analyzing bacterial resistance for polymicrobial dis- eases, such as endodontic infections, is not currently available.

In a molecular study, Jungermann et al. (2011) stud- ied both culture and sensitivity, and identification of nine important antibiotic resistance genes was per- formed. The results showed reasonable correlation between the two methods. This study also revealed that primary infections contained significantly more β-lactam resistance genes than persistent infection. This finding is consistent with the general finding in numerous studies that showed that β-lactamases tend to be expressed by Gram-negative anaerobic bacte- ria, such as Prevotella spp. (Kuriyama et al. 2001), which are more prevalent in primary than persistent infections. This study also showed that an impor- tant tetracycline resistance gene, tet(M), was resis- tant to root canal instrumentation and medication techniques.

12.7 Antibiotic toxicities, allergies, and superinfections

There are many side effects that accompany the use of systemic antibiotics. These side effects pertain to the toxicities of individual drugs (which was discussed briefly for the agents described in this chapter) or aller- gies, and superinfections or secondary infections.

The prevalence of allergy to penicillin-class medi- cations is approximately 0.7–10%. These medications are the most frequent medication-related cause of ana- phylaxis in humans, causing about 75% of fatal ana- phylaxis cases in the USA each year (Neugut et al. 2001).

Oral intake of antibiotics can be associated with var- ious gasterointestinal disturbances, and may suppress important gut flora that is necessary for vitamin K production, thus resulting in problems with hemosta- sis. However, of particular concern to the use of oral antibiotics is the development of C. difficile infection. In 2011 alone, there were 453,000 cases of C. dif- ficile infection and 29,000 deaths in the USA, and approximately one-quarter of those infections were community-acquired (Lessa et al. 2015). The ability of different antibiotics to cause C. difficile infections is shown in Table 12.4.

The progression of bacterial resistance has become one of the most important public health crises of mod- ern times. It is well known that this has been a result of indiscriminate prescriptions to patients for condi- tions that do not warrant and would not benefit from antibiotics, dramatic increase in nosocomial infections

Systemic Antibiotics in Endodontic Infections 277

Table 12.1 Antibiotic resistance of bacteria from endodontic infections

Study Source Sample

size Resistant bacteria cultured Antibiotic resistance Notes

Kuriyama et al. (2001)

Orofacial infections (including 73 dentoalveolar infections)

93 P. melaninogenica P. oralis P. buccae Prevotella intermedia

β-lactamase-positive (71%) β-lactamase-positive (41%) β-lactamase-positive (24%) β-lactamase-positive (23%)

Only Prevotella spp. were positive

Dahlen et al. (2000)

Infected root canals

29 26 E. faecalis and 3 E. faecium

Benzylpenicillin Clindamycin Metronidazole Tetracycline Ampicillin

Sensitive to erythromycin and vancomycin

Pinheiro et al. (2003)

Infected root canals

30 Enterococci (37%) from nonhealing cases

Erythromycin Azithromycin

Sensitive to Benzylpenicillin, amoxicillin and amoxicillin + clavulanic acid

Baumgartner and Xia (2003)

Pus aspirated from abscesses

12 98 anaerobic or facultative strains of bacteria

Metronidazole (55%) Penicillin (15%) Amoxicillin (9%) Penicillin + m Clindamycin (4%) Augmentin, amoxicillin +

metronidazole (0–1%) Jungermann

et al. (2011) 24 strains root

canals of teeth with 9 primary and 7 persistent infections

16 4 aerobe and 20 anaerobe strains

Metronidazole (38%) Clindamycin (25%) Amifloxacin (17% Tetracycline (13%) Amoxicillin, doxycycline (4%) Augmentin, tigecycline (0%)

Sousa et al. (2013)

Root canals with periapical abscess

60 Anaerococcus prevotii (22/60), Parvimonas micra (19/60), Fusobacterium necrophorum (19/60), Prevotella intermedia/ nigrescens (16/60) Streptococcus constellatus (12/60), and Fusobacterium nucleatum (12/60).

A. prevotii and Fusobacterium species to azithromycin and erythromycin, as well as A. prevotii and F. necrophorum to metronidazole

0–13 bacterial species identified per canal. No β-lactamase found

Lins et al. (2013) 20 strains from root canals with primary infections

43 Enterococcus faecalis Tetracycline (70%) Vancomycin (45% borderline) Penicillin, erythromycin (0%)

Al-Ahmad et al. (2014)

Infected root canals

21 Cultured 47 strains of 32 different bacterial species

Metronidazole (49%) Gentamicin (36%) Fosfomycin (30%) Vancomycin (28%) Doxycycline, tetracycline

(13%) Clindamycin (11%) Rifampicin (4%) Penicillin G, ciprofloxacin

(2%) Amoxicillin, moxifloxacin

(0%)

278 Endodontic Microbiology

Table 12.2 Antibiotic resistance genes identified in endodontic infections

Study Source Sample

size Antibiotic resistance genes Notes

Jungermann et al. (2011)

Root canals of 30 primary and 15 persistent infections, preoperatively and after root canal instrumentation and medication

45 blaTEM-1 (33%) Significantly more in primary than persistent infections. Significantly reduced after instrumentation and medication

blaZ (11%) Eliminated after instrumentation and medication

cfxA (2%) Eliminated after instrumentation and medication

tetW (18%) Significantly reduced after instrumentation and medication

tetM (18%) Prevalence not affected by instrumentation and medication

tetQ (9%) Eliminated after instrumentation and medication

van(A), van(D) and van(E) (0%) No vancomycin resistance detected

Rôças and Siqueira (2012)

41 bacterial strains cultured from infected root canals

26 blaTEM (17%) tetW (10%) ermC (10%) tetM (5%) cfxA (2%) tetS (2%)

32% of isolates were positive to 1–3 of 14 genes tested

Rôças and Siqueira (2013)

Abscesses and asymptomatic infected root canals

25 blaTEM Abscess (24%) Asymptomatic (0%)

cfxA Abscess (0%) Asymptomatic (0%)

ermC Abscess (24%) Asymptomatic (25%)

tetM Abscess (8%) Asymptomatic (42%)

tetW Abscess (12%) Asymptomatic (29%)

tetQ Abscess (0%) Asymptomatic (0%)

All eliminated after treatment except tetM (13%) and tetW (8%)

Lins et al. (2013) 20 Enterococcus faecalis strains from root canals with primary infections

43 tetM (60%) tetL (20%)

Systemic Antibiotics in Endodontic Infections 279

Table 12.3 Common β-lactamases in Gram-negative oral bacteria and the bacteria that harbor them

CblA Bacteroides uniformis CepA Bacteroides fragilis CfiA Bacteroides fragilis CSP-1 Capnocytophaga sputigena TEM17 Capnocytophaga ochracea FUS-1 Fusobacterium nucleatum CfxA Bacteroides distasonis CfxA B. fragilis, B. vulgatus CfxA2 Prevotella, Capnocytophaga spp. CfxA3 Capnocytophaga spp. PEN-Y Fusobacterium

Source: Adapted from Dupin et al. (2015). Reproduced with permis- sion of Elsevier.

with resistant bacteria fueled by antibiotic use in hos- pitals, and the massive use of antibiotics in raising farm animals. It is well known that bacterial resistance to penicillins increases in patients who have taken peni- cillins previously (Kuriyama et al. 2000). Therefore, it is essential for the dentist to consider the best available evidence for patient care and use the minimal amount of antibiotics that would result in effective manage- ment of their patients. This practice would assure an adequate balance of risk and benefit for the use of these important drugs.

Table 12.4 Commonly used antibiotic classes and their association with Clostridium difficile infection

Class Association with C. difficile infection

Clindamycin Very common Ampicillin Very common Amoxicillin Very common Cephalosporins Very common Fluoroquinolones Very common Other penicillins Somewhat common Macrolides Somewhat common Metronidazole Uncommon Rifampin Uncommon Tetracyclines Uncommon Carbapenems Uncommon Daptomycin Uncommon Tigecycline Uncommon

Source: Adapted from Leffler and Lamont (2015). Reproduced with permission of Massachusetts Medical Society.

12.8 Role of antibiotics in clinical management of endodontic infections

Endodontic disease is fundamentally a form of infec- tious disease. Therefore, the use of adjunctive antibi- otics in the management of endodontic patients, par- ticularly those with spreading infections, is commonly advocated. However, and as noted previously, antibi- otics are not without side effects and complications themselves. Therefore, the prudent practitioner should use existing best evidence to make decisions on the risk and benefits of using adjunctive antibiotics in the management of patients.

It is common practice in the treatment of other infec- tious diseases to prescribe empirical or specific adjunc- tive antibiotics, to aid in the abatement of the infection. In endodontics, there are a number of factors that influ- ence this decision, which are different from other oral or systemic infections. The source of the infection in endodontics is the infected pulp space, which contains bacterial biofilms. Antibiotics delivered systemically can diffuse into the vital dental pulp and periradicu- lar tissues (Akimoto et al. 1986; Wang et al. 1988). However, they cannot diffuse into the root canal space of a necrotic pulp (Jaouni et al. 2010). There is also little diffusion of antibiotics into abscesses or fascial space infections, as these areas are not adequately vas- cularized. Therefore, the ability of antibiotics to aug- ment healing of endodontic infections that range from pulpitis to abscesses, whether symptomatic or asymp- tomatic, has been the subject of much debate and a number of clinical studies.

Older literature suggested that adjunctive antibiotics may assist in the resolution of acute endodontic infec- tions or the prevention of endodontic flareups (Mata et al. 1985; Morse et al. 1987; Abbott et al. 1988). How- ever, many of these studies did not adequately control for the placebo effect or were not adequately random- ized. Studies using contemporary methodologies for clinical trials have reached different conclusions.

Randomized clinical trials (RCTs) provide the best study design to show cause and effect, and control for various study variables. The following decribes the important RCTs in this area. In the treatment of symp- tomatic irreversible pulpitis, the mere administration of penicillin did not result in improvement compared with placebo (Nagle et al. 2000). Preoperative peni- cillin or amoxicillin in conjunction with adequate root canal instrumentation did not differ from placebo in

280 Endodontic Microbiology

preventing postoperative flareups in patients with pulp necrosis and asymptomatic apical periodontitis (Wal- ton and Chiappinelli 1993; Pickenpaugh et al. 2001). In the treatment of symptomatic apical periodontitis or localized acute apical abscess, penicillin did not dif- fer from placebo in resolving postoperative pain and/or swelling, following adequate instrumentation and inci- sion for drainage (Fouad et al. 1996; Henry et al. 2001). One study found that using erythromycin reduced post- operative pain (Torabinejad et al. 1994); however, as noted before, this antibiotic has many side effects, and is not currently not often used in dentistry. A number of systematic reviews (Matthews et al. 2003; Matthews and Sutherland 2004; Aminoshariae and Kulild 2016), and two Cochrane reviews (Keenan et al. 2005; Cope et al. 2014) have confirmed these findings.

It is important to note that the studies evaluating the efficacy of supplemental antibiotics in patients with pulp necrosis have always employed a methodol- ogy in which the source of the infection (the necrotic pulp space) was adequately debrided and medicated, and the localized swelling, if present, was drained. Most patients in these studies were also systemically healthy. Observational studies have shown that patients on antibiotics frequently experience flareups at the same rate as, or higher than, patients not on antibi- otics, as the rate of flareups is generally considered to depend on preoperative pain (Walton and Fouad 1992). However, the clinician frequently faces a situation in which the patient cannot be adequately treated because of time, complexity of the case, or resources. In addi- tion, there are many situations in which the patient has other systemic diseases that may suppress the immune response, or make them at risk for secondary infec- tions. As no RCTs are available for guidance in these situations, the clinician must use his/her best judgment in triaging and treating the patient, and may prescribe supplemental antibiotics if he/she believes this may enhance patient safety and comfort.

In the realm of endodontic surgery, there are scarce data on the efficacy of antibiotics in preventing post- operative infections. One relatively large RCT showed that clindamycin was no different from placebo in preventing postoperative infections (Lindeboom et al. 2005). However, the overall incidence of postoperative infections in this study was very small and may not be representative of surgery in other settings.

Adjunctive antibiotics are clearly essential in con- trolling spreading endodontic infections, which are characterized by fever, malaise, lymphadenopathy,

fascial space involvement, especially when the patient’s immune response is compromised because of other comorbidities. As noted in Chapter 10, endodon- tic infections can easily spread from the site of primary infection to other sites in the body. Many studies have compared the efficacy of existing antibiotics. Aug- mentin has been shown to be superior to penicillin V in treating spreading dentoalveolar abscesses (Lewis et al. 1993). In this study, patients receiving Aug- mentin (co-amoxiclav) recorded a significantly greater decrease in pain during the second and third days post- operatively. More recently, another study reported on the activity, safety, and tolerability of the azithromycin (500 mg, once daily) with that of Augmentin (625 mg, three times daily) in the treatment of acute periapi- cal abscesses in adults, in an open, randomized, mul- ticenter trial (Adriaenssen 1998). They reported no difference between patients receiving azithromycin and those receiving Augmentin. It is believed by some clinicians that azithromycin may be advanta- geous compared to Augmentin considering the com- pliance and cost effectiveness. However, as noted before, azithromycin was recently reported to be asso- ciated with an increase in cardiovascular deaths seen in patients with a high baseline risk of cardiovascular disease during a 5-day course of azithromycin ther- apy (Ray et al. 2012). Severe endodontic infections that result in fascial space involvement and signifi- cant morbidity are typically treated more aggressively with a number of antibiotics, which include a β-lactam such as IV oxicillin, a quinolone such as moxifloxacin and metronidazole (see Chapter 10). A general recom- mendation for antibiotic usage in symptomatic cases was previously published (Fouad 2002) and has been updated here (Figure 12.4).

12.9 Indications for prophylactic antibiotic therapy

It has been known for decades that endodontic treat- ment procedures can induce systemic bacteremia at a rate of about 17–30% (Bender et al. 1960; Baumgart- ner et al. 1976; Heimdahl et al. 1990; Savarrio et al. 2005), which increases to 50% if instruments in an infected canal are introduced into the periapical lesion (Debelian et al. 1995). The incidence of bacteremia following endodontic surgery is about 33–83% (Baumgartner et al. 1977). In addition, bacteremia was also associate with procedures such as rubber dam

Systemic Antibiotics in Endodontic Infections 281

Fig. 12.4 Recommendations for antibiotic therapy in cases of symptomatic endodontic infections. Source: Fouad (2002). Reproduced with permission of John Wiley and Sons.

isolation (29%) and periodontal ligament anesthesia (97%) (Roberts 1999). Thus, endodontic procedures are included in dental procedures for which prophylac- tic antibiotics in the patient at risk may be indicated.

The American Heart Association has revised its guidelines for prophylaxis of patients at risk of infec- tive endocarditis many times. In the last revision (Wilson et al. 2007), the following conditions for which the prophylaxis was necessary were identified:

� Prosthetic cardiac valve � Previous infective endocarditis � Congenital heart disease (CHD) ◦ Unrepaired cyanotic CHD, including palliative

shunts and conduits ◦ Completely repaired congenital heart defect with

prosthetic material or device, whether placed by surgery or by catheter intervention, during the first 6 months after the procedure

◦ Repaired CHD with residual defects at the site or adjacent to the site of a prosthetic patch or pros- thetic device (which interferes with endothelial- ization)

� Cardiac transplantation recipients who develop car- diac valvulopathy.

The guidelines for patients with prosthetic joints have also been subject to much scrutiny. In 2012, the American Dental Association and the American Asso- ciation of Orthopedic Surgeons published joint guide- lines based on a systematic review of the literature (AAOS and ADA 2012). This report’s main conclusion was that “the practitioner might consider discontinu- ing the practice of routinely prescribing prophylactic antibiotics” for these patients. More recently, the lit- erature was reexamined in light of newer information (Sollecito et al. 2015). In that report, the statement changed to “In general, for patients with prosthetic joint implants, prophylactic antibiotics are not recom- mended prior to dental procedures to prevent prosthetic joint infection.”

Clearly, it remains controversial whether antibiotic prophylaxis really protects the patient against sec- ondary infections from a hematogenous source fol- lowing dental procedures. The reason for this is that direct evidence for situations in which the antibi- otics were protective against these major infections are lacking, and the experimental testing of this would not be considered ethical in patients who truly need to be protected. In the UK, the routine prophylaxis of dental patients for protection against infective

282 Endodontic Microbiology

0 Baseline

N = 290; RCT design; 16S rDNA analysis

Percent Positive Blood Culture

Extraction Alone

Extraction + Amoxicillin

Brushing

1.5 min 5 min 20 min 40 min 60 min

30 Fig. 12.5 Results of a randomized clinical trial (RCT) showing the incidence of bacteremia following single extraction and extraction with amoxicillin versus tooth brushing. Molecular analysis of bacteria was undertaken. Source: Adapted from Lockhart et al. (2008). Reproduced with permission of American Heart Association.

endocarditis was eliminated in 2008. Initial studies following this change revealed no changes in the over- all risk of infective endocarditis (Thornhill et al. 2011). However, more comprehensive recent analysis showed a significant increase in the incidence of infective

endocarditis since the change, although not associated with any change in mortality from this disease (Dayer et al. 2015). It was shown in this study that 277 antibi- otic prescriptions would be needed to prevent one case of infective endocarditis.

Table 12.5 Prophylactic regimens for dental procedures

Situation Agent Regimen: single dose 30–60 minutes before procedure

Standard general prophylaxis—oral Amoxicillin Adults: 2.0 g Children: 50 mg/kg

Unable to take oral medications Ampicillin Adults: 2.0 g IM or IV Children: 50 mg/kg IM or IV

or Cefazolina,b or Adults: 2 g IM or IV

Ceftriaxone Adults: 1 g IM or IV Children: 50 mg/kg IM or IV

Allergic to penicillins Clindamycin Adults: 600 mg Children: 20 mg/kg

or Cephalexina,b Adults: 2.0 g

Children: 50 mg/kg

Azithromycin or clarithromycin

Adults: 500 mg Children: 15 mg/kg

Allergic to penicillin and unable to take oral medications

Clindamycin Adults: 600 mg IM or IV Children: 20 mg/kg IM or IV

or Cefazolina,b or

ceftriaxone Adults: 1.0 g IM or IV Children: 50 mg/kg IM or IV

IM, intramuscularly; IV, intravenously. aCephalosporins should not be used in patients with a history of anaphylaxis, angioedema, or urticaria with penicillins or ampicillin. bOr other first or second generation oral cephalosporin in equivalent adult to pediatric dosage. Source: Adapted from Wilson et al. (2007). Reproduced with permission of American Heart Association.

Systemic Antibiotics in Endodontic Infections 283

In addition to dental procedures, patients at risk of infective endocarditis or prosthetic joint replacement have many other sources of bacteremia. One source related to the oral cavity is tooth brushing. In one study, bacteremia following tooth brushing was compared to that which results after tooth extraction, with and without amoxicillin, given according to the American Heart Association guidelines (Lockhart et al. 2008). The results showed that while amoxicillin adminis- tration lowered the incidence of bacteremia following extraction, tooth brushing was also associated with a sustained bacteremia in about 10% of the population (Figure 12.5).

Among the available antibiotics for prophylaxis, amoxicillin is still the agent of choice for the nonal- lergic patient (Table 12.5). In one study, the incidence of bacteremia following tooth extraction was 96% in controls, 85% following 600 mg clindamycin, 57% following 400 mg moxifloxacin and 46% following 2 g amoxicillin (Diz Dios et al. 2006).

With respect to the prophylaxis for patients with prosthetic joint replacement, it was shown in the AAOS and ADA reviews that patients who are espe- cially at risk of a hematogenous joint infection include:

� Patients with previous (late) artificial joint infection; � Increased morbidity associated with joint surgery

(e.g., wound drainage/hematoma); � Patients undergoing treatment of severe and spread-

ing oral infections (e.g., cellulitis).

Antibiotics are themselves associated with signifi- cant side effects, and from a societal perspective the risk–benefit of such blanket guidelines need to be care- fully considered. Most practitioners agree though that patients in the following groups may benefit from antibiotic prophylaxis:

� Patients with increased susceptibility for systemic infection: ◦ Congenital or acquired immunodeficiency ◦ Patients on immunosuppressive medications ◦ Diabetics with poor glycemic control ◦ Patients with systemic immunocompromising dis-

orders (e.g., rheumatoid arthiritis, lupus erythe- matosus);

� Patients in whom extensive and invasive procedures are planned.

12.10 References

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Al-Ahmad A, Ameen H, Pelz K, et al. 2014. Antibiotic resis- tance and capacity for biofilm formation of different bac- teria isolated from endodontic infections associated with root-filled teeth. J Endod 40: 223–230.

Aminoshariae A, Kulild JC. 2016. Evidence-based recom- mendations for antibiotic usage to treat endodontic infec- tions and pain: a systematic review of randomized con- trolled trials. J Am Dent Assoc 147: 186–191.

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Baumgartner JC, Heggers JP, Harrison JW. 1977. Incidence of bacteremias related to endodontic procedures. II. Sur- gical endodontics. J Endod 3: 399–402.

Baumgartner JC, Xia T. 2003. Antibiotic susceptibility of bacteria associated with endodontic abscesses. J Endod 29: 44–47.

Bender IB, Seltzer S, Yermish M. 1960. Incidence of bac- teremia in endodontic manipulation. Oral Surg Oral Med Oral Pathol 13: 353–356.

Bengtsson-Palme J, Angelin M, Huss M, et al. 2015. The Human Gut Microbiome as a Transporter of Antibiotic Resistance Genes between Continents. Antimicrob Agents Chemother 59: 6551–6560.

Cope A, Francis N, Wood F, Mann MK, Chestnutt IG. 2014. Systemic antibiotics for symptomatic apical periodontitis and acute apical abscess in adults. Cochrane Database Syst Rev 6: CD010136.

Dahlen G, Samuelsson W, Molander A, Reit C. 2000. Iden- tification and antimicrobial susceptibility of enterococci isolated from the root canal. Oral Microbiol Immunol 15: 309–312.

Dayer MJ, Jones S, Prendergast B, Baddour LM, Lockhart PB, Thornhill MH. 2015. Infective endocarditis and antibi- otic prophylaxis: Authors’ reply. Lancet 386: 531–532.

Debelian GJ, Olsen I, Tronstad L. 1995. Bacteremia in con- junction with endodontic therapy. Endod Dent Traumatol 11: 142–149.

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Fouad AF. 2002. Are antibiotics effective for endodontic use? an evidence-based review. Endod Topics 3: 52–66.

Fouad AF, Rivera EM, Walton RE. 1996. Penicillin as a supplement in resolving the localized acute apical abscess. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 81: 590–595.

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Heimdahl A, Hall G, Hedberg M, et al. 1990. Detection and quantitation by lysis-filtration of bacteremia after different oral surgical procedures. J Clin Microbiol 28: 2205–2209.

Henry M, Reader A, Beck M. 2001. Effect of penicillin on postoperative endodontic pain and swelling in symp- tomatic necrotic teeth. J Endod 27: 117–123.

Jaouni M, Tolba M, Romberg E, Nemieboka N, Fouad AF. 2010. Penetration of systemic antibiotics into necrotic pulp space. J Endod 36: 558.

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Jungermann GB, Burns K, Nandakumar R, Tolba M, Venezia RA, Fouad AF. 2011. Antibiotic resistance in primary and persistent endodontic infections. J Endod 37: 1337–1344.

Kalant H, Roschlau WHE (eds). 1998. Principles of Medical Pharmacology. New York, NY: Oxford University Press.

Keenan J, Farman A, Fedorowicz Z, Newton J. 2005. Antibi- otic use for irreversible pulpitis. Cochrane Database Syst Rev 2: CD004969.

Kuriyama T, Karasawa T, Nakagawa K, Yamamoto E, Naka- mura S. 2001. Incidence of b-lactamase production and antimicrobial susceptibility of anaerobic Gram-negative rods isolated from pus specimens of orofacial odontogenic infections. Oral Microbiol Immunol 16: 10–15.

Kuriyama T, Nakagawa K, Karasawa T, Saiki Y, Yamamoto E, Nakamura S. 2000. Past administration of beta- lactam antibiotics and increase in the emergence of beta- lactamase-producing bacteria in patients with orofacial odontogenic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 89: 186–192.

Leffler DA, Lamont JT. 2015. Clostridium difficile infection. N Engl J Med 372: 1539–1548.

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Lewis MA, Carmichael F, MacFarlane TW, Milligan SG. 1993. A randomised trial of co-amoxiclav (Augmentin) versus penicillin V in the treatment of acute dentoalveolar abscess. Br Dent J 175: 169–174.

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Chapter 13 Topical Antimicrobials in Endodontics Anil Kishen

13.1 Introduction 13.1.1 Targeting biofilm in endodontic

disinfection 13.1.2 Therapeutic strategies against

root canal biofilm 13.2 Challenges for topical antimicrobials in

root canal disinfection 13.2.1 Root canal anatomy 13.2.2 Structure and composition of

dentin 13.2.3 Fluid (irrigation) dynamics in

root canal 13.3 Requirements of endodontic topical

antimicrobials

13.4 Classification of topical antimicrobials in root canal therapy 13.4.1 Chemical-based antimicrobials 13.4.2 Nonchemical-based

antimicrobials 13.4.3 Antimicrobial photodynamic

therapy 13.4.4 Laser-assisted root canal

disinfection 13.4.5 Ozone

13.5 Conclusions 13.6 References

13.1 Introduction

Bacterial infection of the root canal system is com- monly associated with deep dental caries, cracks, or fractures that result in pathways in dental hard tissue enabling microbial invasion of the pulp. However, the degree of microbial infection of the pulp tissue is not related to the pathway of microbial invasion. The cur- rent knowledge in endodontic microbiology stresses that endodontic disease is a bacterial biofilm-mediated disease (Ricucci and Siqueira 2010). Therefore, the major objectives that needs to be considered in root canal treatment are (i) to thoroughly eliminate or sub- stantially reduce root canal biofilms, and (ii) to prevent recontamination of the treated root canal systems.

The organization of microbial communities in infected root canals showed marked consistency with

a biofilm mode of growth, although no unique pat- tern of microbial biofilm was associated with any type of endodontic infection (Ramachandran Nair 1987; Nair et al. 1990; Sundqvist and Figdor 2003; Nair 2006; Ricucci and Siqueira 2010). Generally, the degree of microbial infection differs for a tooth with reversible or irreversible pulpitis when compared to a tooth with pulp necrosis. In pulpitis, the pres- ence of vital pulp tissue with active host-immune cells results in varying degrees of noninfected pulp space, whereas the pulp space of teeth with necrotic pulp with radiographic signs of periapical rarefaction will typi- cally harbor higher levels of microorganisms (Zehn- der 2006; Siqueira and Ricucci 2010). Consequently, any effort to eliminate microbial biofilm is more chal- lenging in teeth with necrotic pulp than in teeth with pulpitis.

287

288 Endodontic Microbiology

13.1.1 Targeting biofilm in endodontic disinfection

Endodontic biofilms harbor a polymicrobial popula- tion which, depending upon the endodontic microenvi- ronment, consist of obligate anaerobes and facultative anaerobes that are Gram-positive and Gram-negative bacteria (Ramachandran Nair 1987; Nair et al. 1990; Sundqvist and Figdor 2003; Nair 2006; Ricucci and Siqueira 2010). Typically, a mature biofilm is com- posed of multiple layers of bacteria embedded in a self-made matrix formed of extracellular polymeric substance (EPS). Bacteria in a biofilm are safeguarded against antimicrobials by different mechanisms, stem- ming from the uniqueness associated with the develop- ment and structure of biofilm. It is important to recog- nize that there is no single mechanism that can account for the resistance offered by the biofilm bacteria to antimicrobials.

Different mechanisms have been proposed to explain antimicrobial resistance in biofilm bacteria. They include factors associated with: (i) extracellu- lar polymeric matrix; (ii) growth rate and nutrient availability; and (iii) adoption of resistance phenotype. These mechanisms are expected to act in a concerted manner, amplifying any variation that is associated with the susceptibility of phenotypes to offer enhanced antimicrobial resistance to resident bacteria (Costerton 1999; Prince 2002; del Pozo and Patel 2007).

The EPS has the potential to protect the resident bacteria (i) by acting as a physical barrier against the antimicrobials and (ii) by neutralizing the chemical effect of antimicrobials (del Pozo and Patel 2007). In addition, the bacteria in a biofilm are exposed to different nutrient and growth conditions. The spatial arrangement of bacteria will expose the bacterial cells in the deeper aspects of the biofilm to fewer nutrients

and redox potential than the bacteria on the sur- face. The resistance associated with biofilm bacteria is attributed to the slow growth and starvation of bac- terial cells (Baumgartner et al. 2008). As the degree of nutrient and gas gradients increases with thickness and maturity of a biofilm, the influence of growth rate and oxygen on the antimicrobial resistance is partic- ularly marked in aged biofilm (Lewis 2005). Further, bacterial cells growing in a biofilm community, when exposed to unfavorable stress or low-level antimicro- bials, form specialized survivor cells called persister cells (Brooun et al. 2000). The persister cells are non- growing phenotypic variants of the bacterial cell pop- ulation. When the unfavorable stresses are reversed, these persister cells grow rapidly in the presence of nutrients. Biofilm populations are rich in persister cells; these cells could conceivably survive endodontic treatment procedures and proliferate in the posttreat- ment phase (Lewis 2005). Biofilm bacteria upregulate the expression of stress-response genes, shock pro- teins, and multidrug pumps, which will switch them to more resistant phenotypes (Vrany et al. 1997). Thus, the structure of biofilm and physiologic characteristics of resident microorganisms offer biofilm bacteria resis- tance to antimicrobials (Johnston et al. 2000; Prince 2002) (Figure 13.1).

13.1.2 Therapeutic strategies against root canal biofilm

The presence of EPS and localized high density of bac- terial cells (biomass) in a biofilm has been considered to be a major challenge in endodontics. Therapeutic strategies currently utilized in the treatment of bacte- rial biofilms are shown in Figure 13.2. Generally, the therapeutic strategy against biofilm bacteria focuses

Fig. 13.1 Different methods by which bacteria in a biofilm gain resistance against antimicrobials.

Topical Antimicrobials in Endodontics 289

Fig. 13.2 Strategies to treat biofilm mediated infection.

on: (i) killing the resident bacteria within a biofilm structure, or (ii) disrupting the biofilm structure and simultaneously eliminating the resident bacteria.

Disinfection kinetics of different antimicrobials, including sodium hypochlorite, against bacteria showed that the level of disinfection achieved for a given concentration of antimicrobial was linearly related to the cell density (bacterial biomass). There was a large increase in antibacterial efficacy when smaller inoculum levels were used. However, when higher levels of bacterial inoculum were tested, the efficacies of antimicrobials were severely diminished. The observed variation in antimicrobial efficacy is believed to be produced by the intrinsic self-quenching effect of antimicrobials by the microbial cells during disinfection (Nichols et al. 1989). This observation highlights the significance of using topical antimicro- bials in different stages of endodontic treatment, with different delivery (irrigation) methods, to effectively reduce bacterial biomass and consequently extract the full potential of the antimicrobial selected.

13.2 Challenges for topical antimicrobials in root canal disinfection

Topical antimicrobials used in endodontic therapy are primarily liquid antimicrobials, used to combat estab- lished pathogenic microbial biofilms within the root canals. To successfully achieve this goal, the antimi- crobials are required to counter or compensate for

Fig. 13.3 Challenges in the disinfection of endodontic biofilms.

the challenges present within the root canal system (Figure 13.3).

13.2.1 Root canal anatomy

The root canal system is well established to be a highly complex anatomy presenting challenges to dis- infection using topical antimicrobials. This is not only because of the complex geometries, but also because of the complex features in a root canal system such as accessory canals, lateral canals, apical ramifications, and transverse anastomoses (Gutmann 1992; Cleghorn et al. 2007; Ordinola-Zapata et al. 2013b). The main root canal lumen is sometimes found to communicate with another root canal lumen via an isthmus. These complexities will account for 30–50% of the canal wall left uninstrumented during routine root canal instru- mentation (Peters et al. 2000). Unfortunately, cur- rent topical antimicrobials rely on instrumentation to enhance their efficacy within root canals. The inabil- ity of antimicrobials to penetrate and interact with the bacterial biofilms in the root canal complexities will result in surviving bacterial biofilms in the uninstru- mented portions, apical ramifications, and isthmuses of the root canal system after cleaning and shaping procedures (Vera et al. 2012).

13.2.2 Structure and composition of dentin

Dentin is a porous and hydrated hard tissue. It is made up of an inorganic phase (carbonated hydroxyapatite), an organic phase (collagenous and non-collagenous proteins), and a water phase. The topical antimicrobial used on root dentin can interact with the organic and

290 Endodontic Microbiology

inorganic constituents of the dentin matrix, resulting in a buffering effect. Furthermore, the chemical inter- action and buffering effect is more significant within the root canal, because only a small volume of antimi- crobial is used here. This buffering effect leads to the observed time-dependent and depth effects of chemi- cals in the root dentin (Haapasalo et al. 2010).

The tubular nature of dentin accounts for its porosity and its susceptibility to bacterial invasion (Love 2001). The degree of bacterial penetration varies between dif- ferent regions of the dentin and the numbers of patent dentinal tubules (Love 2001). The inability of antimi- crobials to penetrate infected dentinal tubules results in the survival of bacterial populations within dentin and the infected dentin serving as a reservoir of infection. Berutti et al. (1997) showed that irrigating the canal with sodium hypochlorite (after removing the smear layer), rendered the dentinal tubules bacteria-free only to a depth of 130 μm from the canal lumen, beyond which viable bacteria were detected.

13.2.3 Fluid (irrigation) dynamics in root canal

Irrigation is the process of delivery of irrigant within the root canal. Irrigation dynamics deals with how irrigants flow, penetrate, and exchange within the root canal space and the forces they produce. The process of irrigation (physical effects) and the antibacterial char- acteristics (chemical effects) of the irrigant are critical in root canal disinfection. The physical objectives of irrigation is to allow the flow of irrigant throughout the root canal system in order to detach the biofilm struc- tures as well as to loosen and flush out the debris from the root canals. Physical effectiveness will depend on the ability of fluids to generate optimum streaming forces within the root canal. The physical effects of irrigation will complement the chemical effects of irri- gants such as antibacterial characteristics, ability to inactivate endotoxin, tissue dissolution capacity, and ability to remove debris and smear layer. It should be noted that even the most potent antimicrobial will be ineffective if it cannot penetrate up to the work- ing length, interact with the canal wall, and exchange frequently within the canal space (Gu et al. 2009; Gulabivala et al. 2010).

The tooth roots are embedded in bone sockets and are therefore considered to behave as closed end chan- nels. This can lead to gas entrainment at the closed end during irrigation (vapor lock effect) (Tay et al. 2010).

The delivery of irrigant using a syringe needle results in a slow or passive flow of irrigant at the apical 1– 2 mm from the exit of the needle. Moreover, the shear stresses exerted by the fluid on the canal walls were significantly less when compared with the center of root canal lumen (Boutsioukis et al. 2009, 2010; Chen 2014). In order to circumvent the above challenges, endodontic irrigation should be combined with strate- gies that apply pressure gradients on the irrigants with ultrasonic or sonic agitation or apical negative pressure irrigation. Application of pressure gradients on an irri- gant can improve the fluid flow dynamics within the root canals and subsequently improve the efficacy of a topical antimicrobial (Moser and Heuer 1982; Nielsen and Craig Baumgartner 2007; Basrani 2011).

13.3 Requirements of endodontic topical antimicrobials

The requirements of topical antimicrobials in root canal treatment can be categorized into primary and secondary requirements.

Primary requirements: � To debride and disinfect the root canal system effec-

tively by killing bacteria and fungi in a biofilm, and inactivate viruses and bacterial by-products.

Secondary requirements: � Flow into the entire root canal space and penetrate

the dentinal tubules; � Provide long-term antibacterial effect (substantiv-

ity); � Provide therapeutic efficacy in the presence of dentin

(inorganic/organic constituents) and pulp (organic) tissue remnants;

� Produce no adverse effects on dentin or on the seal- ing ability of restorative materials;

� Produce no cytotoxic effects on vital periapical tis- sues; and

� Preferably, cost-effective and convenient to use with different irrigation devices.

13.4 Classification of topical antimicrobials in root canal therapy

Topical antimicrobials in root canal therapy can be categorized as (chemical-based antimicrobials or nonchemical-based antimicrobials.

Topical Antimicrobials in Endodontics 291

13.4.1 Chemical-based antimicrobials

13.4.1.1 Sodium hypochlorite

Sodium hypochlorite was introduced as a disinfecting agent in the nineteenth century (Zehnder 2006). It was Dakin (1915) who advocated a buffered solution of 0.5% sodium hypochlorite to irrigate infected wounds. It possesses broad-spectrum antibacterial, virucidal, and sporicidal properties (McDonnell and Russell 1999). Experiments showed that sodium hypochlo- rite has the ability to disrupt biofilm matrices (Bryce et al. 2009), while older studies have shown the capac- ity of sodium hypochlorite to dissolve necrotic tissue (Austin and Taylor 1918). It was reported that 5% sodium hypochlorite solution dissolves tissue in 20 minutes to 2 hours (Grossman and Meiman 1941). The ability of sodium hypochlorite to destroy a broad spectrum of bacteria nonspecifically in combination with its capacity to dissolve necrotic tissue make it a topical antimicrobial irrigant of choice in endodontic therapy (Zehnder 2006).

13.4.1.1.1 Mechanism of action The antibacterial effect of sodium hypochlorite is attributed to a combination of mechanisms or chemi- cal injury to the bacteria (Estrela et al. 1995). Sodium hypochlorite in a solution occurs in a dynamic equi- librium (Estrela et al. 2002). The effect of sodium hypochlorite on bacteria and tissue remnants is explained by different chemical reactions (Spano et al. 2001; 2002).

NaOCl + H2O ↔ NaOH + HOCl ↔ Na +

+ OH− + H+ + OCl−

1. Chloramination reaction: hypochlorous acid (HOCl) and hypochlorite ions (OCl–) formed in the hypochlorite solution will react with proteins result- ing in their degradation and hydrolysis. The sol- vent action of hypochlorous acid with organic tissue results in the release of chlorine ions, which com- bines with amino groups of the protein molecules to form chloramines. The chloramines will interfere with bacterial cell metabolism. In addition, chlo- rine will irreversibly oxidize the sulfhydryl group of bacterial enzymes (Estrela et al. 2002).

2. Neutralization reaction: sodium hypochlorite is a strong base with a pH of >11, and neutraliza- tion reaction is the effect of pH. The high pH

enables sodium hypochlorite to produce antibacte- rial effects by different mechanisms. The hydroxyl ions denature and alter the bacterial membrane permeability, inhibit enzymatic activity, alter cell metabolism, and degrade phospholipids resulting in cell death. The hydroxyl ions can be neutralized by amino acids, leading to a decrease in the pH. This neutralization reaction reduces the efficacy of basic antimicrobials. The initial concentration of sodium hypochlorite determines the degree of neutraliza- tion effect, because the concentration is inversely related to the degree of neutralization (Estrela et al. 1995; Spano et al. 2001).

3. Saponification reaction: sodium hypochlorite alters bacterial membrane permeability by degrading the fatty acids in the bacterial cell membranes (Estrela et al. 2002).

13.4.1.1.2 Physicochemical characteristics and relevance to endodontics Sodium hypochlorite is usually applied at concentra- tions between 0.5% and >6% for root canal disin- fection. There is no general consensus on the opti- mum concentration of sodium hypochlorite required for root canal disinfection. Clinical studies showed that both low and high concentrations are equally effec- tive in reducing root canal bacteria (Cvek et al. 1976; Bystrom and Sundqvist 1981). Nevertheless, in vitro studies showed that sodium hypochlorite solution in higher concentration is more effective against bacterial and fungal species (Waltimo et al. 1999; Gomes et al. 2001; Radcliffe et al. 2004). In addition to antibacte- rial effect, sodium hypochlorite dissolves pulp tissue, and the rate of dissolution of pulp remnants was pro- portional to the concentration of sodium hypochlorite used (Moorer and Wesselink 1982; Spano et al. 2001). After mixing, the tissue dissolving ability of 2.6% and 1% sodium hypochlorite was found to remain stable for a period of 1 week, while a significant decrease was seen after 2 weeks and beyond (Johnson and Remeikis 1993). The addition of surfactant was found to nega- tively affect the ability of sodium hypochlorite to dis- solve pulp remnants (Siqueira et al. 2000; Spano et al. 2001).

An in vitro study has shown that heating of sodium hypochlorite (20◦C to 45◦C) produced a 100-fold increase in bacterial killing (Sirtes et al. 2005). However, Stojicic et al. (2010) showed that agita- tion of irrigants within the root canal produced an enhanced antibacterial effect that was greater than

292 Endodontic Microbiology

that of raised temperature. According to the manu- facturer, Chlor-XTRA (Vista Dental Products) is a 6% sodium hypochlorite solution containing a wet- ting agent, proprietary surface modifiers, and alkylat- ing agents to enhance the electrical capacity of the solution. However, experiments have not shown sig- nificantly enhanced antibacterial efficacy or vital tissue dissolving capacity with Chlor-XTRA when compared with 6% sodium hypochlorite (Williamson et al. 2009; De-Deus et al. 2013).

In root canals, the volume of the topical antimicro- bials used has a critical role in their ability to achieve antibacterial efficacy (Moorer and Wesselink 1982; Siqueira et al. 2000). Thus, if a lower concentration of intracanal irrigation is employed, it is recommended that the solution is used in larger volume by frequently replenishing to compensate for the limitations of lower concentrations (Siqueira et al. 2000). This is an impor- tant consideration, because a high concentration of sodium hypochlorite is more reactive and toxic than a lower concentration (Gernhardt et al. 2004). The con- fined root canal anatomy allows the use of higher con- centrations of irrigant with a low incidence of mishaps (Shuping et al. 2000; Peters 2004; McGurkin-Smith et al. 2005).

13.4.1.2 Chlorhexidine

Chlorhexidine was developed by Imperial Chemi- cal Industries Ltd., UK (Davies et al. 1954; Zehn- der 2006). It is widely used for the treatment of skin, eye, and throat infections. It is also used to control plaque in the oral cavity (Greenstein et al. 1986). Chlorhexidine is generally indicated to have wide-spectrum antimicrobial properties against Gram- positive bacteria, Gram-negative bacteria, and yeasts. However, some reports suggest limited effectiveness of chlorhexidine against certain Gram-negative bacte- ria when compared to Gram-positive bacteria (Hugo and Longworth 1964, 1966; Asboe-Jorgensen et al. 1974; Southard et al. 1989). A study that investigated the antibacterial effect of a chlorhexidine-containing mouthwash on microbial ecology of dental plaque microcosms showed that Actinomyces naeslundi, Veil- lonella dispar, Prevotella nigrescens, and streptococci were highly susceptible to chlorhexidine. However, Lactobacillus rhamnosus, Fusobacterium nucleatum, and Neisseria subflava were less susceptible. Exposure to chlorhexidine would shift the distribution of bacte- rial populations towards those that are less susceptible

to chlorhexidine. This shift in plaque microcosms fol- lowing repeated exposure to chlorhexidine resulted in the inhibition of the most susceptible species and the expansion of less susceptible species (McBain et al. 2003). Previous studies showed that the antibacterial efficacy of chlorhexidine was markedly limited against clinical isolates (Suwa et al. 2013) and germinating spores (Penna et al. 2001).

13.4.1.2.1 Mechanism of action The mechanism of action of chlorhexidine is mainly by its cationic nature. The positive charge allows electrostatic binding of chlorhexidine to negatively charged bacterial cells (Davies 1973). This interac- tion results in damage to the outer layers of cell wall, rendering it permeable (Hugo and Longworth 1964, 1966; Hennessey 1973). Depending on the concentra- tion of chlorhexidine, both bacteriostatic and bacte- ricidal effects are observed. The cationic nature also allows chlorhexidine molecules to be adsorbed on to anionic surfaces such as oral mucosa, salivary pellicle, and hydroxyapatite molecules. This reversible bind- ing of chlorhexidine and subsequent release will lead to sustained antimicrobial activity (substantivity). The degree of substantivity depends upon the concentration and duration of application. A longer duration of appli- cation and higher concentration (>0.02%) result in a thicker bound layer of chlorhexidine on the tooth sur- face, which acts as a reservoir releasing chlorhexidine into the environment based on a concentration gradient (McBain et al. 2003; Mohammadi and Abbott 2009). The effective antimicrobial activity, in this case, is observed only when a saturation point is reached after a specific period of interaction with the root dentin (Lin et al. 2003). It was revealed that a 5-minute application of chlorhexidine did not induce substan- tivity, while dentin treated with chlorhexidine for 7 days demonstrated substantivity (Komorowski et al. 2000). Generally, the residual antimicrobial activity of chlorhexidine is suggested to remain in the root dentin for up to 12 weeks (Rosenthal et al. 2004).

13.4.1.2.2 Physicochemical characteristics and relevance to endodontics Chlorhexidine is a polybiguanidine with a structure of two symmetric 4-chlorophenyl rings and a pair of bisguanidine groups connected by a hexamethy- lene chain. It is a strong base, has a cationic poten- tial, and forms a stable salt (Greenstein et al. 1986).

Topical Antimicrobials in Endodontics 293

Chlorhexidine is bacteriostatic in low concentration, producing leakage of cytoplasmic potassium and phos- phorus. In high doses it is bactericidal, causing dam- ages to the cell wall and precipitating cytoplasm. It also produces detrimental effects on bacterial metabolism (Hugo and Longworth 1965; Fardal and Turnbull 1986). Chlorhexidine digluconate salt is easily soluble in water. Aqueous solutions of 0.1–0.2% are suggested for plaque control, while a 2% solution is recom- mended for root canal irrigation (Yesilsoy et al. 1995). CHX-Plus (Vista Dental Products) is a 2% chlorhexi- dine gluconate solution containing proprietary surface modifiers to lower viscosity (Williamson et al. 2009). However, the antibacterial efficacy of CHX-Plus was found to be inferior to 6% sodium hypochlorite (Wang et al. 2012).

Chlorhexidine used between 0.12% and 2.0% con- centration showed low tissue toxicity, both locally and systemically (Loe and Schiott 1970). Two per- cent chlorhexidine used as a subgingival irrigant pro- duced no apparent toxicity on gingival tissues (Loe and Schiott 1970; Southard et al. 1989). Nonetheless, both 2% chlorhexidine and sodium hypochlorite pro- duced inflammatory reactions in the subcutaneous tis- sues of animal models, in which the toxic reaction to chlorhexidine was less than that of sodium hypochlo- rite (Yesilsoy et al. 1995; Oncag et al. 2003). Few cases of allergic and anaphylactic reactions to chlorhexidine have been reported (Okano et al. 1989; Garvey et al. 2001). Furthermore, chlorhexidine interacts with other chemicals such as sodium hypochlorite forming pre- cipitates, which contain parachloroaniline (4-chloro- aniline, p-chloro-aniline, PCA), which can cause tis- sue toxicity (Basrani et al. 2007) and partially block the dentinal tubules in the root dentin.

Chlorhexidine has been utilized as an irrigant and intracanal medicament during root canal treatment. In vitro studies have shown that the antibacterial prop- erty of chlorhexidine as an irrigant depends upon the concentration used. As an irrigant, 2% chlorhexidine has better antibacterial efficacy than 0.12% chlorhex- idine (Basrani et al. 2003). However, clinical studies failed to highlight the additional advantage of using chlorhexidine as a root canal irrigant (Ercan et al. 2004; Siqueira et al. 2007). Studies have highlighted that sodium hypochlorite has not only a higher capac- ity to kill microorganisms, but is also better able to remove bacteria from the root canal (Vianna et al. 2006). However, chlorhexidine has been suggested to be a useful final irrigant to improve periapical

healing (Tanomaru Filho et al. 2002). Despite its use- fulness as a final irrigant, chlorhexidine cannot be advocated as the main irrigant in routine endodontic treatment because it is unable to dissolve necrotic tis- sue remnants (Magnusson and Heyden 1973), exhibits reduced efficacy on certain taxa of Gram-negative microorganisms, and does not disrupt biofilm matrix. Strong value of chlorhexidine as an intracanal medica- ment was also not consistently demonstrated (Paque- tte et al. 2007). It was suggested that the antibacte- rial efficacy of intracanal medication with calcium hydroxide and 2% chlorhexidine gel were compa- rable (Barbosa et al. 1997; Manzur et al. 2007). It was also shown that 7-day intracanal dressing with calcium hydroxide/chlorhexidine paste significantly increased the number of cases yielding negative cul- tures (Siqueira et al. 2007).

A randomized clinical trial showed that 2.5% hypochlorite was significantly more efficient than 0.2% chlorhexidine in achieving negative cultures (Ringel et al. 1982). The difference was significant for obligate anaerobic bacteria while the difference was less significant for facultative anaerobes. In addi- tion, more culture reversals from negative to positive were observed with chlorhexidine than with hypochlo- rite. This culture reversal was attributed to the inability of chlorhexidine to dissolve necrotic tissue remnants, thoroughly disinfect the root canal system, and inabil- ity to disrupt biofilm matrix (Ng et al. 2011). Ng et al. (2011), in a prospective study of the factors affecting outcomes of nonsurgical root canal treatment, con- cluded that irrigation with 2% chlorhexidine as an adjunct to NaOCl would negatively impact periapical healing.

13.4.1.3 MTAD

MTAD is a dual functioning endodontic irrigant, rec- ommended for both removing smear layer and antibac- terial efficacy.

13.4.1.3.1 Physicochemical characteristics MTAD is an aqueous solution of 3% doxycycline hyclate (a broad-spectrum antibiotic), 4.25% citric acid (a demineralizing agent), and a polysorbate deter- gent (Tween 80) (Singla et al. 2011). The demineral- izing agent in the irrigant solution is expected to con- tribute towards debris and smear layer removal, while the broad-spectrum antibiotic provides the antimicro- bial properties for the solution.

294 Endodontic Microbiology

13.4.1.3.2 Relevance to endodontics Earlier studies showed more favorable properties with MTAD than conventional irrigants (sodium hypochlo- rite/EDTA) (Shabahang and Torabinejad 2003; Torabinejad et al. 2003; Newberry et al. 2007). How- ever, subsequent in vitro study showed no difference between 5.25% NaOCl/15% EDTA in comparison with 1.3% sodium hypochlorite/Biopure MTAD in the apical 5 mm of roots infected with Enterococcus faecalis (Kho and Baumgartner 2006). Another study showed that 1.3% sodium hypochlorite/MTAD left nearly 50% of the canals contaminated with E. faecalis, while 5.25% sodium hypochlorite/15% EDTA consistently disinfected the entire root canals (Johal et al. 2007). It was also shown that 6% sodium hypochlorite and 2% chlorhexidine were equally effec- tive and both superior to MTAD in antifungal activity (Ruff et al. 2006).

A strong advantage of using MTAD as a topical antimicrobial in endodontics has not been demon- strated. Based on an in vitro analysis using a bovine dentin model, it was reported that sodium hypochlo- rite and doxycycline were equally effective in killing E. faecalis at the superficial layers of dentin, but at the deeper levels only sodium hypochlorite was supe- rior (Krause et al. 2007). The survival of tetracycline- resistant bacterial strains in infected root canals irrigated with solution containing tetracycline was reported (Rossi-Fedele and Roberts 2007). In addition, experimental studies highlighted the ability of dentin and bovine serum albumin to delay the antibacterial efficacy of MTAD (Portenier et al. 2006). The poten- tial for iatrogenic tetracycline staining with the appli- cation of MTAD should also be taken into account (Tay et al. 2006). A clinical study showed that a final rinse with MTAD and medication with chlorhexidine did not reduce bacterial counts beyond the levels achieved by root canal instrumentation combined with sodium hypochlorite irrigation (Malkhassian et al. 2009).

13.4.1.4 QMiX® 2 in 1

QMiX® is a relatively new, dual functioning, endodon- tic irrigant recommended for its ability to remove the smear layer and its antibacterial effects (Stojicic et al. 2012). It is recommended that QMiX should be used as a final irrigant owing to its advantages both as an effi- cient antibacterial as well as an effective smear layer removing agent (Ma et al. 2011; Stojicic et al. 2012, 2013; Wang et al. 2012, 2013).

13.4.1.4.1 Physicochemical characteristics QMiX® is an irrigating solution that contains EDTA (chelating agent), chlorhexidine (antibacterial agent), and a detergent (surface active agent). The pH of the solution is slightly above neutral. The surface active agent in the solution reduces the surface tension of QmiX, allowing greater wettability and penetration into the root canal (Giardino et al. 2006).

The biocompatibility of QMiX® is better than that of NaOCl. When tested in vivo on subcutaneous tissues in rats, QMiX® produced fewer inflammatory reactions than the other tested endodontic irrigants (3% NaOCl, 2% chlorhexidine, and 17% EDTA) for various time points (Chandrasekhar et al. 2013). Although all the tested irrigants showed the highest inflammatory cell infiltrate at 48 hours, the healing rate was found to be faster for QMiX® on days 14 and 30.

13.4.1.4.2 Relevance to endodontics QMiX®, used either as a single irrigant or in com- bination with 6% NaOCl, reduced bacteria from the dentinal tubules even in presence of smear layer (Wang et al. 2013). The chelating property of QMiX® enables its antibacterial activity even in the presence of smear layer. Another key feature highlighted by the manufac- turers is the lack of dentin erosion by QMiX®, unlike that observed with EDTA and NaOCl irrigation. A time-dependent antibacterial activity of QMiX® was demonstrated inside the dentinal tubules in vitro (Wang et al. 2012, 2013).

In vitro tests on 3-week-old biofilms of E. fae- calis on dentin models demonstrated that a combina- tion of 6% NaOCl and QMiX® eliminated up to 62% of bacteria. This combination penetrated 300 μm into the dentinal tubules (Wang et al. 2012). Few studies have reported inferior antibiofilm efficacy of QMiX® compared to the 2.5–5.25% NaOCl/EDTA combina- tions (Morgental et al. 2013; Ordinola-Zapata et al. 2013a). At the same time, the chelating agent con- taining antibacterial agents were reported to be less effective in eliminating biofilms grown in situ on dentin blocks (Ordinola-Zapata et al. 2013a). It was suggested that irrigants such as MTAD and QMiX®, which are combinations of chelating agents with anti- septics or antibiotics, do not disrupt biofilms even after 5 minutes of exposure. Furthermore, the presence of organic components of pulp tissue or dentin could inactivate the antibacterial efficacy for these topical antimicrobials (Portenier et al. 2002; Morgental et al. 2013).

Topical Antimicrobials in Endodontics 295

13.4.1.5 Calcium hydroxide

Calcium hydroxide is a highly caustic chemical, which was launched as a potential replacement to severely tis- sue toxic and potentially allergenic intracanal medica- ments frequently used in the early nineteenth century. According to Staehle and Kindler (1990), calcium hydroxide was used in root canal therapy long before Hermann had used it for disinfecting root canals. Her- mann (1920) was the first to stress not only its antimi- crobial effects, but also its compatibility with periapi- cal tissue (Hermann 1920; Staehle and Kindler 1990). From a histologic viewpoint, it was highlighted that the placement of calcium hydroxide paste (Calxyl) was to induce apical root closure with cementum or dentin- like hard tissue. This development preceded the obser- vation that calcium hydroxide filling in root canals of teeth with suppurating periapical lesions could cease the exudative process. In a series of reports, Bystrom and Sundqvist (1981, 1983, 1985); Bystrom et al. (1985) supported the benefits of calcium hydroxide as an intracanal medicament. They demonstrated that intracanal medication with calcium hydroxide over a period of time presented significant benefit in reducing cultivable bacteria from the root canal.

13.4.1.5.1 Physicochemical characteristics The high pH of calcium hydroxide (>11) serves as a deterrent for the growth of residual or contaminating bacteria. An important mechanism attributed to this effect is the physical blockade created by the material against the seepage of inflammatory exudate from the periapical tissue into the root canal space. This in turn impedes the nutritional supply for the infecting prote- olytic microorganisms. Thus, the bacterial activity is dampened, and the release of inflammatory elements that mediated periapical response fade away (Heither- say 1975; Caliskan 2004).

13.4.1.5.2 Relevance to endodontics In vitro studies Numerous in vitro experiments have been carried out to examine the effect of calcium hydroxide on bacterial cells and bacterial by-products. Using an in vitro model, it was shown that calcium hydroxide-treated roots contained significantly fewer viable bacterial cells than nonmedicated roots (Parmar et al. 2011). Haapasalo and Örstavik (1987) observed early on that calcium hydroxide placed in the root canal failed to eliminate bacteria from dentinal tubules. A subsequent study highlighted that at least 10-day

application of calcium hydroxide was required to reduce E. faecalis infection within dentinal tubules (Ørstavik and Haapasalo 1990). Although in vitro experiments have demonstrated antibacterial efficacy of calcium hydroxide on endodontic pathogens such as Actinomyces israelii (Barnard et al. 1996), calcium hydroxide did not show significant antimicrobial effect after 2, 3, and 7 days against E. faecalis, Staphy- lococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa in infected dentinal tubules (Estrela et al. 1999). Anaerobic Gram-negative bacteria were sug- gested to be more susceptible to calcium hydroxide paste than facultative Gram-positive microorganisms (Gomes et al. 2002). In addition, an in vitro study has shown the inability of calcium hydroxide to disrupt biofilm matrix (Upadya et al. 2011).

The effect of calcium hydroxide on bacteria and bacterial by-products—lipopolysaccharide (LPS) and lipoteichoic acid (LTA)—has been examined. Calcium hydroxide was found to inactivate LTA from E. fae- calis through deacylation of the lipid moiety (Baik et al. 2011). However, calcium hydroxide hydrolyzed the ester bonds of the lipid-A moiety in bacterial LPS, resulting in the release of free hydroxyl fatty acids (Safavi and Nichols 1993; Buck et al. 2001). Thus, calcium hydroxide can alter the biologic properties of LPS and LTA (Safavi and Nichols 1994). This is considered to be an advantage of calcium hydrox- ide, because sodium hypochlorite and chlorhexidine irrigation could not detoxify bacterial endotoxins (de Oliveira et al. 2007).

Clinical observations A meta-analysis of nine studies (Ørstavik et al. 1991; Sjogren et al. 1991; Yared and Dagher 1994; Shuping et al. 2000; Peters and Wes- selink 2002; Kvist et al. 2004; McGurkin-Smith et al. 2005; Waltimo et al. 2005; Sathorn et al. 2007) con- cluded that calcium hydroxide has only limited effi- cacy in eliminating cultivable bacteria from treated root canals. Although all the included studies in this systematic review except two (Peters and Wesselink 2002; Waltimo et al. 2005) had observed a reduced rate of culture positive samples in comparison with the control procedure. One must conclude that reports employing a culture-dependent result as an outcome parameter have yielded insufficient support for the use of calcium.

Molecular methods have recognized many bacterial species in infected root canals that are not cultivable and have not been characterized previously (Sakamoto

296 Endodontic Microbiology

et al. 2006; Ribeiro et al. 2011). Thus, traditional bacteriologic culture-based analysis of clinical sam- ples may not necessarily reflect all microorganisms present in the sample (Siqueira and Rôças 2005). Fur- thermore, calcium hydroxide is only weakly soluble, and therefore produces limited antimicrobial effects in distant sites, such as anatomic complexities of the root canal system, where mechanical instrumentation has not reached and antimicrobial irrigation has not penetrated.

In a recent survey conducted by Swedish Council on Health Technology Assessment (SBU), two sys- tematic studies were identified to satisfy strict criteria for a moderate level of evidence (Weiger et al. 2000; Molander et al. 2007). Both the studies reported similar frequencies of periapical healing regardless of canals being medicated with calcium hydroxide or root filled in one sitting. SBU concluded that there is a lack of sci- entific support to determine whether calcium hydrox- ide has any effect on the outcome of root canal treat- ment. In conclusion, it could be suggested that despite numerous studies, there is relatively weak evidence to support the use of calcium hydroxide in the manage- ment of root canal infection.

13.4.2 Nonchemical-based antimicrobials

13.4.2.1 Antibacterial nanoparticles

Nanoparticles are particles with at least one or more of their external dimensions in the nanoscale. Typically, nanoscale size ranges from 1 to 100 nm.

13.4.2.1.1 Mechanism of action Metal ions are known to have adverse effects on bac- terial cell functions (Stohs and Bagchi 1995; Reddy et al. 2007; Yoon et al. 2007). Copper ions have been shown to induce oxidative stresses (Cioffi et al. 2005), and affect the redox cycling. These effects produced by copper ions would result in cell membrane and DNA damage. Zinc ions in concentrations exceed- ing the essential threshold would inhibit bacterial enzymes, including dehydrogenase, which impedes the metabolic activity of the bacterial cell (Beard et al. 1995). Silver ions have been known to inactivate pro- teins and inhibit the ability of bacterial DNA to repli- cate (Feng et al. 2000). The metal ions in nanosize exert more potent antibacterial and biologic character- istics. The quantum size effect and increased surface area of nanoparticles provides them with the unique

physicochemical properties that are different from their bulkier counterparts (micro or powder forms).

The key feature of antibacterial nanoparticles is their broad spectrum of antimicrobial activity and far lower propensity to induce microbial resistance. The antibacterial activity of nanoparticles also depends on the material from which the particle is synthe- sized (i.e., organic/polymeric or inorganic/metallic), the size/surface area of the particle, and their charge density (Sawai et al. 1998). The electrostatic interac- tion/adherence of positively charged nanoparticles on bacterial cell surfaces has been associated with the rapid loss of membrane permeability and functions.

13.4.2.1.2 Relevance to endodontics Bacterial adherence is the primary interaction of microbes with a tissue or biomaterial surface. It is recognized to be an important step in the establish- ment of a biofilm-mediated infection (Busscher and van der Mei 1997; An and Friedman 1998; Jefferson 2004). Antimicrobial irrigants that alter the physico- chemical properties of dentin can modulate the nature of bacterial adherence/adhesion force to dentin. Final irrigation with EDTA following sodium hypochlorite (5.2%) irrigation produced minimal reduction (33%) in the bacterial adherence to root dentin (Marshall et al. 1995; Habelitz et al. 2002; Basrani et al. 2007; Kishen et al. 2008b). In such cases, an aqueous suspension of cationic nanoparticles can be applied to root dentin. This nanoparticle treatment can markedly impede bac- terial recolonization and biofilm formation (Kishen et al. 2008a).

13.4.2.1.3 Nanoparticle-based irrigant/ medicament Bioactive, natural, polymeric nanoparticles of chitosan have been researched extensively owing to their excel- lent bioactivity, antimicrobial, and antifungal activi- ties. Chitosan is a natural nontoxic polymer derived by the deacetylation of chitin. The exact mechanisms of antibacterial effect of chitosan and its derivatives are still not well established; however, the electro- static interaction between positively charged chitosan nanoparticles and negatively charged bacterial cell membrane is believed to alter bacterial cell perme- ability, leading to cell death (Rabea et al. 2003). In one study, planktonic E. faecalis were totally eliminated, in contrast to biofilm bacteria (Shrestha et al. 2010). Longer duration of interaction and higher concentra- tion of nanoparticles were required to reduce biofilm

Topical Antimicrobials in Endodontics 297

bacteria in significant numbers (Shrestha et al. 2010). This suggests that the antibacterial nanoparticles may potentially be more useful as an intracanal medicament than traditional medicaments. Furthermore, because of the difference in uptake mechanisms of nanoparti- cles by the prokaryotic and eukaryotic cells, they are capable of producing more targeted bacterial killing without significant cytotoxicity to host cells (Shrestha et al. 2014).

A recent in vitro study showed that syringe irriga- tion with 0.1% AgNP (silver nanoparticle) solution did not disrupt the biofilm structure, and the proportion of viable bacteria in the biofilm structures was not dif- ferent from that of the saline group (Wu et al. 2014). The biofilms treated with 0.02% AgNP gel as medica- ment significantly disrupted the biofilm structure and resulted in the lowest levels of residual viable E. fae- calis cells compared with 0.01% AgNP gel and cal- cium hydroxide groups. These findings further stressed the roles of concentration and duration of interaction on the antibiofilm efficacy of AgNPs. AgNPs as an intracanal medicament, and not as an irrigant, showed potential to eliminate residual bacterial biofilms during root canal disinfection.

13.4.2.1.4 Nanoparticle-based root canal sealers A study has examined the antimicrobial properties of ZnO and resin-based root canal sealers loaded with chitosan and ZnO nanoparticles (Kishen et al. 2008a). This study highlighted that the addition of antibacterial nanoparticles in sealers improved the direct antibacte- rial effect (killing bacteria that come in direct con- tact with the sealer) and diffusible antibacterial effect (killing bacteria by diffusion of antibacterial compo- nents from the sealer) of root canal sealers (Buck et al. 2001). Thus, addition of different antibacterial nanoparticles should improve the antibacterial prop- erties of the root canal sealers (Kishen et al. 2008a). However, in this situation, it is imperative to assess the cytotoxicity of the applied nanoparticles on host cells.

Bioactive glass, as both micro- and nanoparticles, has received some attention in root canal disinfection. Bioactive glass consists of SiO2, Na2O, CaO2, and P2O5 at different concentrations. Their antibacterial activity has been attributed to several factors such as high pH, increase in osmotic effect, and Ca/P precipi- tation (Stoor et al. 1998). 45S5 Bioactive glass suspen- sions or slurries for root canal disinfection were sug- gested to combine the ability to induce high pH with the capacity to continuously release alkaline species

(Waltimo et al. 2009). However, when tested in vitro they showed drastically less antibacterial effect than calcium hydroxide (Zehnder et al. 2004, 2006; Gubler et al. 2008), and ineffectiveness in preventing recon- tamination of instrumented root canals (Gubler et al. 2008). The slurry of bioactive glass nanoparticles with a 12-fold higher surface area than the microparti- cles was expected to possess improved antibacterial properties. Nevertheless, the microparticles of bioac- tive glass produced considerably higher alkalinity and antimicrobial efficacy. This finding did not support the previous report that showed higher antibacterial effi- cacy with a shift from micron- to nano-sized materials (Waltimo et al. 2007).

In summary, most tested antibacterial nanoparticles showed promise for endodontic applications such as intracanal medicament and as a component in root canal sealers. It was noted that these nanoparticles are not very effective over a short duration as is the requirement for a root canal irrigant. However, they may be effective with longer durations of action. They have the ability to impart antimicrobial effect deep into the dentinal tubules; hence their application should be directed towards intracanal medicaments or to poten- tiate the antibacterial efficacy in root canal sealers. Additional research is required to confirm the abil- ity of antibacterial nanoparticles to inactivate bacteria within the dentinal tubules and eliminate biofilms in the anatomic complexities and uninstrumented por- tion of the root canal system. Their interaction with host tissues and immune cells also requires further investigation.

13.4.3 Antimicrobial photodynamic therapy

Photodynamic therapy applies a photosensitizer (i.e., a light-sensitive chemical at a nontoxic concentration), which, when activated with a specific wavelength of light, produces activated oxygen radicals that cause toxic effects on bacterial cells.

13.4.3.1 Mechanism of action

Antimicrobial photodynamic therapy (APDT) involves two specific steps. The first step is the pho- tosensitization stage, during which a photosensitizer is allowed to bind to bacterial cells. The second step is the photoactivation stage, which involves the application of a low-energy light to irradiate the photosensitized tissue. The light employed should

298 Endodontic Microbiology

Fig. 13.4 The principles of photodynamic effect.

have a wavelength that corresponds to the maximum absorption wavelength of the photosensitizer. The light-activated photosensitizer is extremely reactive; it reacts further by one or both of the following pathways to destroy the cell.

1. Type I reaction: the activated photosensitizer triplet state can react with a target, other than oxygen, by hydrogen or electron transfer resulting in radi- cal ions that can react with oxygen, yielding cyto- toxic species such as hydrogen peroxide, super- oxide anion, hydroxyl, and lipid-derived radicals (Figure 13.4).

2. Type II reaction: the photosensitizer triplet state can transfer the excitation energy to ground state molecular oxygen to produce excited state singlet oxygen (Dai et al. 2009) (Figure 13.4).

Singlet oxygen is a strong oxidizing agent and thus highly reactive, with a lifetime of <0.04 μs in a biologic environment and a radius of action of <0.02 μm (Moan and Berg 1991). The reactions of singlet oxygen with the cellular targets lead to cell death. These two typical mechanisms have been proposed to account for DNA damage and cytoplasmic membrane damage lethal to the bacterial cell (Menezes et al. 1990; Bertoloni et al. 2000). Nevertheless, studies have shown that pho- tooxidative effects caused by most photosensitizers in bacteria could lead to damage of multiple targets in bacterial cells such as DNA (Menezes et al. 1990),

membrane integrity (Wakayama et al. 1980), protease activity, and LPS (Komerik et al. 2000; George and Kishen 2008a,b). These findings support the hypothe- sis that APDT can be a feasible alternative to antibi- otics because the bacteria are not known to gain resis- tance against this treatment.

The light sources used for APDT can be a coherent light source (lasers) or a noncoherent light (incandes- cent or halogen lamps). The choice of light source is dictated by the location, required light dose, and the choice of photosensitizer. Laser provides monochro- matic, coherent, and collimated light, offering a wide range of output power. Laser light can be easily coupled into a fiber-optic cable, which can serve as a delivery system (probe) while irradiating complex anatomy such as a root canal. Neodymium-doped yttrium aluminum garnet (Nd:YAG), potassium titanyl phosphate (KTP), helium–neon (HeNe), gal- lium aluminum arsenide (GaAlAs), and diode lasers, light-emitting diodes (LEDs), and xenon-arc lamps have been employed for APDT. The superiority of one type of light source over another has not been clearly demonstrated and hence the use of lasers or lamps depends on the particular application (Prasad 2003).

Over the last decade, several compounds based on the phenothiazinium chromophore are emerging as promising candidates for use as photodynamic antimi- crobial agents. The phenothiazinium group of photo- sensitizers such as methylene blue (MB) and toluidine blue O (TBO) are generally accepted photosensitizers

Topical Antimicrobials in Endodontics 299

for clinical applications (Kishen 2012). Phenoth- iaziniums are generally cationic molecules with a core structure composed of a planar tricyclic aromatic ring system, which functions as a chromophore of these compounds. In addition, cationic porphyrins, phthalo- cyanines, and chlorins have also gained popularity as antimicrobial photosensitizers. Currently, a number of photosensitizers such as MB, TBO, rose bengal, erythrosine, chlorin, and hematoporphyrin have been investigated for their antimicrobial potential against oral pathogens (Hamblin and Hasan 2004).

13.4.3.2 Relevance to endodontics

In endodontics, several studies have tested the effi- cacy of APDT in root canal disinfection (George and Kishen 2007a,b, 2008a,b; Meire et al. 2009). Gener- ally, in vitro experiments reported marked antibacterial efficacy of APDT on a range of endodontic pathogens (Soukos et al. 2006; Williams et al. 2006). The selec- tivity of APDT towards microbial cell killing over the host cells, at the suggested photosensitization periods and light fluence (amount of optical energy delivered across a unit area (joules per square centimeter)), is an obvious advantage of APDT over most other antibac- terials. Soukos et al. (1996) compared the effect of APDT using a combination of TBO and red light against S. sanguis and human gingival keratinocytes and fibroblasts. They reported no reduction in the human cell viability whereas the bacteria were effec- tively killed. Soncin et al. (2002) reported the selective killing of S. aureus over human fibroblasts and ker- atinocytes (four- to sixfold) when subjected to APDT using cationic pthalocyanine and relatively low light fluencies. George and Kishen (2007a) demonstrated 97.7% killing of Enterococcus faecalis compared to a 30% human fibroblast dysfunction following MB- mediated APDT. All these in vitro studies suggest the targeted bacterial killing efficacy of APDT.

The tissue-specific challenges in the application of APDT for endodontic disinfection are as follow:

1. The penetration of light energy into the infected tissue;

2. Penetration of optimum photosensitizer concentra- tion into the infected tissue;

3. Limited availability of environmental oxygen in the infected tissue;

4. The ability of excess photosensitizer to induce dis- coloration of dentin; and

5. Reduction or neutralization of antimicrobial activ- ity caused by the presence of tissue and serum within root canals.

In an approach to improve the antimicrobial effi- cacy of APDT in the root canal system (tissue- specific approach), George and Kishen (2007b) dis- solved MB in different formulations: water, 70% glyc- erol, 70% polyethylene glycol, and a mixture of glyc- erol : ethanol : water (MIX) in a ratio of 30 : 20 : 50. They showed that aggregation of MB molecules was significantly higher with water than other for- mulations. The MIX-based photosensitizer formula- tion had effective penetration into dentinal tubules and enhanced singlet oxygen generation, which in turn improved bactericidal action (George and Kishen 2008a). The same group also showed that the incorpo- ration of an oxidizer and oxygen carrier with the pho- tosensitizer formulation in the form of an emulsion would produce significant photooxidation capabili- ties, which in turn facilitated comprehensive disrup- tion of matured endodontic biofilm structure (George and Kishen 2008b).

Conjugating photosensitizer to various agents or chemical moieties can result in improved photosen- sitizers for APDT. These modified formulations are expected to bind to the outer membrane of bacteria and, upon activation, generate reactive oxygen species, which can then diffuse into the cells, resulting in cell death. Therefore, photo-generated oxidative species are confined to the cell wall and its vicinity, which is a highly susceptible domain for photodynamic action. Gross et al. (1997) and Soukos et al. (1997) covalently conjugated photosensitizer to a poly-l-lysine molecule to produce a bacteria-targeted photosensitizer delivery vehicle. Photosensitizer conjugated with a positively charged bioactive chitosan molecule has also been shown to be highly effective in removing biofilms of Gram-positive and Gram-negative bacteria (Shrestha and Kishen 2012a). Shrestha et al. (2012) showed that rose bengal conjugated chitosan nanoparticles pre- sented a synergistic antibacterial effect produced by the cationic biopolymeric nanoparticles (chitosan) and singlet oxygen generated following photoactivation of the photosensitizer (rose bengal).

Tissues remnants and serum fluid compromised the antimicrobial efficacy of not only the common endodontic irrigants (Portenier et al. 2002), but also the efficacy of APDT (Soukos et al. 2006; Foschi et al. 2007; Shrestha and Kishen 2012b). It has been reported

300 Endodontic Microbiology

that the components of infected root canals such as tissue remnants (pulp), serum, and dentin matrix sig- nificantly reduced the efficacy of APDT (Shrestha and Kishen 2012b). This inhibitory effect is either due to cross-linking action or the compromised half-life of singlet oxygen in the presence of proteins. Further, the hypoxic environment prevailing in the apical third of the root canal may also compromise the antibacterial efficacy of APDT (Kishen 2012).

Other in vivo studies have examined the efficacy of APDT in root canal disinfection (Bonsor et al. 2006a,b; Garcez et al. 2008, 2010). These studies concluded that a combination of chemomechanical preparation (instrumentation plus irrigation with active irrigant) and APDT would bring about maximum reduction in microbial loads. Thus, it is mandatory to improve the antibiofilm efficacy of APDT in the presence of tissue inhibitors, optimize light delivery within the root canal, and develop new photosensitizers and/or formulations in order to achieve predictable microbial elimination within the root canal system.

13.4.4 Laser-assisted root canal disinfection

A laser is a form of electromagnetic radiation emitted through a process of light amplification by stimulated emission of photons. The light emitted from a laser device has all the photons in a coherent state (usually with the same frequency and phase) and produces a lethal effect on bacteria.

13.4.4.1 Mechanism of action

A laser’s effects on a tissue will depend upon its inter- action with the tissue and its depth of penetration. Different types of lasers produce different effects on a tissue, and the same laser can have distinct effects on different tissues. The nature of laser–tissue interac- tion is influenced by: (i) the properties of lasers such as wavelength, energy density, rate/duration of irradia- tion (continuous/pulse repetition), and mode of deliv- ery (direct/indirect tissue contact); (ii) optical proper- ties of the tissue such as absorption, transmission, and scattering characteristics to different wavelengths of light (Miserendino and Pick 1995).

13.4.4.2 Relevance to endodontics

The primary objective of applying laser-assisted disin- fection in endodontic therapy is to enhance the degree

of microbial elimination during the cleaning and shap- ing procedures. Laser-assisted root canal disinfection requires the root canals to be shaped as the parame- ters of the laser used for disinfection are not expected to produce marked ablative effects on dentin tissue (Miserendino and Pick 1995; Moshonov et al. 1995). Infrared lasers such as CO2, Nd:YAG, diode, and erbium lasers have been applied for endodontic disin- fection. The bactericidal effect of lasers in most cases is brought about by the photothermal effects. The laser- induced thermal effect will produce alteration of the bacterial cell wall, leading to changes in osmotic gradi- ents, swelling, and cell death. Gram-negative bacteria showed a higher resistance against laser irradiation than Gram-positive bacteria. The higher resistance of Gram-negative bacteria is attributed to their cell wall characteristics (Miserendino and Pick 1995). Clini- cal studies have examined the antimicrobial efficacy of high-power lasers in endodontics (Dostalova et al. 2002; Leonardo et al. 2005). Generally, it could be concluded from these studies that there is no strong evidence currently available to support the application of high-power lasers in endodontic disinfection.

The delivery of laser energy throughout the root canal system and absorption of laser energy by dentin tissue are important issues to consider in laser-assisted root canal disinfection (Schoop et al. 2004). This will influence the degree of structural alteration in dentin and elimination of bacterial biofilm from the root canal system. Black Indian ink or 38% silver ammonium solution was applied on the root canal before irradiat- ing with pulsed Nd:YAG laser (1064 nm) to enhance the effect of disinfection within the root canal (Rooney et al. 1994). Schoop et al. (2004) showed that the Nd:YAG laser presented a bacterial reduction of 85% at 1 mm depth within the dentin when compared to diode laser (810 nm) which produced 63% bacterial reduction at a depth of 750 μm into the dentin.

The thermal energy produced by erbium lasers is absorbed mostly by the surface structure because of their high affinity to water molecules, thus exerting higher bactericidal effect on the surface of root dentin (Wang et al. 2007). It was reported that Er:YAG irra- diation reduced the number of viable bacteria, but did not completely eliminate biofilm structure or bacte- ria. As bacteria decalcify the hydroxyapatite discs and invade the porous decalcified layer, the inability of the Er:YAG laser to penetrate this layer may have con- tributed to the limited efficiency (Noiri et al. 2008). It is important to realize that microbial penetration

Topical Antimicrobials in Endodontics 301

into degraded dentin tissue and anatomic complexities is a common observation in endodontically infected teeth. Other studies have suggested that Er:YAG laser treatment (100 mJ pulses) (Meire et al. 2012) and Er,Cr:YSGG laser irradiation (2 W and 3 W output powers for 16 s) showed antibacterial properties, but their effectiveness was less significant than sodium hypochlorite solution (Yavari et al. 2010). The bacte- ricidal effect of erbium lasers in the root canal in vitro was also observed to be inferior to that of Nd:YAG lasers. However, a contradictory finding supporting the superior antibacterial efficacy of Er:YAG laser in combination with sodium hypochlorite, when com- pared with Nd:YAG laser has been reported (Cheng et al. 2012).

Bergmans et al. (2006) tried to define the role of lasers as a disinfecting tool by using Nd:YAG laser irra- diation on certain endodontic pathogens in vitro. They concluded that Nd:YAG laser irradiation is not an alter- native but a possible adjunct to existing protocols for root canal disinfection (Bergmans et al. 2006). Despite all these studies, it was suggested that endodontic pathogens that grow as biofilms are difficult targets to eradicate, even upon direct laser exposure. In addi- tion, there are several limitations associated with the intracanal use of high-power lasers that cannot be over- looked. The emissions of laser energy from the tip of the optical fiber or laser guide is directed along the root canal and not necessarily lateral to the root canal walls. Thus, it is a challenging task to obtain uniform coverage of the root canal surface using a laser (Goodis 2002; Stabholz 2003). The safety of such a procedure is another limitation, because potential thermal dam- age to the periapical tissue is possible. Direct emission of laser irradiation from the tip of the optical fiber in the vicinity of the apical foramen may result in the transmission of irradiation beyond the apical foramen, which can adversely affect the supporting periapical tissue. This effect can be hazardous in teeth with close proximity to the mental or the inferior alveolar nerves (Stabholz 2003).

Laser-activated irrigation and photon-initiated pho- toacoustic streaming (PIPS) aim to enhance the antibacterial efficacy of root canal disinfection by utilizing the characteristic high absorption of certain lasers with liquid irrigant (Blanken et al. 2009; De Moor et al. 2009; Kimura et al. 2011). The mech- anism of interaction of erbium, chromium: YSGG (Er,Cr:YSGG) laser with liquid irrigant in the root canal was attributed to the efficient absorption of the

mid-infrared wavelength light by water. The thermal component during this interaction is moderate and is not a major concern in PIPS (Blanken et al. 2009; De Moor et al. 2009). The efficient absorption of the erbium laser by liquid leads to vaporization and forma- tion of vapor bubbles. The creation and the dynamics of bubbles within the root canal is observed to enhance the efficacy of the root canal irrigant or antimicrobial. It is also suggested that the Er:YAG laser dissociates water and generates hydroxyl radicals (Lubart et al. 2005). The bubble dynamics achieved are identical in water, sodium hypochlorite, and EDTA solution and, subsequently, may be used to enhance the efficacy of sodium hypochlorite or EDTA. However, PIPS may cause extrusion of irrigant (George and Walsh 2008). Current studies do not consistently demonstrate an advantage for PIPS in eliminating root canal biofilms (Peters et al. 2011; Pedulla et al. 2012; Sahar-Helft et al. 2013; Zhu et al. 2013). PIPS may have the poten- tial to improve fluid dynamics within the root canal but further research is required to establish its clinical efficacy.

13.4.5 Ozone

Ozone (O3) is an energized, unstable, gaseous form of oxygen (O2), which dissociates readily back into oxygen, and in doing so liberates a reactive form of oxygen, the singlet oxygen (O1), capable of oxidizing nearby cells.

13.4.5.1 Mechanism of action

Ozone is an unstable allotrope of oxygen that is formed naturally in the ozone layer from atmospheric oxygen by electric discharge or exposure to ultraviolet radi- ation. Ozone presents a broad range of antibacterial efficacy without developing drug resistance (Restaino et al. 1995; Paraskeva and Graham 2002).

13.4.5.2 Relevance to endodontics

The results of various studies on the efficacy of ozone on endodontic disinfection have been inconsistent. This inconsistency is attributed to the lack of infor- mation about the optimum duration of application and concentration to be used within the root canal (Nagayoshi et al. 2004; Arita et al. 2005; Hems et al. 2005). The ozone gas concentration currently used in endodontics is 4 g/m3. This concentration has been

302 Endodontic Microbiology

shown to be slightly less cytotoxic than NaOCl (2.5%). The aqueous ozone (up to 20 μg/mL) showed essen- tially no toxicity to oral cells in vitro (Ebensberger et al. 2002; Estrela et al. 2007; Noguchi et al. 2009).

Ozone demonstrated substantial antibacterial effect in vitro when tested against E. faecalis. Hems et al. (2005) tested ozone against E. faecalis in both plank- tonic and biofilm cultures (48-hour-old biofilm grown on cellulose nitrate membrane). Different interaction time ranging from 30 to 240 s was allowed in both cultures. It was concluded that ozone had an antibac- terial effect on planktonic E. faecalis cells and those suspended in fluid, but little effect when embedded in a biofilm structure (Hems et al. 2005; Estrela et al. 2007). Another study assessed the antimicro- bial efficacy of aqueous (1.25–20 μg/mL) and gaseous ozone (1–53 g/m3) as an alternative antiseptic against endodontic pathogens in suspension and a biofilm model (Huth et al. 2006). E. faecalis, Candida albi- cans, Peptostreptococcus micros (Parvimonas micra) and P. aeruginosa were grown in planktonic culture or in monospecies biofilms in root canals for 3 weeks. The antibacterial efficacy of ozone in gaseous and aqueous forms depended on the concentration, type of bacte- rial strains, growth modes of microbes (planktonic or biofilm), and duration of application.

Pseudomonas fluorescens in planktonic form and biofilms were tested in another study. The planktonic form was completely eliminated by low concentrations of ozone (0.1 ± 0.3 ppm) within 15 or 30 min contact time. While the disinfectant action of ozone on biofilm models was less effective, with only a decrease of two orders of magnitude, the increased contact time was not found to be helpful (Viera et al. 1999). A recent study has claimed that ozone dissolved in oil can be used as an intracanal medicament (Silveira et al. 2007). Studies on the effect of surface tension on the flow characteristics of ozonized oil, chemical stability of ozonized oil, and their interaction with root dentin and obturating material is justified before ozone can be applied in endodontics (Guinesi et al. 2011).

The reduced effectiveness of ozone against sessile bacteria when compared with planktonic bacteria is due to the characteristics of biofilm (Viera et al. 1999). The polymeric layer may form a physical–chemical barrier preventing deeper penetration of the dissolved ozone into the biofilm structure (Stoodley et al. 1994). In addition, blockage of water channels in biofilm by the oxidation products of ozone may impede the fur- ther penetration of ozone to the inner layers of biofilm

structure (Lawrence et al. 1994). In a systematic review by Azarpazhooh and Limeback (2008), ozone has been highlighted for its biocompatibility with human oral epithelial cells, gingival fibroblasts, and periodontal cells, although conflicting evidence of antimicrobial efficacy of ozone in root canal disinfection cannot be overlooked.

13.5 Conclusions

Endodontic disease is a biofilm-mediated infection. The elimination of bacterial biofilm from the root canal system remains the primary challenge in the manage- ment of endodontic disease. The key to the successful application of topical antimicrobials for root canal dis- infection is to address all the challenges present in the root canal system in their entirety rather than focus- ing only on the antibacterial aspect. In this regard it is important to combine an optimal concentration and volume of topical antimicrobial with good irriga- tion strategy in a stratified manner to achieve optimal reduction of microbial population. In the haste to intro- duce newer antibacterial strategies it is important not to neglect potential constraining factors associated with their application in root canals.

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Zhu X, Yin X, Chang JW, Wang Y, Cheung GS, Zhang C. 2013. Comparison of the antibacterial effect and smear layer removal using photon-initiated photoacoustic streaming aided irrigation versus a conventional irrigation in single-rooted canals: an in vitro study. Photomed Laser Surg 31(8): 371–377.

Chapter 14 Endodontic Infections in Incompletely Developed Teeth George T.J. Huang, Domenico Ricucci, and Louis M. Lin

14.1 Introduction 14.2 Review of tooth development as it

relates to endodontic pathosis 14.3 Etiology, prevalence, and pathogenesis

of pulp disease in incompletely developed teeth

14.4 Microbiology of endodontic infections in teeth from pediatric patients

14.5 Management of immature teeth 14.5.1 Diagnosis 14.5.2 Clinical management

14.6 Orthodontic considerations in pathologically involved incompletely formed teeth

14.7 Stem cells for pulp and periodontal tissue regeneration

14.8 Recent innovations on the regeneration of tooth form

14.9 Conclusions and prospects 14.10 References

14.1 Introduction

Two specific aspects are encountered when perform- ing endodontic treatment on immature teeth: large canal space and open apex resulting from incomplete root formation. These aspects necessitate special pro- cedures during treatment and affect the outcome of treatment. A conservative approach is the preferred treatment option as long as there is the possibility of complete root maturation. When pulp is totally necrotic and infected, disinfection of the wide open canal is a challenge. Routine practice calls for provid- ing an appropriate environment to form an apical cal- cific barrier for these teeth, to ensure effective instru- mentation and filling of the canal space.

Generally, the clinical diagnosis for immature teeth follows the same criteria as mature teeth. Vital pulp therapy is an important treatment of choice for imma- ture teeth with a vital pulp. When diseased pulp tissue

is partially removed by pulpotomy, continued root for- mation, or apexogenesis, is initiated. When teeth are diagnosed with nonvital pulp, the entire canal system is debrided, cleansed, and apexification is contemplated. However, the clinical definition of pulp vitality is deter- mined by diagnostic tools that show sensibility, rather than vitality, and the histologic condition of the pulp cannot be clinically determined. Before 2000, there were a number of case reports in the literature that suggest the potential for immature teeth to complete root formation even after being clinically diagnosed with nonvital pulps.

During the early twenty-first century, more convinc- ing and well-presented clinical studies further demon- strate that despite the formation of periapical abscesses with extensive periradicular bone resorption as the result of root canal infection in immature teeth, con- servative treatment that involves disinfection of the canal space can allow root development to maturation

311

312 Endodontic Microbiology

(Iwaya et al. 2001; Banchs and Trope 2004; Chueh and Huang 2006). The interest generated from this clinical observation appears to be underscored by the discov- ery of various dental stem cells, including dental pulp stem cells (DPSC) (Gronthos et al. 2000), which may shed light on the understanding of the repair potential of the pulpodentin complex. Furthermore, a report on the discovery of dental stem cells from apical papilla (SCAP) (Sonoyama et al. 2006, 2008) may explain the mechanisms underlying the continued maturation of roots after endodontic treatment of immature teeth.

This chapter emphasizes this new perspective of the management of immature teeth in addition to provid- ing an overview of more traditional protocols. Further- more, in order to manage clinical cases from an in- depth biologic point of view, it is important to review the recent advancement in stem cell biology, especially dental tissue-derived stem cells. This new understand- ing may reshape our traditional way of decision mak- ing in the management of immature teeth. In light of the fact that the discovery and characterization of dental stem cells extends not just to facilitate our clin- ical endodontic treatment planning, this chapter also introduces the potential applications of these stem cells for regenerative endodontics; for example, dentin/pulp regeneration and generation of bio-roots.

The immature permanent tooth has a higher progno- sis for healing than teeth with completely formed apex. This is observed clinically when comparing the pulpal and periodontal healing of teeth with open apex and fully mature teeth in response to caries, luxation and avulsion injuries, transplantation, or simple operative procedures (Skoglund et al. 1978; Skoglund 1981). This is also observed in the remarkable potential for pulpal regeneration that has been described in classic studies, but has more recently been documented and explored clinically. This regenerative ability may be because of the higher prevalence of progenitor or stem cells in the young pulp, greater primary and collateral circulation from the open apex, or the stronger ability of the inflamed pulp to cope with fluctuations in pul- pal blood flow or neuropeptide content given its large communication with apical soft tissues. Therefore, the pathogenesis of endodontic infections tends to take a different route in these teeth, despite the lack of any evidence of a difference in microbial or other host- related differences in endodontic infections in these patients. Clearly, if untreated, these infections can take a dramatic route and lead to significant morbidity. However, the clinical presentation generally tends to

show greater healing potential in teeth with immature apex than in mature teeth. In this chapter, a description of the unique presentation of endodontic infections in immature teeth and their treatment, as well as the recent interest in pulpal regeneration in these teeth is presented. The immature tooth may be more biolog- ically suited to deal with infection, but it tends to be structurally weaker, and its development is necessary for jawbone development. Thus, there is a significant clinical interest in reversing the course of endodontic infections in immature teeth, in order to allow them to fully maturate.

14.2 Review of tooth development as it relates to endodontic pathosis

Tooth is a unique and complex structure developed via interactions between dental epithelium and mesoderm. From the formation of dental lamina in the epithelium, teeth develop in sequential stages including lamina, bud, cap, early bell, and late bell stages (Bhasker 1991; D’Souza 2002). The dental lamina induces underlying ecto-mesenchyme to form the dental papilla. At the cap stage, the entire tooth organ is surrounded by den- tal follicle made of mesenchymal cells. At the early bell stage, the dental epithelium is stratified and the junction where the internal and external dental epithe- lial layers meet is termed cervical loop, which has an important role in root development. Odontoblasts dif- ferentiated from the mesenchymal cells in the dental papilla produce dentin in the late bell stage. As the coronal dentin encases the dental papilla, it evolves to dental pulp. During this stage, the crown is developed followed by root formation as the epithelial cells from the cervical loop proliferate apically and influence the differentiation of odontoblasts from the dental papilla and cementoblasts from follicle mesenchyme. This apically extending, two-layered epithelial wall (merg- ing of the inner and outer enamel epithelium) form the Hertwig’s epithelial root sheath (HERS) which is responsible for determining the shape of the root(s). The epithelial diaphragm surrounds the apical open- ing to the pulp and eventually becomes the apical fora- men. When the first layer of dentin has been laid down, HERS begins to disintegrate leaving behind discontin- ued epithelial cell rests of Malassez in the periodontal ligament.

The apical portion of the dental papilla during the stage of root development was not well described

Endodontic Infections in Incompletely Developed Teeth 313

(a) (b)

(c)

Fig. 14.1 Anatomy of apical papilla. (a) An extracted human third molar depicting root attached to the root apical papilla (open arrows) at developmental stage. (b) Hematoxylin and eosin (H&E) staining of human developing root (R) depicting epithelial diaphragm (open arrows) and apical cell rich zone (open arrowheads). (c) Harvested root apical papilla for stem cell isolation. Source: Adapted from Sonoyama et al. (2008). Reproduced with permission of Elsevier.

in the literature until 2008. The physical and histo- logic characteristics of the dental papilla located at the apex of developing human permanent teeth have been defined and this tissue is termed apical papilla (Huang et al. 2008; Sonoyama et al. 2008). The tis- sue is loosely attached to the apex of the developing root and can be easily detached with a pair of tweezers (Figure 14.1). Apical papilla is apical to the epithelial diaphragm and there is an apical cell-rich zone lying between the apical papilla and the pulp. Importantly, there are stem/progenitor cells located in both dental pulp and apical papilla, but they have somewhat dif- ferent characteristics (Sonoyama et al. 2006, 2008). Because of the apical location of the apical papilla, this tissue may be benefited by its collateral circulation which enables it to survive during the process of pulp necrosis.

Generally, the roots are half to two-thirds formed at the eruption with wide open apices and the pulp volume is still large with thin dentin walls. Three years after tooth eruption is normally needed for fur-

ther deposition of dentin and maturation of the apex. However, as the eruption takes place, the tooth is sus- ceptible to trauma and caries invasion, both of which may threaten the viability and functionality of the cells involved in root development. Damage of the pulp, apical papilla, and/or the HERS resulting in cell death impedes root formation. Loss of HERS ceases the continued root apical extension because odonto- blasts can no longer be differentiated from the api- cal papilla. However, hard tissue can still be formed by cementoblasts which are normally present in the apical region and by mesenchymal cells in the den- tal follicle which may differentiate after the injury into hard tissue-producing cells (Torneck 1982). Cooke and Robotham (1988) advised avoidance of trauma to the tissue around the apex. They speculated that the rem- nants of the survived HERS at the apices of immature teeth, after disinfection, may organize the apical meso- dermal tissue into root components. This mesodermal tissue is now considered to be the apical papilla as mentioned above.

314 Endodontic Microbiology

The fate of human pulp tissue after dental trauma has been observed in clinical radiographs. Excellent radio- graphic images have been reported demonstrating the ingrowth of bone and periodontal ligament (PDL; next to the inner dentinal wall) into the canal space with arrested root formation after replantation of avulsed maxillary incisors (Kling et al. 1986; Andreasen et al. 1995a,b), suggesting a complete loss of the viability of pulp, apical papilla, and HERS. Some patients demon- strated partial formation of root accompanied with ingrowth of bone and PDL into the canal space and in some cases the roots continued to develop to their completion, suggesting at least partial pulp or apical papilla survival after the replantation. It is noted, how- ever, that severe narrowing of canal space is usually associated with survived pulp.

14.3 Etiology, prevalence, and pathogenesis of pulp disease in incompletely developed teeth

Children and young adults aged 6–18 years have sequential permanent tooth eruption and maturation of the roots. Any factors that impinge upon the vital- ity of the pulp can interfere with the completion of root development. Traumatic injuries to young per- manent teeth affect 30% of children (Andreasen and Ravn 1972; Andreasen and Andreasen 1994). Anterior teeth are particularly susceptible to this type of dam- age. First molars erupt around 6–7 years of age when oral hygiene is more difficult to maintain than it is for older children.

The prevalence of untreated dental caries is highest in children aged 6–8 years among all age groups (NIDCR/CDC 2002). This age group is similar to traumatic injury to the teeth in children (Andreasen and Andreasen 1994). Dental anomalies such as dens evaginatus or dens invaginatus leading to pulp infection are also frequently seen in children (Chueh and Huang 2006; Chen et al. 2012). Therefore, children have a high prevalence of pulp disease, especially in those families under the federal poverty level (NIDCR/CDC 2002). Losing immature teeth is difficult to manage as the jaws are still in develop- ment, and any restorative procedure is likely to be a temporary measure. Because of the important role of HERS in continued root development, after pulpal injury, every effort should be made to maintain its viability (Rafter 2005).

The studies of microbial ecology related to the infected, incompletely developed permanent teeth are scarce. Nevertheless, the endodontic microbiology in incompletely developed teeth and mature teeth is likely comparable, because the main source of infection—oral microflora—does not appear to be significantly affected by age. However, the number and depth of dentinal tubules invaded by bacteria are higher and deeper in the infected, incompletely devel- oped teeth, or teeth in young compared to older people (Carrigan et al. 1984; Kakoli et al. 2009). The bacteria form biofilm on the canal walls (Figure 14.2a–d), and penetrate into the root dentinal tubules (Fig- ure 14.2a,b), irregularities of the canal wall (Fig- ure 14.2c), resorption areas (Figure 14.2e,f), lateral/ accessory canals (Figure 14.2g,h), apical ramifications

� Fig. 14.2 (a) Immature maxillary central incisor with necrotic pulp. The apical portion of the canal is filled with a thick bacterial biofilm. Note the varying bacterial concentration (Taylor’s modified Brown and Brenn, original magnification ×25). (b) Magnification of the rectangular area of the left root canal wall in (a). The apparently empty space between the dentin wall and the thick bacterial biofilm is a shrinkage artifact. Note the severe bacterial colonization of in dentinal tubules, some of which show bacteria up to the resorbed external radicular surface (original magnification ×100, inset ×400). (c) High power view from an area of the left canal wall coronal to the point indicated by the arrow in (a). The biofilm is filling the irregularities between the original calcospherites. No predentin can be seen (original magnification ×400). (d) Longitudinal section from a maxillary lateral incisor with necrotic and infected pulp, passing through the transition from predentin to dentin. Predentin appears colonized by some bacterial aggregations (original magnification ×400). (e) Apical third of a mandibular second premolar with necrotic and infected pulp. A thick bacterial biofilm is filling the canal as well as several areas of resorption present on the right canal wall (original magnification ×50). (f) High power view of the area demarcated by the rectangle in (e) (original magnification ×400). (g) Mesial root of a mandibular first molar exhibiting a large periapical radiolucency. Section of a mesio-distal plane showing lateral canals in the apical third (original magnification ×16). (h) Detail of the entrance of the lateral canals in (g). A bacterial biofilm is present on the walls of the main canal and of the lateral canals (original magnification ×50). (i) Detail of the apical root canal showing an apical ramification, colonized by bacteria (original magnification ×100). (j) Cross-cut section taken at the middle third of the root shown in (g). A wide isthmus connecting the two mesial canals is present, exhibiting a thick bacterial biofilm (original magnification ×16).

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j)

316 Endodontic Microbiology

(Figure 14.2i), and the isthmuses (Figure 14.2j) of the infected incompletely developed teeth. These bacteria are impossible to completely eradicate with mechan- ical debridement, antiseptic irrigation, and antimicro- bial intracanal medication. In addition, they are not accessible to sampling technique for bacteriologic examination by culture or molecular methods.

Two distinct clinical situations should be well defined before a clinical treatment approach can be determined, because their clinical outcomes are dra- matically different. The first is the immature tooth with vital pulp. Apexogenesis is a clinical treatment of immature teeth with vital pulp to preserve remain- ing normal vital tissue and to allow completion of root formation and apical maturation. The second situa- tion is an immature tooth with totally necrotic, and frequently infected, pulp. Apexification is then under- taken, defined as the clinical treatment of immature teeth with nonvital pulp by inducing a calcific bar- rier at the open apex (Goldstein et al. 1999; Rafter 2005). After apexogenesis, teeth with vital pulp ther- apy develop a normal thickness of dentin, root length, and apical morphology. Teeth receiving apexification normally gain only an apical mineralized tissue bridge without further development of root.

14.4 Microbiology of endodontic infections in teeth from pediatric patients

In primary or deciduous teeth with infected necrotic pulp, using the checkerboard DNA–DNA hybridiza- tion technique it was detected that the frequency of bacterial species present in the infected root canals were Fusobacterium nucleatum subspecies nucleatum followed by Fusobacterium periodonticum, Prevotella melaninogenica, Prevotella nigrescens, and Prevotella intermedia (Triches et al. 2014). In another study, the most prevalent bacterial species were Prevotella inter- media (96.9%), Neisseria mucosa (65.2%), Prevotella nigrescens (56.2%), Tannerella forsythia (56.2%), Prevotella denticola (53.1%), and Fusobacterium nucleatum subspecies vincentii (50%) (Tavares et al. 2011). Therefore, the infected root canals of primary teeth also had a diverse bacterial infection.

The microbiology in permanent mature teeth with infected necrotic pulps and/or apical periodontitis is well established (Siqueira and Roas 2009). Most bacteria isolated from infected root canals of per- manent teeth were strict anaerobes (70%), with

Peptostreptococcus micros (35%), Fusobacterium necrophorum (23.3%), Fusobacterium nucleatum (11.7%), Prevotella intermedia/nigrescens (16.7%), Porphyromonas gingivalis (6.7%), and Porphy- romonas endodontalis (5%) (Gomes et al. 2004). In immature permanent teeth with pulp necrosis, the most prevalent bacteria detected in the infected root canals were Actinomyces naeslundii (66.7%), followed by Porphyromonas endodontalis, Parvimonas micra, and Fusobacterium nucleatum (33.34%). It appears that the microbial profile of infected immature permanent teeth is similar to that of primarily infected mature permanent teeth (Nagata et al. 2014).

14.5 Management of immature teeth

14.5.1 Diagnosis

Advanced technologies for endodontic diagnostic methods such as laser Doppler flowmetry tests have been shown to be promising (Yanpiset et al. 2001; Roeykens et al. 2002); however, traditional sensitivity testing by cold, heat, and electrical pulp testing remain popular diagnostic tools to differentiate vital from non- vital pulps. The accuracy of these tests in mature teeth was found to be 86% for the cold test, 71% for the heat test, and 81% for the electrical test (Petersson et al. 1999). Whether a negative response means a totally or partially necrotic pulp, or simply a severely calcified pulp, cannot be determined. Inflammation and necro- sis of pulp is a progressive process and it has long been established that there is no close correlation between the results of these clinical tests and the histologic diagnosis (Seltzer et al. 1963). Teeth that respond nor- mally to cold and electrical pulp tests compared with control teeth are clinically considered to have a normal pulp, whereas those responding to these tests with a brief hypersensitive reaction are considered to have a reversible pulpitis. Teeth with spontaneous or linger- ing pain following stimulation are considered to have an irreversible pulpitis.

The reliability of the diagnosis for immature teeth is complicated by the fact that the sensory plexus of nerves in the subodontoblastic region is not well developed and that not all pulpal nerves end amongst the odontoblasts, or into predentin or dentin, as in fully developed teeth in occlusion (Bernick 1964; Fulling and Andreasen 1976; Klein 1978; Fuss et al. 1986). Therefore, the clinical diagnosis of reversible

Endodontic Infections in Incompletely Developed Teeth 317

or irreversible pulpitis, or vital or nonvital pulp can be very challenging in incompletely developed perma- nent teeth with immature nerve innervation. Cold, heat, and electric current are used to stimulate the response of sensory nerve fibers in the pulp and not to deter- mine the extent of pulp injury or pulp vitality, which is more related to the disturbance of blood circulation. There is no good correlation between nerve response and tissue injury. Pulp vitality is the function of pulp blood circulation.

History of symptoms, radiographic examination, and information on the duration and characteristics of the pain are of critical importance. Other clinical tests including visual examination for color change or cracks and percussion testing should be conducted. The presence of a swelling, sinus tract, and extensive radiolucency associated with the tooth is more indica- tive of pulp necrosis and periapical infection for mature teeth than is the case for immature teeth (Iwaya et al. 2001; Chueh and Huang 2006). Therefore, it may be helpful in cases where treatment is indicated to con- firm the final diagnosis after the tooth is accessed and the pulp space directly observed.

14.5.2 Clinical management

14.5.2.1 Vital pulp therapy

Vital pulp therapy includes indirect and direct pulp capping, pulpotomy, and apexogenesis. It is important to preserve the vital pulp tissue as much as possible to encourage continued root maturation in incompletely developed permanent teeth.

14.5.2.1.1 Apexogenesis Apexogenesis treatment procedure falls into the cate- gory of vital pulp therapy for cases with open apices. The common clinical indications are cases demon- strating signs of typical reversible pulpitis, or if the pulp is simply exposed by fracture from trauma or by caries removal. Direct pulp capping can be carried out as needed. A shallow pulpotomy (Cvek pulpotomy) is performed when the pulp is minimally exposed by mechanical reason or trauma. In principle, immature teeth should be treated as conservatively as practi- cal to allow apexogenesis to occur (Weisleder and Benitez 2003). Therefore, pulp capping is the first line treatment option if the pulp is considered largely nor- mal. For cases having more extensive trauma or caries, pulpotomy is the next alternative approach.

14.5.2.1.2 Microbiology and infection control Indirect pulp capping. Teeth undergoing operative procedures should be isolated with a rubber dam. Indi- rect pulp capping is to remove infected dentin, leav- ing behind partially decalcified but not infected dentin (Leksell et al. 1996; Bjornal and Kidd 2005), and to place a layer of calcium hydroxide cement with- out exposing the pulp. The problem is the difficulty in determining clinically what is decalcified but not infected dentin (Alacam 1985), and there has been a scarcity of studies using caries staining agent for indi- rect pulp capping. However, the goal is to provide the pulp with an opportunity to lay down new dentin underneath the caries removal site in order to pre- vent pulp exposure. Approximately 6–8 weeks later the tooth is accessed and examined for the forma- tion of new dentin. Recent reports indicate success rates of 83–96% at 2–4 years’ follow-up in primary teeth that received indirect pulp capping treated with calcium hydroxide or adhesive resin systems (Falster et al. 2002; Marchi et al. 2006). Although there is a lack of clinical studies and reports on the outcome of indirect pulp capping of immature permanent teeth, it may be considered as a clinical option for treating immature permanent teeth. Alternatively, more aggres- sive removal of caries at the cost of pulp exposure and treatment with direct pulp capping may be undertaken.

Direct pulp capping. The pulp is capable of repair after treatment if contaminated by oral flora only briefly after mechanical exposure. Longer exposure to oral cavity (1–7 days) mounts pronounced inflamma- tory infiltrates based on studies in monkeys (Cox et al. 1982). Clinically, when pulp is exposed, either as a result of mechanical nature or from caries removal, the exposed site should be kept clean and disinfected with 5.25% or 6% NaOCl, which also achieves hemosta- sis. The cavity is then carefully rinsed with normal saline and dried with wetted sterile cotton pellets or paper points. Mixed mineral trioxide aggregate (MTA) cement is placed over the exposed spot followed by overlaying a wetted cotton pellet and the tooth is pro- visionally restored with unbonded resin-based com- posite. A final restoration is then placed within 5–10 days if the tooth is asymptomatic and responds nor- mally to cold (Bogen et al. 2008). It was reported that hard tissue bridge formation is more complete after MTA treatment than BMP-7 treatment of pulp exposure in rats (Andelin et al. 2003). The diameter of the exposure site is normally in the range of 1 mm.

318 Endodontic Microbiology

Theoretically, larger sizes of exposure should not affect outcome so long as the management is properly per- formed. It is suggested that there should be good hem- orrhage control and prevention of blood clot formation before capping (Stanley 1989). The blood clot may subject the exposed site to secondary infection. An older study has shown that the success rate of direct pulp capping is 92.9% with mechanical exposure and 33.3% with carious exposure based on at least 3 years of follow-up (Al-Hiyasat et al. 2006). Another study revealed 44.5% failures in 5 years and 79.7% failures in 10-year follow-up for capping of carious exposure (Barthel et al. 2000). These two studies did not specify mature or immature teeth. Farsi et al. (2006) found a success rate of 93% with evidence of continued root growth after using MTA as direct pulp capping to treat carious exposure in young permanent teeth. Bogen et al. (2008) found a 97.96% success rate of direct pulp capping after removal of deep caries with an observa- tion period of 9 years. Among 49 teeth they treated, at follow-up 9 teeth were presumably immature teeth based on the documentation of patients’ age and tooth type. However, Mente et al. (2010, 2014) observed the outcomes of direct pulp capping with MTA or calcium hydroxide after complete removal of caries of vital teeth in patients aged 7–78 years, with a mean age of 44 years. They found that the overall success rates were 80.5% of teeth in the MTA group and 59% of teeth in the calcium hydroxide group after a long-term follow-up (mean 42 months). MTA appeared to pro- vide better long-term results after direct pulp capping than calcium hydroxide (Mente et al. 2010, 2014; Li et al. 2015).

Pulpotomy. Pulpotomy requires aggressive removal of the inflamed pulp tissue, although clinically it is dif- ficult to determine which part of the pulp is actually inflamed. Cvek (1978) reported a 96% success rate of mature and immature teeth treated with partial pulpo- tomy and capping with calcium hydroxide for pulp exposure resulting from crown fracture. The interval between accident and treatment varied from 1 hour to 90 days. A 0.5% chlorhexidine solution was used to clean the teeth and the partial pulpotomy removed approximately 2 mm in depth into the pulp tissue at the exposed site. Using the monkey as a study model, it was found that pulp inflammation extends only approx- imately 2 mm even up to 7 days after pulp exposure by fracture and the teeth left open to oral environment (Cvek et al. 1982). Fong and Davis (2002) reviewed

the success of partial pulpotomy for immature perma- nent teeth that had trauma, carious exposure, or coronal pulpitis. The success rates ranged from 83% to 100% (Fong and Davis 2002). Trope et al.’s (2002) group reported that, in a canine study model, partial pulpo- tomy with calcium hydroxide capping with a bacteria- tight coronal restoration is a viable approach to treat inflamed pulp.

Complete pulpotomy removes the entire pulp tissue in the pulp chamber down to approximately the orifice levels. Hemorrhage control is accomplished by pres- sure on the canal orifices, with a hemostatic agent such as ferric sulfate, the use of a cotton pellet with sodium hypochlorite, or the use of calcium hydroxide powder. This is followed by placing either calcium hydroxide or MTA in the same way as for direct pulp capping (El-Meligy and Avery 2006; Witherspoon 2008).

The understanding of the effects of certain growth factors such as transforming growth factor-β (TGF-β) and bone morphogenic proteins (BMPs) on dentino- genesis provides a potentially potent vital pulp ther- apy approach via biologically directed tissue repair (Rutherford and Gu 2000; Tziafas et al. 2000). The delivery of growth factors such as BMP-7 and growth/ differentiation factor 11 (Gdf11) by ex vivo trans- duced cells or stem cells implanted into exposed pulp to induce reparative dentin formation has also been tested in animal models (Iohara et al. 2004; Nakashima et al. 2004; Rutherford 2001). Cell-based therapy with the combination of growth factors may become future mode of vital pulp treatment approaches.

14.5.2.2 Nonvital pulp therapy

14.5.2.2.1 Apexification When performing calcium hydroxide apexification, obturation of the root canal normally takes place after the apical calcified barrier is formed and resolution of periapical lesion is evident which indicate the suc- cess of disinfection (Figure 14.3). One- or two-visit MTA apexification is different from multiple-visit cal- cium hydroxide apexification in treatment procedures. MTA apexification does not require evidence of res- olution of apical periodontitis before completion of the treatment procedure. Systematic review and meta- analysis of calcium hydroxide and MTA apexification showed that the clinical success of both treatments was comparable (Chala et al. 2011). This does not imply that control of root canal infection is not paramount in apexification. The high success rate of one- or two-visit

Endodontic Infections in Incompletely Developed Teeth 319

(a) (b) (c)

Fig. 14.3 Traditional apexification with calcium hydroxide. (a) Tooth 8 of an 11-year-old was necrotic with open apex. (b) The canal was cleaned and shaped and filled with calcium hydroxide every 3 months until an apical stop was detected. (c) The apical half of the canal was filled with gutta-percha and the remaining canal with composite. Source: Adapted from Huang (2008). Reproduced with permission of Elsevier.

MTA apexification is likely due to bacteria-tight seal of the apical foramen by the MTA plug (Torabinejad and Parirokh 2010). Even though some residual bac- teria may remain in the canal after chemomechanical debridement in MTA apexification, apical periodon- titis will not persist or develop because there is no pathway connecting the canal to the periapical tissues (Molander et al. 1998).

14.5.2.2.2 Microbiology and infection control Disinfection of nonvital immature teeth is generally similar to that of mature teeth, except that the nature of canal size and the divergent canal shape toward apex render the shaping procedure somewhat differ- ent. Circumferential filing with hand instruments may be more effective than rotary instrumentation. To reach the blunderbuss apical canal, precurved stainless steel files may be used. Depending on the level of the tooth maturation, the less tooth structure there is, the less aggressive filing should be undertaken to preserve as much dentin as possible. Therefore, chemical cleans- ing will have a more important role to disinfect the canal. Cvek et al. (1976) found that antimicrobial

effect by mechanical cleansing is very low for mature and even lower for immature teeth. Irrigation with up to 5% NaOCl enhances the antimicrobial effect but is still inadequate. Current chemical agents used for canal irrigation besides NaOCl include chlorhexidine, EDTA, IKI (iodine-potassium iodide) (Safavi et al. 1985), and triple antibiotic paste consisting of metron- idazole, ciprofloxacin, and minocycline. Sato’s group first described the use of the mixture of these three antibiotics against Escherichia coli infected dentin in vitro (Sato et al. 1996). The same group also tested their bactericidal efficacy against microbes from cari- ous dentin and infected pulp and found them to be suf- ficiently potent to eradicate the bacteria. The respec- tive antibiotic alone substantially decreases bacterial recovery, but cannot kill all the bacteria as does the mixture of antibiotics (Hoshino et al. 1996a). It was found that this triple antibiotic paste is more effective than 1.25% NaOCl in disinfecting immature teeth in vivo in a dog study model (Windley et al. 2005). The triple antibiotic paste was mixed at a concentration of 20 mg of each antibiotic per milliliter, inserted into canal with a sterile Lentulo spiral filler, and left in

320 Endodontic Microbiology

the canal for a period of 2 weeks. It is generally dif- ficult to test the antimicrobial effectiveness of antibi- otics in endodontic models, because of the absence of an inactivator to prevent the carry-over effect. Fur- thermore, the effectiveness of bactericidal and bacte- riostatic antibiotic combinations, and the discoloring effects of tetracycline analogs, such as minocycline, raise some concerns about whether this is the most suitable antibiotic combination for this purpose.

To date, the most commonly used medication asso- ciated with apexification is calcium hydroxide paste (Rafter 2005). The purpose of its use is twofold: disin- fection and induction of apical calcific barrier. When first introduced (Kaiser 1964), it was proposed to mix calcium hydroxide with camphorated parachlorophe- nol (CMCP). To avoid the potential cytotoxicity of CMCP, calcium hydroxide was later tested for its effi- cacy by mixing with just saline or sterile water and was found to have similar clinical success (Michanow- icz and Michanowicz 1967; Binnie and Rowe 1973). Calcium hydroxide is antimicrobial because of its release of hydroxyl ions which can cause damage to the bacterial cellular components. The best example is the demonstration of its effect on lipopolysaccha- ride (LPS). Calcium hydroxide chemically alters LPS which affects its various biologic properties (Safavi and Nichols 1993, 1994; Barthel et al. 1997; Nelson- Filho et al. 2002; Jiang et al. 2003).

Traditional calcium hydroxide apexification It has been unclear whether calcium hydroxide possesses the ability to induce apical barrier or whether its antimicro- bial activity provides an environment where mineral- ized tissue is able to develop. Early in vivo studies have suggested the ectopic induction of bone by calcium hydroxide (Mitchell and Shankwalker 1958). It was considered that the layer of firm necrosis created by the contact of calcium hydroxide generates a low-grade irritation of the underlying tissue sufficient to pro- duce a matrix that mineralizes (Schroder and Granath 1971; Holland et al. 1977). Calcium is attracted to the area and mineralization of newly formed collagenous matrix is initiated from the calcified foci. High pH was considered an important factor for its ability to induce hard tissue formation (Javelet et al. 1985). The time required for apical barrier formation in apexification using calcium hydroxide varies from 3 to 20 months (Yates 1988; Sheehy and Roberts 1997; Finucane and Kinirons 1999). Other conditions such as age and pres- ence of symptoms or periradicular radiolucencies may

have a role in the time needed to form apical barrier (Cvek 1972; Ghose et al. 1987; Mackie et al. 1988). Refreshing the calcium hydroxide paste usually takes place every 3 months.

Obturation of the root canal takes place normally when the apical calcific barrier is formed (Figure 14.3). The mineralized barrier provides a matrix against which traditional gutta-percha filling material can be condensed. The barrier may be verified radiographi- cally and by clinically detecting the apical stop with endodontic files.

MTA apexification In recent years, the use of MTA to fill the apical third without the need for calcific bar- rier formation has been shown to be successful. MTA was first introduced in 1993 by Torabinejed and his coworkers (Lee et al. 1993; Torabinejad et al. 1993) and approved by the Food and Drug Administration (FDA) in 1998. This material has demonstrated good biocompatibility and antimicrobial activity. It has a pH of 12.5 after setting, which is similar to that of cal- cium hydroxide (Torabinejad et al. 1995a). The first popular use of MTA was perforation repairs in roots (Lee et al. 1993) or furcations (Ford et al. 1995; Arens and Torabinejad 1996) for its excellent tissue com- patibility. Use then extended to direct pulp capping, root-end retrograde filling, and apical plug in apex- ification (Ford et al. 1996; Torabinejad et al. 1997; Shabahang and Torabinejad 2000). Additionally, MTA appears to exhibit antimicrobial properties (Torabine- jad et al. 1995b). In comparison to calcium hydrox- ide on hard tissue induction, MTA appears to have a greater consistency based on in vivo studies in dogs (Shabahang et al. 1999).

Some reports have shown that using MTA for apex- ification may shorten the treatment period with more favorable results. After 1 week’s calcium hydroxide medication in the canal, MTA is condensed down to the apical canal by plugging motion with pluggers or with the blunt end of gutta-percha points, after a biocom- patible collagen matrix, such as CollaCote, is placed in the apical region. The sealing and apical exten- sion is carefully controlled and monitored radiograph- ically against the matrix material. Favorable results have been reported (Maroto et al. 2003; El-Meligy and Avery 2006; Pace et al. 2007). One-visit apexifi- cation using MTA has also been reported (Witherspoon and Ham 2001). Some authors considered that lengthy treatment protocols are inconvenient and may lead to ultimate failure simply because patients cannot return

Endodontic Infections in Incompletely Developed Teeth 321

for the numerous visits each months apart. They pro- posed a one-visit apexification protocol with MTA as an alternative to the traditional treatment practices with calcium hydroxide (Steinig et al. 2003). This expedient cleaning and shaping of the root canal system followed by its apical seal with MTA makes an immediate place- ment with a bonded core within the root canal in one visit possible, which may prevent potential fractures of immature teeth. One-visit or two-visit approach con- tradicts the traditional concept in that an apical barrier must be first formed in order to obtain a good stop. As the nature of placing the MTA to the apex is quite dif- ferent from that of gutta-percha, different techniques have been developed to make MTA apical plug possi- ble. The sealing ability is superior when MTA is used as a root-end filling material and condensed against a physical barrier rather than as an orthograde api- cal plug. A thickness of 1–4 mm for the MTA apical plug provides adequate retention (Hachmeister et al. 2002; de Leimburg et al. 2004). In immature teeth with necrotic pulps and periapical bone resorption, irregu- lar dentin walls, divergent apices, and lack of physical barrier make the adaptation and control of MTA apical placement more difficult. Some authors suggested that hand condensation resulted in better adaptation and fewer voids than ultrasonic compaction (Aminoshariae et al. 2003). As noted before, the use of resorbable material such as freeze-dried bone, collagen plug, or other biocompatible materials packed into the apical region to serve as a physical barrier or matrix against which MTA may be condensed has been suggested (reviewed by Rafter 2005). Proper apical placement of MTA into a tooth with open apex should lead to a favor- able outcome (Figure 14.4). Recent meta-analysis of direct pulp capping with calcium hydroxide or min- eral trioxide aggregate showed that MTA had a higher success rate than calcium hydroxide (Li et al. 2015). Mente et al. (2013) found that the presence of preoper- ative apical periodontitis was an important prognostic factor of MTA apexification.

14.5.2.3 Outcome of endodontic therapy on young permanent teeth

14.5.2.3.1 Outcome of vital pulp therapy on young permanent teeth A systemic review of vital pulp therapy (partial and full pulpotomy) in vital permanent teeth with cariously exposed pulp showed that the success rate of vital

pulp therapy was over 99% after 3 years of follow- up examination (Aguilar and Linsuwanont 2011). The authors suggested that complete removal of coronal infected or inflamed pulp leaving uninflamed healthy pulp was more critical than the status of the root apex in vital pulp therapy.

The success of pulp capping following mechanical or traumatic exposure is high, therefore this procedure should be the first choice for immature teeth if condi- tions allow. In fact, because the immature teeth have great potential to heal and regenerate, efforts should be made toward creating a favorable environment for root maturation to complete. Considering the high levels of success for direct pulp capping with MTA (Bogen et al. 2008), pulp capping and pulpotomy for apexoge- nesis may be a favorable alternative to apexification for young permanent teeth, although there has been a lack of clinical studies on pulp capping success focusing on immature permanent teeth. Failure of pulp capping cases has been attributed to infection left behind in the pulp which normally should become noticeable early, or attributed to microleakage. It was shown in studies using monkeys that the formation of dentin bridge does not secure the leakage-proofing as most dentin bridges contain multiple tunnel defects, which remain open to the underlying pulp from the medicament interface. The recurring pulp inflammation observed after 1- and 2-years direct pulp capping is associated with bacterial contamination (Cox et al. 1985).

With respect to the use of calcium hydroxide for the induction of apical barrier formation and healing in immature permanent teeth, the successful rate is 74–100% in terms of apical barrier formation, irre- spective of the proprietary brand used, as reviewed by Sheehy and Roberts (1997). In the traditional apexifi- cation treatment, apical closure is accomplished by the formation of cementum-like barrier of various thick- nesses. The hard tissue barrier has been described as a cap, bridge, or ingrown wedge which is composed of cementum, dentin, bone, or so-called osteodentin, which can deposit on the inner walls of the canal (Steiner and Van Hassel 1971; Torneck et al. 1973; Lieberman and Trowbridge 1983; Ghose et al. 1987; Walia et al. 2000; Ritter et al. 2004; Rafter 2005). The apical hard tissue bridge is generally described as consisting of cementum, dentin, bone, and osteo- dentin. Histologically, the distinction of cementum and bone requires careful observation. Cementum for- mation can proceed from the periphery of the apex

322 Endodontic Microbiology

(a) (b) (c)

(d) (e) (f) (g)

6 month 3 yr 5 yr 14.5 yr

Fig. 14.4 An open apex 8 with apex filled with mineral trioxide aggregate (MTA). (a) Before treatment. (b) Canal cleaned and shaped. (c) Apex filled with MTA plug. (d–g) Six-month, 3-year, 5-year, and 14.5-yr follow-up, respectively. Courtesy Dr. G. Bogen.

towards the center in decreasing concentric circles. Formation of dentin requires the presence of odonto- blasts. The remaining soft tissue at the apical region of nonvital immature teeth cannot be easily identified clinically. If there are viable odontoblasts, formation of some dentin structure is certainly a possibility.

In contrast to apexogenesis, apexification treatment (traditional or MTA) does not generally lead to an additional formation of root dentin, leaving behind a weak root that is susceptible to fracture. Moreover, traditional apexification has been associated with a high incidence of cervical root fracture (Cvek 1991), which is attributed to the weakening effect of calcium

hydroxide on dentin (Andreasen et al. 2002). Filling the canal from mid-root to coronal third with resin bonding to strengthen the root has been advocated after the completion of apexification (Rabie et al. 1985; Katebzadeh et al. 1998; Pene et al. 2001; Goldberg et al. 2002).

A clinical case was reported of apical bridge for- mation and the tooth was extracted for orthodontic reasons. The histologic examination showed that the apical barrier is basically made of cementum that not only forms a bridge, but also extends coronally along the dentinal wall. Sharpey’s fibers were not explic- itly described by the authors, but appeared to form

Endodontic Infections in Incompletely Developed Teeth 323

between the cementum inside the canal and the soft tissue (Lieberman and Trowbridge 1983).

14.5.2.4 Paradigm shift

While the decision for teeth to undergo apexogenesis or apexification has been determined by the result of pulp vitality, recent well-presented reports have shown that this straightforward dichotomized approach may sacrifice certain cases that still have the potential to undergo apexogenesis even when clinical condition qualifies for an apexification treatment. These case reports have shown convincingly that immature teeth clinically diagnosed with nonvital pulp and apical peri- odontitis or apical abscess can undergo apexogenesis (Iwaya et al. 2001; Banchs and Trope 2004; Chueh and Huang 2006; Thibodeau and Trope 2007). In fact, before these recent reports that provoked our tradi- tional treatment decisions, there had been sporadic case reports in the literature documenting this obser- vation (Rule and Winter 1966; Nevins et al. 1977; Saad 1988; Matusow 1991a,b; Whittle 2000; Selden 2002).

The recent reports stimulated a new perspective as to how we determine the treatment plans for these cases (Huang 2008). A common aspect of the reported cases is that those teeth showing continual maturation of root and apex had developed extensive periapical lesions, some with sinus tract formation before the treatment, which is a condition normally considered to have total necrosis and infection of the pulp requiring apexification (Figures 14.5, 14.6, and 14.7) (Iwaya et al. 2001; Banchs and Trope 2004; Chueh and Huang 2006).

14.5.2.4.1 Clinical diagnosis of partially vital pulp Clinical diagnosis of the vitality of pulp is crude. It was considered in the past that the presence of radiolucent lesions at the apices confirms a total necrosis and infec- tion of the pulp, until Lin et al. (1984) biopsied pulp tissue from mature permanent teeth associated with periapical radiolucency and found that vital tissues were present in some cases, even within the pulp cham- bers in rare cases. The size of the lesions in that study varied significantly, and some had little change from normal radiographic appearance, while others could be described as moderately extensive. In the case of immature teeth, association of periapical lesions with

remaining vital tissues within the canal could be more common, although there is a lack of histologic evi- dence for this. However, recent case reports of imma- ture teeth that all have radiolucent lesions, some with quite extensive purulence in the periradicular region, showed remarkable root maturation after revascular- ization or revitalization treatment (Iwaya et al. 2001; Banchs and Trope 2004; Chueh and Huang 2006). This suggests that vital pulp tissue may have remained in the canals or the ingrowth of new connective tissue filling the canal space. Iwaya et al. (2001) reported that during the treatment of an immature second mandibular pre- molar with an apical abscess, the patient felt the inser- tion of a smooth broach into the canal before reaching the apex, indicating the partial sensitivity in the pulp space. Thirty-five months after the treatment, the root formation was complete and the tooth responded to an electrical pulp test. Similarly, Chueh and Huang (2006) reported four cases of immature teeth with api- cal periodontitis or apical abscess. In some of these cases, either the patients felt the entry of instrument into the pulp chamber or vital pulp tissue and hemor- rhage were observed.

14.5.2.4.2 Disinfection protocol The management of these immature teeth in the case reports mentioned here has the following common fea- tures: (i) minimal or no instrumentation; (ii) irrigation with 2.5–5.25% NaOCl, 3% hydrogen peroxide and/or Peridex; and (iii) medication with calcium hydrox- ide or antimicrobial agents consisting of metronida- zol, minocycline, and ciprofloxacin in paste form. This clinical protocol has been termed “revitalization,” or a less descriptive term “revascularization” (Huang and Lin 2008). “Revitalization” is used in the text hereafter.

It was noted by some authors that calcium hydrox- ide is not recommended as intracanal medicament, so as not to damage the remaining pulp tissue and Her- twig epithelial root sheath (Banchs and Trope 2004; Chueh and Huang 2006). Recent studies, however, found that calcium hydroxide is the least toxic to stem cells compared to the antibiotics (Ruparel et al. 2012). The antibiotics are only nontoxic at 0.1 mg/mL whereas calcium hydroxide is nontoxic at as high as 100 mg/mL. When calcium hydroxide is at 1 mg/mL, it actually increases the stem cell proliferation. Based on these case reports, a clinical protocol to treat immature teeth, after a thorough irrigation with 1.5%

324 Endodontic Microbiology

(a) (b)

Fig. 14.5 (a) Radiograph showing a lower premolar of an 11-year-old patient having an extensive periradicular lesion. (b) Twenty-four-month radiograph after treatment showing complete root development. Source: Adapted from Banchs and Trope (2004). Reproduced with permission of Elsevier.

hypochlorite, and possibly with 17% EDTA to remove the smear layer, is summarized as the following:

1. The antimicrobial paste each at 0.1 mg/mL pre- pared in an injectable and resorbable gel material is introduced into the canal and the accessed cavity sealed with 2 mm cavit as an inner layer and 2 mm durable cement such as glass ionomer as the outer layer. If calcium hydroxide is to be used, 1 mg/mL

is injected into the canal. The patient has to return in 2 weeks; otherwise, apexification will result.

2. After 2 weeks, the patient should return for eval- uation. If asymptomatic and there are no signs of intraoral pathology, the canal will be re-accessed to visually examine the condition under the micro- scope. If the canal is clean, it will be irrigated again with NaOCl followed by sterile normal saline or 17% EDTA (Galler et al. 2011, 2015) and then

Endodontic Infections in Incompletely Developed Teeth 325

(a) (b)

Fig. 14.6 Clinical case of a 10-year-old patient. (a) Radiograph showing an immature root of tooth 29 with an open apex and an extensive radiolucency at the periapical and mesial regions of the root. (b) Seven months after the initial treatment showing complete maturation of the root apex, healing of the periradicular bone, a significant increase of the calcified tissue in the root, decrease of root canal space, and the calcified coronal third of the root canal. Source: Adapted from Chueh and Huang (2006). Reproduced with permission of Elsevier.

dried. Banchs and Trope (2004) induced hemor- rhage by an exploration penetrating slightly into the periapical tissue allowing the blood clot to form in the canal and stopped at a level 3 mm below that of the cemento-enamel junction (CEJ). MTA was then placed over the blood clot. They considered the blood clot as a scaffold and source of growth factors to facilitate the regeneration and repair of tissues into the canal. Currently, there is neither his- tologic evidence showing that blood clot is required for the formation of repairing tissues, nor are there systematic clinical studies to show that this approach is optimal. However, these cases reports

at least provide some guidelines as to the extent of healing potential in these immature teeth.

The accessed cavity is sealed with glass ionomer cement or composite and the tooth should be followed up periodically to observe the maturation of the root. If after several rounds of intracanal irrigation and medi- cation the clinical symptoms show no sign of improve- ment (i.e., persistent presence of sinus tract, swelling, and/or pain), apexification procedure or apical surgery may be considered before extraction as the final option.

Bacteria in biofilm firmly attached to the canal walls and in the canal dentinal tubules are very hard to

326 Endodontic Microbiology

(a) (b)

Fig. 14.7 Clinical case of a 10-year-old patient. (a) Radiograph showing a radiolucent lesion at the periapical area of tooth 20 with a wide open apex (a gutta-percha point into the sinus tract). (b) Thirty-five months after the initial treatment revealing a market reduction of the root canal space and maturation of the root apex. Source: Adapted from Chueh and Huang (2006). Reproduced with permission of Elsevier.

eliminate by canal irrigants (sodium hypochlorite solu- tion) and intracanal medicaments (calcium hydroxide) during root canal therapy (Haapasalo and Orstavik 1987; Orstavik and Haapasalo 1990; Svensäter and Bergenholtz 2004; Estrela et al. 2009; Chavez de Paz et al. 2010). In addition, bacteria in biofilm can evade the host innate and adaptive defense mecha- nisms and resist antimicrobial chemotherapy (Stewart and Costerton 2001). Although triple antibiotic paste has been shown to be effective in controlling bac- terial infection in vitro (Hoshino et al. 1996b; Sato et al. 1996) and in incompletely developed necrotic teeth with apical periodontitis in vivo animal model by bacteriological culture (Windley et al. 2005), these experiments do not simulate the clinical condition of bacterial colonization in the root canal system of long- standing, infected, incompletely developed, necrotic teeth with apical periodontitis. It was shown that revi- talization procedures of human immature permanent teeth with pulp necrosis treated with triple antibiotic paste or calcium hydroxide and 6% sodium hypochlo- rite or 2% chlohexidine were efficient but not com- plete in reducing viable bacteria in the canals by cul- turing method (Nagata et al. 2014). It is not known if the canals of infected incompletely developed per- manent teeth could be thoroughly disinfected with- out mechanical debridement to disrupt the biofilm, allowing the antiseptic irrigant and antimicrobial intra- canal medicament to penetrate into the biofilm to kill the bacteria (Fouad 2011; Fouad and Nosrat 2013). Revitalization therapy has been shown to have the

potential to increase the thickness of the canal walls; therefore it might be more beneficial to disrupt the biofilm by performing a certain degree of mechani- cal debridement during revitalization procedures (Lin et al. 2013, 2014). The residual bacteria remaining in the dentinal tubules, lateral/accessory canals, api- cal ramifications, and biofilm after root canal dis- infection in the revitalized teeth are different from those in root-filled teeth after nonsurgical root canal therapy. The residual bacteria in the revitalized teeth may survive for a long period of time because they can obtain nutrients from the newly formed tissue in the canal (Lin et al. 2013, 2014). Due to the root canal anatomy of incompletely developed permanent teeth and prevention of toxic antiseptic irrigants to gain access to the periapical tissues to damage stem cells and vasculature, Endo-Vac (Kerr Corp. Orange CA), ultrasonic irrigation, or photodynamic disinfec- tion have been suggested as a part of root canal disin- fection procedures (Burleson et al. 2007; Carver et al. 2007; da Silva et al. 2010; Ng et al. 2011; Fouad and Nosrat 2013; Al Shahrani et al. 2014). If root canal infection is not under control, neither repair nor regeneration can occur, despite the presence of stem cells in the inflamed periapical tissues retain- ing regenerative potential in the immature permanent teeth (Liao et al. 2011). The root canal disinfection protocol of revascularization or revitalization therapy should be evaluated in a long-term follow-up exami- nation to improve the clinical success rate (Lin et al. 2013).

Endodontic Infections in Incompletely Developed Teeth 327

14.5.2.4.3 Outcome Given the right conditions, many tissues are pro- grammed and capable of self-regeneration to repair the damaged portion. The literature is replete with the finding that if disinfection can be performed effec- tively, pulp tissue can heal and new dentin bridge will form. Pulp tissue in immature teeth with open apex has a rich blood supply and therefore it is more capable of resisting infection and healing following irritation. As an example, pulp polyp is normally seen in imma- ture teeth when the pulp is widely exposed to the oral cavity. The newly formed epithelial layer helps defend against the invading microbes and keeps the underly- ing tissue alive except the tissue is no longer normal and filled with inflammatory infiltrates.

As already noted, teeth inherit a thin and weak root after successful apexification susceptible to fracture. Shifting apexification to apexogenesis even for non- vital teeth with apical periodontitis or apical abscess is a clinically beneficial approach for patients. The aforementioned case reports all show that the partially survived pulp and apical tissue of immature teeth have the potential to regenerate and complete root forma- tion. The infection had not recurred during the period of follow-up observation.

Whether the thickened root is formed by pulp tis- sue from the remaining vital pulp tissue at the apical region, or formed by PDL tissue which has grown into the root canal from the apical foramen and deposited the cementum onto the inner surface of the root dentin, has been of great interest to the endodontic community. It was speculated that some pulp tissue survived and allowed apexogenesis to occur (Huang 2009). DPSC have been identified to exist in permanent teeth (Gron- thos et al. 2000, 2002; Shi and Gronthos 2003). The apical papilla contains SCAP that have been described to be more robust stem cells than DPSC (Sonoyama et al. 2006, 2008). The SCAP may survive the infection to allow root maturation while the surviving DPSC in the remaining vital pulp rebuild the lost pulp tissue in the canal (Huang 2009). Iwaya et al. (2001) suggested that the open apex provides good communication from pulp space to the periapical tissues, therefore it may be possible for periapical disease to occur while the pulp is only partially necrotic and infected.

Animal studies focusing on the changes in pulp tis- sue after replantation showed that various hard tissues including dentin, cementum, and bone may form in pulp space, depending on the level of pulp recov- ery (Skoglund and Tronstad 1981; Kvinnsland and Heyeraas 1989; Ritter et al. 2004). By tracing the

migration of periodontal cells after pulpectomy in immature teeth, Vojinovic and Vojinovic (1993) found that periodontal cells migrate into the apical pulp space during the repair process (Vojinovic and Vojinovic 1993). Therefore, if one assumes the total loss of pulp tissue but remaining in a sterile condition, the outcome is the ingrowth of periodontal tissues. This may explain the increased thickness of the canal wall and the severe shrinkage of canal space. Taken together, if pulp tis- sue is totally lost, the canal space may be occupied by cementum, PDL, and bone. In this situation, it is diffi- cult to identify clinically via radiographs because the canal space may well be PDL tissue and the thickened root structure be cementum. Recent studies in dogs and ferrets have revealed that endodontically involved immature permanent teeth undergoing revitalization treatments resulted in having the root canal space filled with periodontal tissues rather than pulp (Wang et al. 2010; Torabinejad et al. 2015). These tissues showed cementum grown into the canal dentinal walls and the canal space filled with bone-like, periodontal ligament- like, and fibrous connective tissues (Figure 14.8). A number of reports also showed histologic analysis of the tissues in the canal space in human immature per- manent teeth after revitalization treatments. Similar to the findings in dogs, mainly cementum and fibrous tis- sue were grown in the canal space (Shimizu et al. 2013; Becerra et al. 2014; Nosrat et al. 2015) (Figure 14.9). Two case reports, however, demonstrated pulp-like or fibrous connective tissue regeneration in human teeth after revitalization procedures (Shimizu et al. 2012; Torabinejad and Faras 2012) (Figure 14.10). It should be noted that it is difficult to identify the pulp con- dition clinically therefore it is likely that the regener- ated pulp-like tissues were the remaining pulp tissue in the canal at the time of treatment. One case in the dog study showed that the remaining pulp tissue after pulpectomy, infection, and disinfection may survive and retain its original tissue structure (Wang et al. 2010) (Figure 14.11).

14.6 Orthodontic considerations in pathologically involved incompletely formed teeth

Orthodontic treatment often initiates at ages ranging from 12 to 16 years, during which time the dentition is mixed with immature teeth (Anthony 1986). Young individuals in this age group are also more suscepti- ble to dental injuries. Therefore, it is not uncommon

328 Endodontic Microbiology

Fig. 14.8 Histologic study of vital tissues formed in dog teeth after revitalization procedures. Thickened root resulting from the deposition of intracanal cementum (IC) onto dentin. Intracanl bone-like tissues (IB) scattered in the root canal space along with intracanal PDL-like tissues (IPL). (a) A sample showing vital tissues in the canal. A magnified view of the boxed region is shown in Inset a. Black arrows indicate cementocyte-like cells in IC; blue arrowheads indicate cementoblast-like cells lining against the IC. (b) A sample showing well-generated IPL extending from extracanal periodontal ligament (PL). (Inset b) Magnified view of the boxed region in (b). The yellow arrowheads indicate Sharpey’s fibers. Yellow dashed lines, angles of ligament fibers. The black arrows indicate cementocyte-like cells in IC. Scale bars: a, Inset a, and b, 500 μm; Inset b, 200 μm. Source: Adapted from Wang et al. (2010). Reproduced with permission of Elsevier.

to encounter immature teeth that require endodontic therapy while under orthodontic treatment, which can result in root resorption. Continuous forces seem to produce more resorptions than discontinuous forces (Weiland 2006). Orthodontic forces are traumatic to the periodontal tissues and cause the expression of proinflammatory cytokines and release of mediators by periodontal tissue cells leading to the resorption of bone and cementum. The degree of the induced inflammation depends on various factors including the amount of orthodontic forces and individual vari- able biological responses or genetic influences (Al- Qawasmi et al. 2006; Filho et al. 2006). Increased angiogenesis and inflammatory response in pulp also occurs (Derringer et al. 1996; Vandevska-Radunovic 1999). Endodontically treated teeth under orthodon- tic treatment do not show more apical root resorption than normal vital teeth (Esteves et al. 2007). Proper

orthodontic treatment does not affect the development of normal immature teeth. When immature teeth under orthodontic forces develop pathosis, teeth appear to have a doubled infliction and therefore the course of healing after endodontic treatment may be compro- mised. Clinical observations of immature teeth hav- ing apexification, however, do not show retardation or inhibition of the deposition of a calcified barrier at the root apex during an active orthodontic move- ment in children (Anthony 1986) or in adults (Fava 1999). In the case of transplantation of immature teeth followed by orthodontic treatment, studies have shown that orthodontic movement of the transplants appeared to have a tendency to shorten the final root length (Lagerstrom and Kristerson 1986).

Active orthodontic movement does not appear to affect the apexification procedures of immature perma- nent teeth with necrotic pulp and apical periodontitis

Endodontic Infections in Incompletely Developed Teeth 329

Fig. 14.9 Histologic study of a human tooth after revitalization treatment. (A) (a) Preoperative radiograph: tooth 9 exhibits incomplete formation of the root. Periapical radiolucency is present. (b) Postoperative radiograph: the periapical radiolucent area appears to be larger than the preoperative lesion, with sharp margins. (c) Follow-up radiograph taken 12 months after revitalization: the periapical radiolucency has resolved with only slight thickening of the periodontal ligament around the root apex. The canal space is reduced in size and the thickness of the canal walls is increased. (d) The patient presented after 25 months with complete crown fracture. Thickening of the root canal walls increased further. The periapical lesion completely resolved. (B) Histologic study of the tooth after extraction due to nonrestorability. (a) Section passing approximately at the center of the root canal. A mineralized tissue fills the apical portion of the canal (H&E; original magnification x16). (b) Detail of the apical portion in (a). An island of soft tissue is present in the calcified tissue apically (original magnification x25). The inset shows magnification of the ramification indicated by the lower arrow. Its lumen contains uninflamed connective tissue (original magnification x400). (c) A high-power view of the apical soft tissue in (b). Vital connective tissue with fibroblasts and abundance of collagen fibers. Absence of inflammatory cells (original magnification x400). (d) Magnification of the area indicated by the upper arrow in (b). The calcified tissue filling the apical canal is irregular and is demarcated apically by a cementum-like tissue, with some osteoblast-like lacunae. Increased root length is caused by deposition of cementum-like tissue (original magnification x400). (e) A high-power view of the area of the root canal wall indicated by the left arrow in (a). From left to right: area with high concentration of dentinal tubules (tubules are cut transversally by the microtome blade), area with less tubules, and calcified tissue with no dentin tubules (original magnification x400). (f) A high-power view of the area of the root canal wall indicated by the right arrow in (a). From right to left: area with high concentration of dentin tubules (cut obliquely by the microtome blade), area with only few tubules, and calcified tissue with no dentin tubules (original magnification x400). Source: Adapted from Shimizu et al. (2013). Reproduced with permission of Elsevier.

330 Endodontic Microbiology

Fig. 14.10 Histologic study of a human tooth after revitalizatioin treatment. (A) (a) Peoperative radiograph of tooth 9. (b) A radiograph of the fractured tooth 3.5 weeks after revascularization. (c) A photograph of the extracted tooth. Note a small mass of soft tissue attached to the root apex (arrow). M, mineral trioxide aggregate plug. (B) Histology of the section of extracted revitalized tooth 9. (a) A loose connective tissue with few collagen fibers has filled the canal space up to the coronal MTA plug (H&E, original magnification x 200). The MTA plug was removed before histologic tissue processing. (b) High magnification of the square in (a) (the apical root canal). Flattened odontoblast-like cells lined along the predentin (solid arrows). Many blood vessels filled with red blood cells (open arrows). No mature nerve-like bundles along the blood vessels are observed. Most cells are spindle shaped. (c) High magnification of the rectangle in (a) (the apical foramen). There are fewer blood vessels (arrow) and cellular components at the apical foramen than in the canal. (d) High magnification of the square in (c) (part of the root apex). Layers of epithelial-like HERS (arrow) surrounding the root apex. Spaces in the tissue are artifacts caused by histologic preparation. Source: Adapted from Shimizu et al. (2012). Reproduced with permission of Elsevier.

if root canal infection is under control (Anthony 1986; Alacam and Ucuncu 2002).

14.7 Stem cells for pulp and periodontal tissue regeneration

In light of the recent isolation and characterization of adult dental stem cells as well as the progress of regenerative medicine, clinicians should re-evaluate current clinical protocols when treatment planning for certain cases. This is especially true for immature teeth as they are still at the growing phase of their lifespan.

Stem cell biology has become an important field for the understanding of tissue regeneration. In general, stem cells are defined by having two major proper- ties. First, they are capable of self-renewal. Second, when they divide, some daughter cells give rise to cells

that eventually become terminally differentiated cells. Depending on the type of stem cells and their ability and potency to become different tissues, the following categories of stem cells have been established:

1. Totipotent stem cells: each cell is capable of devel- oping into an entire organism;

2. Pluripotent stem cells: cells from embryos (embry- onic stem cells) that when grown in the right envi- ronment in vivo are capable of forming all types of tissues; and

3. Multipotent stem cells: postnatal stem cells or adult stem cells that are capable of giving rise to multiple lineages of cells.

Dental stem cells belong to the third category (Robey and Bianco 2006).

Embryonic stem cells are potentially immortal when grown in vitro, whereas multipotent or unipotent stem

Endodontic Infections in Incompletely Developed Teeth 331

Fig. 14.11 The survival of remaining pulp in a dog tooth after pulpectomy, root canal infection, disinfection, and receiving revitalization procedures. (a) Thickened inner root canal walls resulting partly from the thickened dentin on one side (left) and the deposition of intracanal cementum (IC) on the other side. (b) Magnified view from the top boxed region in (a). Blue arrowheads demarcate the dentin (D) and IC. The yellow arrowheads show the odontoblasts (od) and cementoblasts-like cells (c). (c) A magnified view of the right boxed region in (a). The IC extending from the dentinal wall toward the opposite of the canal forming a bridge. (d) A higher-magnification view of the odontoblast layer (od) from the left boxed region in (a). (Ea and Eb) Radiographs of the roots (*) showing pre- and post-experimental treatment, respectively (sample from group 2). Scale bars: (a) 500 μm, (b,c) 200 μm, and (d) 50 μm. Source: Adapted from Wang et al. (2010). Reproduced with permission of Elsevier.

cells have a limited life-span and become senescent after a certain number of population doublings in culture. Adult stem cells have been isolated from various tissues. Bone marrow is a source of stem cells of multipotency including mainly hematopoi- etic and mesenchymal stem cells. Mesenchymal stem cells (MSCs), identified in many mesenchymal tis- sues, are capable of becoming many lineages of cells when grown in defined conditions including osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic lineages (Tuan et al. 2003; Baksh et al. 2004).

To date, five types of human dental stem/progenitor cells have been isolated and characterized:

1. Dental pulp stem cells (DPSC) (Gronthos et al. 2000);

2. Stem cells from exfoliated deciduous teeth (SHED) (Miura et al. 2003);

3. Stem cells from apical papilla (SCAP) (Sonoyama et al. 2006);

4. Periodontal ligament stem cells (PDLSC) (Seo et al. 2004); and

5. Follicle precursor cells (DFPC) (Morsczeck et al. 2005).

Among these, all except SHED are from perma- nent teeth. These dental stem cells are considered mesenchymal-like stem cells and possess different potential for becoming specific tissue forming cells. DPSC, SHED, and SCAP are from the developed or developing pulp tissue. These ex vivo expanded cells can differentiate into odontoblast-like cells and pro- duce ectopic dentin-like tissue in vivo (Huang et al. 2009). When grown in cultures and induced under specific conditions, DPSC and SHED can differen- tiate into neuronal and adipogenic cells in addition to dentinogenic cells (Miura et al. 2003; Zhang et al. 2006a). Some reports have shown that DPSC also have chondrogenic, myogenic, and osteogenic potentials (Laino et al. 2005, 2006; Zhang et al. 2006b; d’Aquino et al. 2007). These dental stem cells have been shown

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Endodontic Infections in Incompletely Developed Teeth 333

that they can be utilized for dental tissue regenera- tion (i.e., pulp/dentin and periodontal ligament). Com- plete pulp regeneration using DPSC or SCAP (Huang et al. 2010; Iohara et al. 2011; Nakashima and Huang 2013). PDLSC have been shown to regenerate peri- odontal defects and periodontal ligament in large ani- mal models (Liu et al. 2008; Zhao et al. 2013). More importantly, the identification of these dental stem cells provide a better understanding of the biology of pulp and periodontal ligament tissues and their regenerative potential after tissue damage.

14.8 Recent innovations on the regeneration of tooth form

Dental implants have been a favorable option over bridges or removable dentures to restore lost teeth and functions. Recent isolation of various dental stem cells shed light on the possibility of regeneration of tooth structure. Cells isolated from tooth bud can form ectopic teeth in vivo when seeded onto scaf- folds (Young et al. 2002, 2005; Duailibi et al. 2004; Komine et al. 2007; Nakao et al. 2007). A few reports have demonstrated the orthotopic regeneration of engi- neered teeth. Single cells from the dog tooth buds at the bell stage were isolated and directly seeded onto scaffolds and transplanted back to the original tooth sockets. Dentin structure regeneration was achieved, but not enamel, or root formation (Honda et al. 2006). With a swine model, Chen’s research team utilized ex vivo expanded tooth bud cells (from bell stage), cul- tured onto cylinder-shaped scaffolds and autografted back to the original alveolar sockets. They were able to observe tooth formation with root structures along with periodontium (Kuo et al. 2007). More recently, bio- engineered functional whole tooth erupted to occlusion can be regenerated in a mouse model (Ikeda et al. 2009; Tsuji 2013). The other approach is to use SCAP or DPSC and PDLSC to form a bio-root (Sonoyama et al. 2006; Wei et al. 2013). Using a minipig model, autol- ogous SACP and PDLSC from minipigs were loaded onto hydroxyapatite/tricalcium phosphate (HA/TCP) and gelfoam scaffolds, respectively, and implanted into sockets of the lower jaw. A post channel was precreated to leave space for post insertion. Three months later, the bio-root was exposed and a porcelain crown was inserted (Figure 14.12). This approach is a relatively fast way of creating a root to which an artificial crown can be installed. The bio-root is different from a natural

root in that the root structure is deposited by SCAP in a random manner. Nevertheless, the bio-root is encircled with periodontal ligament tissue and appears to have a natural relationship with the surrounding bone. What remains to be improved is the mechanical strength of the bio-root which is approximately two-thirds that of a natural tooth.

14.9 Conclusions and prospects

Although the healing potential and defense mecha- nisms of pulp have been long recognized, the intensity and the nature of the virulence of infection are still the determining factors for the outcome of pulp recov- ery. Immature teeth, by having a large and young pulp tissue and an open apex to allow good blood supply, show remarkable healing potential in conditions that would not be possible for mature teeth. The discovery and understanding of pulp stem cells provide us with a better insight into the healing potential of immature teeth. Currently, there are ongoing large-scale clinical studies on the success rate of treating immature teeth with periradicular bone resorption using revitalization procedures. The time lapse between pulp infection and endodontic intervention in relation to clinical success is a critical issue. Presumably, the longer duration of an infected pulp in immature teeth, the fewer pulp tis- sue and stem cells survive. Additionally, the longer the infection, the more likelihood of a deeper penetration of microbial colonies into dentinal tubules, which ren- ders the disinfection process more difficult. At present, revitalization procedures have not provided convinc- ing evidence that pulp and dentin can be regenerated in the canal space, whereas introducing exogenous dental stem cells has shown promise that pulp can be fully regenerated along with newly deposited dentin on the canal dentinal walls.

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Chapter 15 Prognosis of Healing in Treated Teeth with Endodontic Infections Shimon Friedman

15.1 Introduction: The critical importance of prognosis

15.2 Outcome measures and criteria in assessment of endodontic prognosis 15.2.1 Clinical outcome measures 15.2.2 Radiographic outcome

measures 15.2.3 Outcome criteria

15.3 Levels of evidence in assessment of endodontic prognosis 15.3.1 Study designs 15.3.2 Methodologic rigor 15.3.3 “Current best evidence” for the

prognosis of endodontic treatment

15.4 Prognosis of primary apical periodontitis after initial treatment 15.4.1 Potential for healing 15.4.2 Time-course of healing 15.4.3 Prognostic variables

15.5 Prognosis of posttreatment apical periodontitis after orthograde retreatment

15.5.1 Potential for healing 15.5.2 Time-course of healing 15.5.3 Prognostic variables

15.6 Prognosis of posttreatment apical periodontitis after apical surgery 15.6.1 Potential for healing 15.6.2 Time-course of healing 15.6.3 Prognostic variables

15.7 Prognosis of posttreatment apical periodontitis after intentional replantation 15.7.1 Dynamics of external root

resorption 15.8 Etiology of persistent apical

periodontitis after endodontic treatment 15.8.1 Persistent infection after

nonsurgical treatment 15.8.2 Persistent infection after apical

surgery 15.9 Conclusions

15.10 References

15.1 Introduction: The critical importance of prognosis

The term prognosis in the context of health care is defined as the forecast of the course of a disease. In the context of endodontic infection, the associated dis- ease is apical periodontitis, and prognosis means the

chance of the affected tissues to heal after treatment of the offending tooth. In common language, however, the term prognosis of treatment is often used as syn- onym for the expected positive outcome.

Prognosis is a critically important element in clinical decision-making, particularly when different alterna- tives are available for treatment of a given disease. The

341

342 Endodontic Microbiology

current ethical concepts of health care delivery require that clinical decision-making be based on sound evi- dence and involve the patient. Evidence to support the benefits and risks of available treatment alternatives is shared with the patient, who then selects a specific treatment based on his/her individual values, priori- ties, and resources (Pellegrino 1994; Ambrosio and Walkerley 1996; Wertz 1998; Schattner and Tal 2002; Fournier 2005). To conform to this concept, health care providers must be well versed in the evidence that supports the prognosis of alternative treatments they suggest to patients. Clinical decision-making in endodontics is no exception to this prevailing concept; therefore, dentists and endodontic specialists should be well informed about the prognosis of different endodontic treatment modalities.

Information about prognosis of endodontic treat- ment is available from over 260 studies on nonsurgical and surgical treatment. While all this evidence appears abundant, it has been highly inconsistent because of methodologic and technical variations, precluding indiscriminate review of the many available studies. Supported by inconsistent evidence, answers to the main questions related to the prognosis of endodontic treatment modalities have remained equivocal (Fried- man 1998). Specifically, careful review is required of the prognosis in teeth with endodontic infections, known to be compromised relative to teeth without infections (Friedman 1998).

It is well established that questions about the prog- nosis following state-of-the-art treatment should be addressed in structured reviews that focus on studies selected according to well-defined criteria. Accord- ingly, this chapter aims to define the prognosis of nonsurgical and surgical endodontic treatment of teeth with preoperative lesions indicative of active infective process, and to identify prognostic variables, based on selected studies.

15.2 Outcome measures and criteria in assessment of endodontic prognosis

Much of the confusion regarding the prognosis of endodontic treatment is caused by inconsistent use of outcome criteria, resulting in highly variable “success” rates among the different studies (Friedman 1998). This inconsistency has affected current studies on initial root canal treatment (Table 15.1), orthograde retreatment (Table 15.2), apical surgery (Table

15.3), and intentional replantation (Table 15.4). The inconsistencies result from use of ambiguous or older terms such as “success” and “failure,” and from lack of calibration in outcome assessment. This section of the chapter focuses on outcome measures, assessment strategies, and criteria for assessment of prognosis after nonsurgical and surgical endodontic treatment.

15.2.1 Clinical outcome measures

Clinical outcome measures have been widely used to assess the health state of endodontically treated teeth. Patients’ reporting on presence or absence of pain (sub- jective measure) and clinical recording of presence or absence of swelling, sinus tract, and tenderness to percussion and palpation (objective measures) have been commonly used. Inasmuch as any one of these clinical outcome measures can be an expression of persistent endodontic infection, they are not specific signs of apical periodontitis; therefore, they have been coupled with radiographic measures in most studies. Nevertheless, in many studies on nonsurgical treat- ment (Ørstavik et al. 1987; Molven and Halse 1988; Murphy et al. 1991; Ørstavik and Hörsted-Bindslev 1993; Smith et al. 1993; Ørstavik 1996; Trope et al. 1999; Heling et al. 2001; Pettiette et al. 2001; Waltimo 2001; Cheung 2002; Huumonen et al. 2003; Peters et al. 2004; Marending et al. 2005), and in at least one apical surgery study (Rapp et al. 1991), only the radiographic appearance was used to assess the out- come without use of clinical outcome measures. In this manner, the “success” rate can be overestimated by inclusion of teeth that appear radiographically nor- mal but that are symptomatic (Friedman 2002b).

15.2.2 Radiographic outcome measures

Assessment of radiographs is subject to bias (Goldman et al. 1972, 1974; Reit and Hollender 1983; Zakariasen et al. 1984; Eckerbom et al. 1986). Calibration and specific observer strategies have been advocated for endodontic studies (Rud et al. 1972a; Reit 1987b; Molven et al. 2002a), to improve the consistency of assessment. A frequently used strategy uses the Periapical Index (PAI) (Ørstavik et al. 1986) for cal- ibration purposes and as reference for assessment of radiographs. Assessed radiographs are compared with five sets of radiographic images and their schematic representations (see Chapter 2). These images are derived from a histologic–radiographic correlation

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an d

Pe te

rs 1 9 9 0

5 0

0 .3

–6 .5

n y

n n

– 9 6

– Sj

ö gr

en et

al . 1 9 9 0

2 0 4

a 8 –1

0 y

y y

y 8 6

– –

M u rp

hy et

al . 1 9 9 1

8 9

0 .3

–2 n

n n

n 4 6

4 8

9 4

Sm it h

et al

. 1 9 9 3

4 8 1

2 –5

n n

n y

– 8 1

– Fr

ie d m

an et

al . 1 9 9 5

1 1 3

0 .5

–1 .5

y y

n n

6 9

2 5

9 4

Ç al

iş ka

n an

d Se

n 1 9 9 6

1 7 2

2 –5

y y

n n

8 1

8 8 9

Ø rs

ta vi

k 1 9 9 6

1 2 6

a, b

4 n

y y

y 7

5 1

3 ≥

8 8

Sj ö gr

en et

al . 1 9 9 7

5 3

≤ 5

y y

8 3

– –

Tr o p e

et al

. 1 9 9 9

7 6

1 n

y n

y 8 0

d –

– W

ei ge

r et

al . 2 0 0 0

6 7

1 –5

y y

n y

7 8

1 6

9 4

W al

ti m

o et

al . 2 0 0 1

– 1 –4

n y

y n

5 0

– –

C h u ga

l et

al . 2 0 0 1

1 7 7

a 4

n n

y y

6 3

– –

Pe ak

et al

. 2 0 0 1

2 8 0

≤ 1

n n

– 8

7 –

Pe tt

ie tt

e et

al .

2 0

0 1

4 0

1 n

y n

n 6

0 –

– H

el in

g et

al . 2 0 0 1

3 1 9

1 –1

2 n

y y

n –

6 5

– Pe

te rs

an d

W es

se li n k

2 0 0 2

3 8

1 –4

.5 n

y y

y 7 6

2 1

9 7

H o sk

in so

n et

al . 2 0 0 2

3 3 5

a 4 –5

n y

7 4

– –

B en

en at

i an

d K

h aj

o ti a

2 0 0 2

8 9 4

0 .5

–7 n

n n

n 6 2

2 9

≥ 9 1

Fr ie

d m

an et

al . 2 0 0 3

7 2

4 –6

y n

y y

7 4

1 1

≥ 8 5

H u u m

o n en

et al

. 2 0 0 3

1 5 6

1 y

y n

n 7 6

2 –

Fo u ad

an d

B u rl

es o n

2 0 0 3

1 7 8

≥ 2

n n

n y

6 5

– –

Ta n i-

Is h ii

an d

Te ra

n ak

a 2 0 0 3

9 4

1 n

y y

n –

– ≥

9 6

Pe te

rs et

al . 2 0 0 4

1 0 2

1 –3

y y

n y

7 6

– –

Fa rz

an eh

et al

. 2 0 0 4 b

2 4 2

b 4 –6

y n

y y

7 9

7 ≥

8 6

Ø rs

ta vi

k et

al . 2 0 0 4

1 2 1

a, b

3 n

y y

y 7

9 –

– Ç

al iş

ka n

2 0 0 4

4 2

2 –1

0 n

y y

n 7 4

1 0

≥ 8 4

C h u

et al

. 2 0 0 5

5 8

3 –4

n y

7 9

– 8 5

M ar

q u is

et al

. 2 0 0 6

2 0 9

b 4 –6

y n

y y

8 0

6 ≥

8 6

A q ra

b aw

i 2 0 0 6

1 7 7

5 n

y y

n 7 9

– –

Su ch

in a

et al

. 2 0 0 6

2 2

e 0

.5 –5

.5 n

y n

n 5

5 3

6 ≥

9 1

C o n n er

et al

. 2 0 0 7

2 9

≥ 1

n n

5 0

2 3

≥ 7 3

M o la

n d er

et al

. 2 0 0 7

1 0 1

2 y

y y

n 7 0

2 0

9 6

Im u

ra et

al .

2 0

0 7

2 9

0 ≥

1 .5

n y

9 1

– –

Z m

en er

an d

Pa m

ei je

r 2 0 0 7

4 8

5 n

y n

n 9 2

– –

(c o n ti n u ed

)

Ta b

le 1

5 .1

(C o n ti n u ed

)

A p p ra

is al

ca te

go ri

es O

u tc

o m

e (%

)

St u dy

C as

es o b se

rv ed

Fo ll o w

-u p

(y ea

rs )

C o h o rt

Ex p o su

re A

ss es

sm en

t A

n al

ys is

H ea

le d

H ea

li n g

Fu n ct

io n al

∗

d e

C h ev

ig n y

et al

. 2 0 0 8 a

2 9 2

b 4 -6

y y

8 2

8 9 5

Pe n

es is

et al

. 2

0 0

8 6

3 1

y y

n n

6 5

2 9

≥ 9 4

Si q u ie

ra et

al 2 0 0 8

1 0 0

1 –4

n y

n n

7 6

1 9

≥ 9 5

C o

tt o

n et

al .

2 0

0 8

5 0

1 –2

n y

y y

7 6

– –

C h eu

n g

an d

Li u

2 0 0 9

8 7

– n

y n

y 6 4

– –

Te rv

it et

al .

2 0 0 9

1 9

2 –4

n y

y y

8 4

– –

Ö ze

r an

d A

kt en

er 2 0 0 9

8 0

3 y

n n

n 8 3

– 9 4

H si

ao et

al . 2 0 0 9

5 9

≥ 0

.6 n

n n

n 8

1 –

– M

et zg

er et

al . 2 0 0 9

3 1

e 0

.5 y

y n

n 7

3 2

– Z

m en

er an

d Pa

m ei

je r

2 0 1 0

4 6

b 8

n y

n n

9 3

– 1 0 0

Se tz

er et

al . 2 0 1 0

1 8

4 –6

+ n

n y

y 2 8

5 0

≥ 7 8

N g

et al

. 2 0 1 1

6 1 4

a ≥

2 y

y y

y 7

6 –

– R

ic u cc

i et

al .

2 0 1 1

2 8 5

5 y

y y

y 8 3

– –

Pa te

l et

al .

2 0

1 2

a 4

8 1

y y

n n

6 9

g 9 0

1 0 0

Li an

g et

al . 2 0 1 2

1 2 5

a 2

n n

5 8

– 9

6 Pa

re d es

-V ie

yr a

an d

En ri

q u ez

2 0 1 2

2 8 2

2 y

y n

n 9 3

5 ≥

9 8

Z m

en er

an d

Pa m

ei je

r 2 0 1 2

4 6

b 1 0

n y

n n

– 9 2

1 0 0

Sa in

i et

al . 2 0 1 2

1 2 9

1 n

y n

y 7 5

– –

B er

n st

ei n

et al

. 2 0 1 2

5 1 1

3 –5

n n

n y

7 2

– –

H al

e et

al . 2 0 1 2

1 3

1 .5

–3 n

y n

y –

6 9

– So

u za

et al

2 0 1 2

4 3

2 n

y n

n 9 0

– –

va n

d er

B o rd

en ,

et al

. 2 0 1 3

7 1

a 1 –3

n y

4 5

g 4 3

≥ 8 8

M ar

ti n s

et al

. 2 0 1 3

1 2

f 0

.5 y

y n

n 6

7 1

6 ≥

8 3

Li an

g et

al . 2 0 1 3

8 4

1 –1

.5 n

y n

y 3 2

g 6 2

≥ 9 4

C as

te lo

t- En

ke l

et al

. 2 0 1 3

6 5

3 .5

n n

y y

8 8

– –

M ar

ti n s

et al

. 2 0 1 4

3 0

1 y

y n

n 9 3

7 1 0 0

P ir

an i et

al . 2 0 1 5

6 6

1 0 –1

9 n

y n

y 6 8

– –

C ra

ve ir

o et

al . 2 0 1 5

2 1 2

– n

y n

y 9 3

– –

A zi

m et

al .

2 0 1 6

1 7 2

a 0 .5

–8 n

n n

y 5 3

1 1

– D

em ir

ci an

d Ç

al iş

ka n

2 0 1 6

1 1 2

2 y

y n

n 9 7

– ≥

9 7

∗ A

sy m

p to

m at

ic ,

w it h o u t o r

w it h

re si

d u al

ra d io

lu ce

n cy

(≥ n o t re

p o rt

ed ; ra

te is

su m

o f h ea

le d

an d

h ea

li n g)

. a R

o o ts

co n si

d er

ed as

u n it

o f ev

al u at

io n ,

ra th

er th

an te

et h .

b In

cl u d es

re p ea

te d

m at

er ia

l. c A

ll ca

n al

s o b li te

ra te

d to

so m

e ex

te n t.

d Te

et h

tr ea

te d

in tw

o se

ss io

n s

w it h o u t in

tr ac

an al

m ed

ic at

io n

ex cl

u d ed

. e H

IV -p

o si

ti ve

p at

ie n ts

. f C

o n ve

n ti o n al

tr ea

tm en

t gr

o u p .

g A

ss es

sm en

t w

it h

p er

ia p ic

al ra

d io

gr ap

h s.

n , D

o es

n o t sa

ti sf

y cr

it er

ia o f ac

ce p ta

b le

q u al

it y.

y, Sa

ti sfi

es cr

it er

ia o f ac

ce p ta

b le

q u al

it y.

Ta b

le 1

5 .2

Fo ll o w

-u p

st u d ie

s p u b li sh

ed si

n ce

1 9 8 8 ,

re p o rt

in g

sp ec

ifi c

d at

a o n

th e

o u tc

o m

e o f o rt

h o gr

ad e

re tr

ea tm

en t

in te

et h

w it h

en d o d o n ti c

in fe

ct io

n . H

ig h li gh

te d

st u d ie

s h av

e b ee

n se

le ct

ed as

“c u rr

en t

b es

t ev

id en

ce ”

A p p ra

is al

ca te

go ri

es O

u tc

o m

e (%

)

St u dy

Ex am

in ed

sa m

p le

Fo ll o w

-u p

(y ea

rs )

C o h o rt

Ex p o su

re A

ss es

sm en

t A

n al

ys is

H ea

le d

H ea

li n g

Fu n ct

io n al

∗

M o lv

en an

d H

al se

1 9 8 8

9 8

a 1 0 –1

7 n

n y

y 7 1

b –

– A

ll en

et al

. 1

9 8

9 3

1 5

c ≥

0 .5

n n

7 3

1 2

≥ 8 5

Sj ö gr

en et

al . 1 9 9 0

9 4

a 8 –1

0 y

y y

y 6 2

– –

va n

N ie

u w

en h u ys

en et

al . 1 9 9 4

5 6 1

c ≥

0 .5

n n

n y

7 8

– –

Fr ie

d m

an et

al . 1 9 9 5

8 6

0 .5

–1 .5

y y

n n

5 6

3 4

≥ 9 0

D an

in et

al 1 9 9 6

1 8

1 n

y n

n 2 8

2 8

≥ 5 6

Su n d q vi

st et

al .

1 9 9 8

5 4

4 n

y y

y 7 4

– –

P ia

to w

sk a

et al

. 1

9 9

7 6

0 c

– n

y n

n 4

3 4

2 ≥

8 5

A b b o tt

1 9 9 9

4 3

c 0 .3

–4 n

n n

n 9 8

1 –

K vi

st an

d R

ei t 1 9 9 9

4 7

4 y

y y

n 5 8

d -

– C

h u ga

l et

al . 2 0 0 1

8 5

a ,c

4 n

n y

y 7

9 –

– H

o sk

in so

n et

al .

2 0

0 2

g 7 6

c 4 –5

n y

7 8

– –

Fa rz

an eh

et al

. 2 0 0 4 a

6 9

4 –6

y n

y y

8 1

5 ≥

8 6

G o rn

i an

d G

ag li an

i 2 0 0 4

4 5 2

c 2

y n

n n

6 5

4 –

Fr is

ta d

et al

. 2

0 0

4 1

1 2

a ,c

,e 2 0 –2

7 n

n y

y 9 6

– –

Im u

ra et

al .

2 0

0 7

4 0

4 1

.5 –5

n y

8 1

– –

Er ca

n et

al . 2 0 0 7

6 4

1 n

y n

n 6 4

1 4

≥ 7 8

d e

C h ev

ig n y

et al

. 2 0 0 8 b

1 4 7

4 -6

y n

y y

8 0

6 9 3

H si

ao et

al . 2 0 0 9

1 3

≥ 0

.6 n

n n

n 7

0 –

– N

g et

al . 2 0 1 1

7 6 3

a ≥

2 y

y y

y 7

4 –

– R

ic u cc

i et

al . 2 0 1 1

7 1

5 y

y y

y 8 3

– –

To u b o u l

et al

. 2 0 1 4

1 0 8

2 -4

n y

8 0

1 1

– A

zi m

et al

. 2 0 1 6

4 1

a 0 .5

–8 n

n n

y 6 8

5 –

∗ A

sy m

p to

m at

ic ,

w it h o u t o r

w it h

re si

d u al

ra d io

lu ce

n cy

(≥ n o t re

p o rt

ed ;

ra te

is su

m o f h ea

le d

an d

h ea

li n g)

. a R

o o ts

co n si

d er

ed as

u n it

o f ev

al u at

io n ,

ra th

er th

an te

et h .

b C

as es

cl as

si fi ed

as “u

n ce

rt ai

n ”

ex cl

u d ed

. c M

ay in

cl u d e

u n sp

ec ifi

ed n u m

b er

o f te

et h

w it h o u t in

fe ct

io n .

d A

p p ro

xi m

at e

fi gu

re d ed

u ct

ed fr

o m

gr ap

h .

e In

cl u d es

re p ea

te d

m at

er ia

l. n , D

o es

n o t sa

ti sf

y cr

it er

ia o f ac

ce p ta

b le

q u al

it y.

y, Sa

ti sfi

es cr

it er

ia o f ac

ce p ta

b le

q u al

it y.

Ta b

le 1

5 .3

Fo ll o w

-u p

st u d ie

s p u b li sh

ed si

n ce

1 9 9 5 ,

re p o rt

in g

sp ec

ifi c

d at

a o n

th e

o u tc

o m

e o f ap

ic al

su rg

er y

in te

et h

w it h

en d o d o n ti c

in fe

ct io

n .

H ig

h li gh

te d

st u d ie

s h av

e b ee

n se

le ct

ed as

“c u rr

en t

b es

t ev

id en

ce ”

A p p ra

is al

ca te

go ri

es O

u tc

o m

e (%

)

St u dy

C as

es o b se

rv ed

Fo ll

o w

-u p

(y ea

rs )

C o h o rt

Ex p o su

re A

ss es

sm en

t A

n al

ys is

H ea

le d

H ea

li n g

Fu n ct

io n al

∗

Je ss

le n

et al

. 1 9 9 5

9 3

5 y

y n

n 5 9

2 8

≥ 8 7

A u gu

st 1 9 9 6

3 9

1 0 –2

3 n

n n

n 7 4

1 5

≥ 8 9

D an

in et

al .

1 9

9 6

1 9

1 n

y y

n 5

8 2

6 ≥

8 4

R u d

et al

.1 9 9 6

3 5 1

a 0

.5 –1

.5 n

y y

n 8

2 1

2 ≥

9 4

Su m

i et

al . 1 9 9 6

1 5 7

0 .5

–3 y

y n

n –

– 9 2

Ja n

ss o

n et

al .

1 9

9 7

6 2

0 .9

–1 .3

y y

n n

3 1

5 5

≥ 8 6

R u

d et

al .

1 9

9 7

b 5 5 1

a 0

.5 –1

.5 n

y y

n 7

9 1

6 ≥

9 5

B ad

er an

d Le

je u n e

1 9 9 8

2 5 4

1 n

y n

n –

– 8 1

D an

in et

al .

1 9 9 9

1 0

1 n

y y

n 5 0

5 0

1 0 0

K vi

st an

d R

ei t 1 9 9 9

4 5

4 y

y n

y 6 0

– –

R u

b in

st ei

n an

d K

im 1

9 9

4 1

.2 n

y n

n 9

7 –

– Te

st o ri

et al

. 1 9 9 9

1 3 4

1 -6

n y

n n

7 8

9 ≥

8 7

vo n

A rx

an d

K u rt

1 9 9 9

4 3

1 y

y n

n 8 2

1 4

≥ 9 6

Z u o lo

et al

. 2 0 0 0

1 0 2

1 –4

y y

y n

9 1

– 9 2

R ah

b ar

an et

al . 2 0 0 1

1 2 9

c ≥

4 y

y y

y 3

7 3 3

≥ 8 0

R u d

et al

2 0 0 1

8 3 4

a 0 .5

–1 2 .5

n y

y n

9 2

1 ≥

9 3

vo n

A rx

et al

. 2 0 0 1

2 5

1 y

y n

n 8 8

8 ≥

9 6

R u

b in

st ei

n an

d K

im 2

0 0

2 d

5 9

5 –7

y y

n n

9 2

– –

Je n se

n et

al . 2 0 0 2

6 0

f 1

y y

n y

7 3

1 7

≥ 9 0

C h o n g

et al

. 2 0 0 3

1 0 8

2 n

y y

y 9 0

6 ≥

9 6

W es

so n

an d

G al

e 2 0 0 3

7 9 0

5 y

y n

n 5 7

5 ≥

6 2

M ad

d al

o n e

an d

G ag

li an

i 2 0 0 3

1 2 0

0 .3

–3 n

y y

n 9 3

3 ≥

9 6

Sc h w

ar tz

-A ra

d et

al . 2 0 0 3

2 6 2

0 .5

–0 .9

n y

4 4

2 1

≥ 6 5

W an

g et

al . 2 0 0 4

9 4

4 -8

y y

7 4

– 9 1

G ag

li an

i et

al .

2 0 0 5

2 3 1

a 5

y y

n y

7 8

1 0

8 9

Li n d eb

o o m

et al

. 2 0 0 5

1 0 0

1 y

y n

y 8 9

1 0

9 9

Ta sc

h ie

ri et

al . 2 0 0 5

2 8

1 n

y n

n –

9 3

≥ 9 3

T se

si s

et al

. 2 0 0 6

(m o d er

n te

ch n iq

u e)

4 5

e 0

.5 –1

.5 y

y n

n 9

1 4

≥ 9 5

Ta sc

h ie

ri et

al .

2 0

0 6

af 7 1

1 y

y n

n 9 3

– –

M ar

in -B

o te

ro 2 0 0 6

3 0

k 1

n y

n n

5 0

3 7

– vo

n A

rx et

al . 2 0 0 7

1 9 1

1 y

y n

y 8 4

1 0

≥ 9 4

Ya zd

i et

al .

2 0 0 7

g 6 0

R p

6 .5

–9 y

y n

y 7 8

1 7

≥ 9 0

Ta sc

h ie

ri et

al .

2 0

0 7

ak 2 8

1 n

y n

n –

9 3

– W

al iv

aa ra

et al

. 2 0 0 7

5 5

1 y

y n

n 8 0

– 9 1

Pe ñ ar

ro ch

a et

al . 2 0 0 7

f 3 3 3

1 y

y n

n 7 4

1 5

≥ 8 9

Ta sc

h ie

ri et

al .

2 0

0 8

k 1 0 0

2 n

y n

n –

9 1

– G

ar ci

a et

al . 2 0 0 8

1 0 6

1 y

n n

n 7 5

1 0

– Sa

u n d er

s 2 0 0 8

2 7 6

0 .3

–6 n

y n

n 5 9

3 0

9 2

Jo n as

so n

et al

. 2 0 0 8

2 1

h 0

.5 –4

n y

n n

/a 8

1 1

4 9

5 K

im et

al . 2 0 0 8

1 9 2

1 –5

n y

n n

9 1

– –

O rt

eg a-

Sá n ch

ez et

al . 2 0 0 9

3 7

0 .5

–1 .5

n n

7 5

1 0

8 8

C h ri

st ia

n se

n et

al . 2 0 0 9

2 5

i 1

n y

n n

9 7

0 ≥

9 7

W äl

iv aa

ra et

al . 2 0 0 9

1 4 7

1 –3

y y

n n

8 7

– –

B ar

o n e

et al

. 2 0 1 0

1 3 4

p 4 –1

0 y

y y

y 7 4

1 0

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Prognosis of Healing in Treated Teeth with Endodontic Infections 349

Table 15.4 Follow-up studies since 1987, reporting specific data on the outcome of intentional replantation in teeth with endodontic infection

Treatment outcome (%)

Study Cases observed Follow-up (years) “Success” Root resorption Persistent infection

Tegsjö et al.a 56 4 86 14 – Warfvinge and

Kahnberg 1989b

26 2 75 0 –

Koenig et al. 1988

177 0.5–4 82 5 11

Kahnberg 1988b 58 2–7 71 0 29 Keller 1990 34 3 91 0 9 Bender and

Rossman 1993

31 0–22 81 6 10

Raghoebar and Vissink 1999

29 1–11 72 14 –

Kahnberg 1996b 21 10 90 0 Çalişkan et al.

1998b 34 0.5–3 95 0

Choi et al. 2014 287 0.5–4.5 90 3 – Cho et al. 2016 159 0.5–12 95 3 5

aTeeth rotated 180◦. bCoronally repositioned teeth.

study (Brynolf 1967). They represent a healthy peri- apex (score 1), minor changes perceived as consistent with a healthy periapex (score 2), and increasing extent and severity of apical periodontitis (scores 3–5). Radiographs are assigned a score according to which of the reference images they match best. Such “blinded” and reference-based assessment reduces bias and improves the sensitivity of assessment, compared with the common assessment of success or failure (Ørstavik et al. 1986, 2004). The PAI has been used mainly in studies on nonsurgical treatment (Ørstavik and Hörsted-Bindslev 1993; Ørstavik 1996; Trope et al. 1999; Waltimo et al. 2001, 2005; Friedman et al. 2003; Huumonen et al. 2003; Farzaneh et al. 2004a,b; Ørstavik et al. 2004; Peters et al. 2004; Marending et al. 2005; Aqrabawi 2006; Marquis et al. 2006; Cotton et al. 2008; de Chevigny et al. 2008a,b; Penesis et al. 2008; Mente et al. 2009; Suter et al. 2009; Tervit et al. 2009; Saini et al. 2012; Cheung et al. 2013; Martins et al. 2013), but also apical surgery studies (Wang et al. 2004; Barone et al. 2010) to minimize bias and to facilitate comparisons with nonsurgical treatment studies from the same group (Friedman et al. 2003; Farzaneh et al. 2004a,b; Marquis et al. 2006; de Chevigny et al. 2008a,b). Although the PAI was

originally designed to observe changes in mean scores as the main outcome, they can be used to dichotomize outcomes as health (scores 1 and 2) and disease (scores 3, 4, and 5) (Trope et al. 1999; Waltimo et al. 2001, 2005; Boucher et al. 2002; Dugas et al. 2003; Friedman et al. 2003; Huumonen et al. 2003; Farzaneh et al. 2004a,b; Peters et al. 2004; de Chevigny et al. 2008a,b; Kirkevang 2006; Marquis et al. 2006).

In recent years, increasing use of cone-beam com- puterized tomography (CBCT) imaging in endodontics has drawn attention to possible use of this novel tech- nology for outcome assessment (Velvart et al. 2001; Lofthag-Hansen et al. 2007; Estrela et al. 2008; Low et al. 2008; Christiansen et al. 2009; Patel 2009; Wu et al. 2009; Liang et al. 2011, 2012, 2013; Patel et al. 2012a,b; Cheung et al. 2013; Esposito et al. 2013; Fernandez et al. 2013; van der Borden et al. 2013; Pope et al. 2014; Tanomaru et al. 2015; von Arx et al. 2015, 2016; Zhang et al. 2015). While interpre- tation of CBCT images allows for excellent repeata- bility and reproducibility (Estrela et al. 2008; von Arx et al. 2016), suggesting it is less prone to bias than two-dimensional periapical radiographs, use of CBCT imaging may result in overdiagnosis and lower inter- vention thresholds in the absence of clear definition of

350 Endodontic Microbiology

the appearance limits of normal, healthy tissues (Pope et al. 2014). In studies where both periapical radio- graphs and CBCT imaging were used to detect periapi- cal lesions associated with apical periodontitis, lesion detection with CBCT was consistently more sensitive and more specific and the proportion of healthy teeth confirmed by CBCT was about 10–30% lower than that observed in periapical radiographs (Velvart et al. 2001; Lofthag-Hansen et al. 2007; Estrela et al. 2008; Low et al. 2008; Christiansen et al. 2009; Patel 2009; Wu et al. 2009; Liang et al. 2011, 2012, 2013; Patel et al. 2012a,b; Cheung et al. 2013; Esposito et al. 2013; Fernandez et al. 2013; van der Borden et al. 2013; Pope et al. 2014; Tanomaru et al. 2015; von Arx et al. 2015, 2016; Zhang et al. 2015). However, it should be noted that, as yet, CBCT is not the gold standard radiographic outcome measure for periapical health; it is not routinely used preoperatively and, therefore, its should not be routinely applied for posttreatment outcome assessment.

15.2.3 Outcome criteria

Criteria and terminology used for outcome assessment in endodontic studies have varied, mainly in the use of “strict” and “lenient” classifications of “success” (Ng et al. 2007, 2008). Whereas in the majority of the studies “success” or “complete healing” is defined as

complete radiographic and clinical normalcy, in many current studies “success” is defined primarily as clin- ical normalcy that may be accompanied by a resid- ual radiolucency, which is either decreased in size or unchanged. The difference in “success” between these two sets of criteria can be approximately 15% (Fried- man et al. 1995; Wang et al. 2004; Ng et al. 2007, 2008, 2011). Adding to the confusion, outcome cate- gories of “uncertain,” “doubtful,” “questionable,” and “improved” have been used inconsistently, to imply uncertainty of the outcome, improved outcomes after nonsurgical treatment and apical surgery, and even nonimproved outcomes after apical surgery. These inconsistencies result for the major part from use of the ambiguous and value-laden terms “success” and “fail- ure” (Ørstavik 1996); therefore, these terms should be replaced with neutral expressions to facilitate commu- nication with patients. The terms used should prefer- ably relate to the specific goals of treatment.

In teeth with endodontic infection, the primary goal of treatment is to heal the tissues affected by api- cal periodontitis (Ørstavik and Pitt Ford 1998). Thus, the primary outcome of treatment should be related to healing (Rud et al. 1972a; Byström et al. 1987; Ørstavik 1996; Friedman 2002b, 2005; Friedman and Mor 2004). The term healed is used for complete clin- ical and radiographic normalcy (no signs, symptoms, residual radiolucency) (Figures 15.1, 15.2, and 15.3).

(a) (b) (c)

Fig. 15.1 Primary infection healed after initial treatment. (a) Maxillary second molar with apical periodontitis extending along the mesial root surface, and associated sinus tract (traced with a gutta-percha cone). (b) Completed treatment. (c) At 8 years, radiographic and clinical normalcy suggest that the tooth has healed. Source: Friedman (2002). Reproduced with permission of Blackwell Munksgaard.

Prognosis of Healing in Treated Teeth with Endodontic Infections 351

(a) (b) (c)

Fig. 15.2 Persistent infection healed after orthograde retreatment. (a) Maxillary first premolar with posttreatment apical periodontitis restored with a cast post and crown. (b) Completed retreatment with the original crown re-cemented in place. (c) At 4 years, radiographic and clinical normalcy suggest that the tooth has healed. Source: Friedman (2002). Reproduced with permission of John Wiley and Sons.

(a) (b) (c) (d) (e)

Fig. 15.3 Persistent infection healed after apical surgery. (a) Maxillary canine with a large excess of sealer and persistent infection. (b) Completed surgery, including root-end filling with MTA. (c) At 3 months, some bone deposition is suggested, but the lesion is not reduced. (d) At 6 months, the tooth is symptom free and the lesion appears to be healing. (e) At 1 year and 8 months, radiographic and clinical normalcy suggest that the tooth has healed. Source: Friedman (2005). Reproduced with permission of John Wiley and Sons.

352 Endodontic Microbiology

(a) (b) (c)

Fig. 15.4 Persistent infection healed by scar formation (incomplete healing) after apical surgery. (a) Maxillary lateral incisor with a root filling extruded beyond the root end, and persistent apical periodontitis. (b) Completed surgery, including root-end filling with Super-EBA. (c) At 1 year, radiographic and clinical normalcy suggest that the tooth has healed with a small scar formed several millimeters from the root end. Courtesy of Dr. Richard Rubinstein. Source: Friedman et al. (2005). Reproduced with permission of John Wiley and Sons.

This category includes the typical appearance of a scar after apical surgery (Andreasen and Rud 1972a,b; Rud et al. 1972a; Molven et al. 1987, 1996) (Figure 15.4). The term healing is used for decreased radiolucency and clinical normalcy after a follow-up period shorter than 4 years (Figure 15.3d). The term nonhealed or persistent apical periodontitis is used for persistent radiolucency regardless of clinical presentation (Fig- ure 15.5), or persistent symptoms. The secondary goal of treatment is to retain the tooth in a symptom-free function. Thus, the respective outcome of treatment should be related to retention of the tooth, and the term functional retention used for clinical normalcy even in presence of persistent radiolucency. Importantly, indi- vidual patients may define elimination of symptoms as their specific treatment goal, particularly when clinical conditions suggest a poor prognosis for healing.

15.3 Levels of evidence in assessment of endodontic prognosis

Reports on prognosis are frequently inconsistent in methodology and in the level of evidence they provide

(Sackett et al. 1991). Consequently, structured analysis of the literature is necessary to differentiate clinical studies according to the level of evidence, and to gather valid evidence from selected studies.

15.3.1 Study designs

Design categories of clinical studies are defined by the Cochrane Collaboration (http://www.cochrane.org/ glossary/5 - letterc) as follows:

� Clinical trial: “An experiment to compare the effects of two or more healthcare interventions. Clinical trial is an umbrella term for a variety of designs of health- care trials, including uncontrolled trials, controlled trials, and randomised controlled trials.” Among the latter, N-of-1 randomized trial is “A randomized trial in an individual to determine the optimum treat- ment for that individual. The individual is given repeated administrations of experimental and con- trol interventions (or of two or more experimental treatments), with the order of the treatments being randomized.”

http://www.cochrane.org/glossary/5 - letterc

Prognosis of Healing in Treated Teeth with Endodontic Infections 353

(a) (b) (c)

Fig. 15.5 Persistent infection after initial treatment. (a) Maxillary lateral incisor with primary infection. (b) Completed treatment. (c) At 1 year, unchanged radiolucency suggests persistence of the infection. Source: Friedman (2002). Reproduced with permission of Blackwell Munksgaard.

� Cohort study: “An observational study in which a defined group of people (the cohort) is followed over time. The outcomes of people in subsets of this cohort are compared, to examine people who were exposed or not exposed (or exposed at dif- ferent levels) to a particular intervention or other factor of interest. A prospective cohort study assem- bles participants and follows them into the future. A retrospective (or historical) cohort study identifies subjects from past records and follows them from the time of those records to the present. Because sub- jects are not allocated by the investigator to different interventions or other exposures, adjusted analysis is usually required to minimize the influence of other factors (confounders).”

� Case–control study: “A study that compares people with a specific disease or outcome of interest (cases) to people from the same population without that dis- ease or outcome (controls), and which seeks to find associations between the outcome and prior expo- sure to particular risk factors. This design is particu- larly useful where the outcome is rare and past expo- sure can be reliably measured. Case–control studies are usually retrospective, but not always.”

� Cross-sectional study: “A study measuring the dis- tribution of some characteristic(s) in a population at a particular point in time.”

� Case series: “A study reporting observations on a series of individuals, usually all receiving the same intervention, with no control group.”

Different designs are appropriate for different assessment aims, such as effectiveness of therapy inter- ventions, prognosis, or risks associated with inter- ventions; therefore, reviews geared to answer specific questions should focus on studies with matched design (Fletcher et al. 1996). While randomized controlled trial (RCT) is the appropriate design for assessing effectiveness of different interventions, the appropriate design for assessment of prognosis is a cohort study (Green and Byar 1984; Fletcher et al. 1996).

15.3.2 Methodologic rigor

A primary concern in clinical studies is different forms of bias. Data may be distorted so that differences are demonstrated between groups that may not really exist, while existing differences may not be shown (Fletcher

354 Endodontic Microbiology

et al. 1996). Bias can occur during assembly of the study cohort, when groups characteristics differ in vari- ables that may influence the outcome or in capacity to heal (Fletcher et al. 1996). Bias can also occur during assessment of the outcome (Fletcher et al. 1996), par- ticularly if assessment is carried out by the providers of treatment, who may be biased towards favorable outcome (Goldman et al. 1972). A structured checklist can be used to identify bias in studies on prognosis so as to determine their internal validity (Department of Clinical Epidemiology and Biostatistics 1981; Sackett et al. 1991; Laupacis et al. 1994; Fletcher et al. 1996; Sutherland 2001):

� Was the study cohort defined, assembled at the incep- tion of the study, at a common point in the course of the disease, described in detail?

� Was the referral pattern described? � Were baseline features measured reproducibly? � Was the follow-up achieved in at least 80% of the

inception cohort, the follow-up period described, and long enough for the outcome of interest to occur?

� Were the criteria used for outcome assessment described, either objective or applied in a blind fashion, clinically important and reproducibly mea- sured?

� Was adjustment for extraneous or important prog- nostic factors carried out?

The checklist criteria can be grouped into four gen- eral categories, used as the basis for appraisal of the endodontic studies below.

15.3.2.1 Cohort, at inception and end-point of the study

The inception cohort should be clearly characterized for variables that can potentially influence the out- come, pattern of referral of treated subjects, type of cases treated, and case selection criteria used (Fletcher et al. 1996). Case selection is likely to deter- mine the results (Ingle et al. 1994), because sub- jects are included or excluded according to perceived prognosis.

At the end-point of the study, failure to examine the majority of treated subjects may skew and invali- date the results (Strindberg 1956; Fletcher et al. 1996); therefore, at least 80% of the treated subjects should be examined (Department of Clinical Epidemiology and Biostatistics 1981; Laupacis et al. 1994; Sutherland

2001). Those who are not examined should be explic- itly accounted as “dropouts” who do not present for follow-up at their own volition (their absence may be related to the outcome of interest) or “discontinuers” who are excluded from the study for accountable rea- sons, for example death or relocation (their absence is not related to the outcome of interest).

The examined sample is a determinant of the study’s validity (Fletcher et al. 1996), and of its statistical power when associations are analyzed between the outcome and different variables. Small differences in outcome require large samples to achieve significance (Fletcher et al. 1996).

15.3.2.2 Exposure (treatment, intervention)

Treatment providers should be characterized, as their expertise may determine the results (Ingle et al. 1994). Treatment procedures performed should be current and explicitly described to avoid the need for interpreta- tion. Studies may be excluded if the treatment proce- dures are considered irrelevant or unacceptable.

15.3.2.3 Outcome assessment

To minimize bias (Fletcher et al. 1996), objective outcome measures should be used consistently in a blinded manner; therefore, examiners should be inde- pendent and calibrated with established reliability. The follow-up period should be long enough to capture the outcome of interest (Figure 15.6). Specifically for endodontic studies, the conclusion of the dynamic healing processes must be captured in the majority of the study sample.

15.3.2.4 Data reporting and analysis

Data pertaining to the study cohort, intervention, out- come assessment, and analysis should be reported in detail to allow identification of potential bias and assessment of validity. Statistical analyses should be designed to minimize bias, and take into account extra- neous factors and their potential confounding effects. Preferably, multivariate analyses should be used to account for all the variables.

15.3.3 “Current best evidence” for the prognosis of endodontic treatment

Evidence-based practice is “the conscientious, explicit and judicious use of current best evidence in making

Prognosis of Healing in Treated Teeth with Endodontic Infections 355

(a) (b) (c)

Fig. 15.6 Healing dynamics after initial treatment. (a) Immediate postoperative radiograph of mandibular first molar with extensive primary infection, included in a clinical study. (b) At 18 months (termination of the study), residual smaller radiolucency at the mesial root tip suggests that healing is incomplete. (c) At 3.5 years, the area is completely healed. Extension of the study to four years would have captured the completion of healing. Adapted from Friedman et al. (1995). Source: Friedman (2002). Reproduced with permission of Elsevier and Blackwell Munksgaard.

decisions about the care of individual patients” (Sack- ett et al. 1991). Accordingly, to support evidence- based endodontic practice, the current best evidence for endodontic prognosis needs to be identified. The level of evidence of clinical studies is determined by the research question, study design, and method- ologic rigor. Studies are ranked by descending hierar- chy of evidence (http://www.cebm.net/index.aspx?o = 5653). Note that the level of evidence may be graded down on the basis of study quality; it may be graded up if there is a large or very large effect.

Evidence for treatment benefits:

� Level 1: systematic review of randomized trials or N-of-1 trials;

� Level 2: randomized trial or observational study with dramatic effect;

� Level 3: nonrandomized controlled cohort/follow-up study;

� Level 4: case-series, case–control or historically con- trolled studies;

� Level 5: mechanism-based reasoning.

Evidence of prognosis:

� Level 1: systematic review of inception cohort stud- ies;

� Level 2: inception cohort studies; � Level 3: cohort study or control arm of randomized

trial; � Level 4: case-series or case–control studies, or poor

quality prognostic cohort study.

Systematic reviews have been published on the out- come of nonsurgical endodontic treatment (Sathorn et al. 2005; Ng et al. 2007, 2008; Peng et al. 2007; Figini et al. 2008; Naito 2008; Panitvisai et al. 2010; Su et al. 2011; McGuigan et al. 2013; Aminoshariae and Kulild 2015; Kang et al. 2015) and apical surgery (Peterson and Gutmann 2001; Niederman and Theo- dosopoulou 2003; Torabinejad et al. 2009; Tsesis et al. 2009, 2011; Del Fabbro and Taschieri 2010; Setzer et al. 2010, 2012; Tang et al. 2010; von Arx et al. 2010; Tsesis et al. 2013; Kang et al. 2015; Serrano- Gimenez et al. 2015), where methodologic rigor was not used as a consideration for inclusion of stud- ies in the review and where the prognosis for teeth with endodontic infections was not always specified. While these reviews satisfy many criteria for system- atic reviews, they do not exclude short-term outcome studies and they do not highlight the current best evi- dence for endodontic prognosis in teeth with apical periodontitis. In contrast, Friedman (2002b, 2005) has

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356 Endodontic Microbiology

been reviewing clinical endodontic studies focusing on methodologic rigor, so as to identify the current best evidence supporting prognosis of endodontic non- surgical and surgical treatment beyond the short-term of 1 year. The methodologic rigor of clinical stud- ies is a crucial consideration (Barton 2000), so much so that rigorous cohort studies can outweigh compro- mised RCTs and structured reviews of rigorous cohort studies can yield consistent conclusions with those of systematic reviews of RCTs (Benson and Hartz 2000; Concato et al. 2000). Accordingly, Friedman’s reviews on prognosis have excluded RCTs and rather included prospective cohort studies that comply with three of the four methodology criteria: cohort, inter- vention, assessment, analysis/reporting. This chapter represents an update on the previous reviews (Fried- man 2002b, 2005) and the data reported in the first edition of this textbook. Inclusion criteria were fur- ther restricted, to samples of at least 50 teeth, without treatment history of orthograde retreatment prior to apical surgery, publication since 1990 to capture cur- rent interventions, follow-up of at least 2 years for nonsurgical treatment and 4 years for apical surgery to avoid short-term outcomes, availability of data on the proportion of healed teeth, and multivariate analysis of outcome predictors.

Excluding studies with samples that have been repeated, eight studies (Table 15.1) represent the cur- rent best evidence for the prognosis of primary apical periodontitis after initial root canal treatment (Sjögren et al. 1990, 1997; Ørstavik 1996; Peters et al. 2004; de Chevigny et al. 2008a; Weiger et al. 2000; Ng et al. 2011; Ricucci et al. 2011). In addition, five studies (Table 15.2) represent the current best evi- dence for the prognosis of posttreatment apical peri- odontitis after orthograde retreatment (Sjögren et al. 1990; Sundqvist et al. 1998; de Chevigny et al. 2008b; Ng et al. 2011; Ricucci et al. 2011) and three stud- ies (Table 15.3) represent the current best evidence for the prognosis of posttreatment apical periodon- titis after apical surgery (Barone et al. 2010; von Arx et al. 2012, 2014). None of the current stud- ies on intentional replantation (Table 15.4) meet the methodology criteria; thus, the level of evidence to support the prognosis of this specific treatment modal- ity is the lowest. Collectively, the current best evi- dence studies form the basis for the prognosis of treatment in teeth with endodontic infections several years after treatment. They also serve as reference for

identifying outcome predictors that significantly influ- ence the prognosis.

15.4 Prognosis of primary apical periodontitis after initial treatment

Treatment providers are expected to advise patients of the prognosis of treatment and to maximize the prog- nosis by using treatment methods based on solid evi- dence. Therefore, the prognosis is reported along with outcome predictors that may influence it. Although the methodology among the eight current studies reviewed in this section (Sjögren et al. 1990, 1997; Ørstavik 1996; Weiger et al. 2000; Peters et al. 2004; de Chevi- gny et al. 2008a; Ng et al. 2011; Ricucci et al. 2011) is rather uniform, they still differ in case selection, study materials, and, consequently, in reported outcomes. Several of these studies have also not controlled for nonendodontic variables that could be of importance in periapical healing. Included among these potential confounding variables are restorative issues, periodon- tal disease, presence of neighboring teeth, the patient’s smoking habits, systemic diseases, bone modula- tory medications, as well as genetic and epigenetic variables.

15.4.1 Potential for healing

The proportion of completely healed teeth after ini- tial treatment ranges from 75% (Ørstavik 1996) to 86% (Sjögren et al. 1990). Considering the uniform outcome assessment among the selected studies, the results may have varied because of differences in tooth types, definition of the tooth or root as the evaluated unit (Friedman 2002b), case selection (Ingle et al. 1994), and restoration.

In addition to the healed teeth, “healing” has been reported in 8% (de Chevigny et al. 2008a) to 16% (Weiger et al. 2000) of the teeth. Typically, the pro- portion of healing captured in a study is inversely pro- portional to the follow-up period, because the healing process often requires years to be completed (Fried- man 2002b). Thus, the potential of teeth with apical periodontitis to heal within 2–4 years after initial treat- ment is 75–86%, while an additional 10–15% may still be healing at this time interval. It also is noteworthy that regardless of whether the periapical tissues heal or not, about 90–95% of the teeth remain symptom-free

Prognosis of Healing in Treated Teeth with Endodontic Infections 357

and functional (Ørstavik 1996; Weiger et al. 2000; de Chevigny et al. 2008a; Ng et al. 2011).

15.4.2 Time-course of healing

The healing process of primary apical periodonti- tis lesions is initiated within the first year after treatment (Reit 1987a; Kvist and Reit 1999); however, its completion often requires longer. Therefore, of all the teeth that heal eventually, only 50–70% appear completely healed by 1 year (Adenubi and Rule 1976; Ng et al. 2011), with the proportion increasing up to 90% by 2–4 years and up to 95% by 6 years (Byström et al. 1987; Sjögren et al. 1990; Ørstavik 1996; Kvist and Reit 1999; Ng et al. 2011). The remaining 5–6% of treated teeth may continue healing for years and appear healed only in the second or third decade after

treatment (Molven et al. 2002b; Fristad et al. 2004). As long as 4–6 years after treatment, about 8–13% of the teeth may still appear healing (Ørstavik 1996; Farzaneh et al. 2004b; Marquis et al. 2006; de Chevi- gny et al. 2008a). Considering the rather lengthy time- course of healing, studies with short follow-up periods underestimate the potential prognosis of the treated sample.

Infrequently after nonsurgical treatment, very exten- sive lesions can heal without total resolution of the radiolucency, when fibrous tissue occupies the peri- apical space (apical scar) (Penick 1961; Bhaskar 1966; Byström et al. 1987; Nair et al. 1999; Selden 1999; Kabak et al. 2005; Saunders 2008; Schulz et al. 2009; Zhang et al. 2015; Çalişkan et al. 2016) (Figure 15.7). Because this occurrence is infrequent, persisting lesions should be considered

(e) (f)(b)

(a) (c) (d) (g)

Fig. 15.7 Primary infection healed by scar formation after initial treatment. (a,b) Mandibular lateral incisor and canine with primary infection associated with an orofacial fistula. (c) Completed treatment. (d,e) At 2 years, the fistula has healed with minimal scarring of the skin. The residual radiolucency may suggest persistence of infection. (f) Clinical view after reflection of a full thickness flap reveals a thick fibrous bundle connecting the periapical lesion and the soft tissues over the chin. Histologic examination of the dissected bundle confirmed it to be fibrous (scar) tissue. (g) At 6 months after surgery, further decreased radiolucency and better defined periodontal ligament space suggest healing in progress. Source: Friedman (2002). Reproduced with permission of Blackwell Munksgaard.

358 Endodontic Microbiology

as persistent apical periodontitis rather than fibrous scars.

Reversal of the healing process is uncommon (Ørstavik 1996; Kvist and Reit 1999), suggesting that extended follow-up of teeth that demonstrate signs of healing after 1 year may be unnecessary (Ørstavik 1996). Nevertheless, root-filled teeth remain constantly at risk of recurrent infection in the long term. For example, over 1% of teeth observed to be healed 10–17 years after treatment reverted to disease a decade later (Molven et al. 2002b). To address this long-term risk, periodic follow-up of root-filled teeth is advocated.

15.4.3 Prognostic variables

Outcome predictors can be divided into those that have been identified in multivariate analyses, those that appear to be nonsignificant, and those that are equivo- cal and require further study. They can also be divided into preoperative variables that inform the projected prognosis before treatment (best derived from cohort studies), and intraoperative variables that are consid- ered during treatment to maximize the prognosis (best derived from RCTs).

15.4.3.1 Significant outcome predictors

Adjunctive irrigation with chlorhexidine (intraopera- tive). One current best evidence study (Ng et al. 2011) suggests a poorer prognosis in teeth were canals are irrigated with chlorhexidine as an adjunct to princi- pal irrigation with sodium hypochlorite (66% healed) compared to canals where chlorhexidine is not used (83% healed). Note that based on a cohort study, the evidence supporting this predictor is weak.

Extruded root filling (intraoperative). Poorer progno- sis (about 67% healed) has been reported in three studies (Sjögren et al. 1990; Ng et al. 2011; Ricucci et al. 2011) for root fillings extruded beyond the root end, compared to adequate filling length (about 86% healed). Although comparable outcomes for ade- quate and extruded root fillings have been reported in two other studies (Weiger et al. 2000; de Chevigny et al. 2008a), the collective evidence supporting the adverse impact appears to outweigh that refuting this impact.

Defective or no restoration (intraoperative). Poorer prognosis (about 50% healed) is reported in one study (Ng et al. 2011) for teeth with defective or missing restorations, compared to adequate restorations (about 80% healed). Although comparable outcomes for ade- quate and defective/missing restorations are reported elsewhere (Ricucci et al. 2011), the evidence support- ing the adverse impact appears to outweigh that refut- ing this impact.

15.4.3.2 Equivocal variables

Systemic health (preoperative). One current best evi- dence study (Ng et al. 2011) reports no association between systemic health and prognosis. Another study where the cohort is not differentiated for teeth without or with infection (Marending et al. 2005) suggests that a compromised nonspecific immune system impairs the prognosis, but this conclusion is not well supported by the statistical data. It is possible that healing in the immune-compromised patients requires a longer time than the 2-year observation in these studies.

Number of roots (preoperative). Two studies (Ng et al. 2011; Ricucci et al. 2011) have shown no association of prognosis with the numbers of roots. Conversely, one study (de Chevigny et al. 2008a) reports on poorer prognosis of multi-rooted teeth (79% healed) com- pared to single-rooted teeth (90% healed), suggesting that the risk of persistent disease in multi-rooted teeth is multiplied by the number of roots.

Radiolucency size (preoperative). Better prognosis in teeth with small lesions (≤5 mm in diameter) than larger lesions has been reported in two current best evidence studies (Weiger et al. 2000; Ng et al. 2011), while comparable outcomes have been reported for small and large lesions in four other studies (Sjögren et al. 1990, 1997; de Chevigny et al. 2008a; Ricucci et al. 2011).

Sinus tract (preoperative). Sinus tracts have not been significantly associated with prognosis in four studies (Sjögren et al. 1990; Weiger et al. 2000; de Chevi- gny et al. 2008a; Ricucci et al. 2011). Conversely, in another study (Ng et al. 2011) a poorer prognosis was reported for teeth with a sinus tract (67% healed) than when no tract was present (85% healed).

Prognosis of Healing in Treated Teeth with Endodontic Infections 359

Flareup (intraoperative). Flareups have not been sig- nificantly associated with prognosis in two studies (Sjögren et al. 1990; de Chevigny et al. 2008a), but in another study (Ng et al. 2011) teeth where flareup occurred had a poorer prognosis (62% healed) than teeth where flareup did not occur (83% healed).

Root filling method (intraoperative). Two studies have suggested no association between the vertically and laterally compacted root fillings and prognosis (Peters et al. 2004; Ng et al. 2011), while The Toronto Study series (de Chevigny et al. 2008a) has reported a better prognosis (87% healed) in teeth treated with flared canal preparation and vertically compacted warm gutta-percha than in teeth treated with step- back instrumentation and lateral compaction of gutta- percha (77% healed). Their finding notwithstanding, the authors emphasize the requirement to validate this variable in a randomized controlled trial (de Chevigny et al. 2008a).

Short root filling (intraoperative). Poorer prognosis has been reported in two studies (Sjögren et al. 1990; Ricucci et al. 2011), while comparable outcomes for adequate and short root fillings have been reported in three other studies (Weiger et al. 2000; Peters et al. 2004; de Chevigny et al. 2008a).

Bacterial culture before root filling. Bacteriologic root canal samples showing no growth before root filling were associated with better prognosis in one study (Sjögren et al. 1997). Nevertheless, the limited apti- tude of root canal bacterial sampling techniques and culture (Paquette et al. 2007) undermine the ability to conclusively answer this research question.

Complications. Perforation, file breakage, and mas- sive extrusion of filling materials have all been sug- gested to impair healing (Sjögren et al. 1990; de Chevi- gny et al. 2008a) and measures must be taken to avoid them. However, when they occur, endodontists cur- rently may successfully manage complications asso- ciated with perforation (Main et al. 2004; Ghoddusi et al. 2007; Pace et al. 2008; Mente et al. 2010; Ree and Schwartz 2012; Krupp et al. 2013; Pontius et al. 2013; Mente et al. 2014; Gorni et al. 2016) and frac- tured instruments (Spili et al. 2005; Panitvisai et al. 2010; Fu et al. 2011; Murad and Murray 2011; Ng et al. 2011; McGuigan et al. 2013; Ungerechts et al. 2014). Thus, the negative influence of mid-treatment

complications on the prognosis may be mitigated by current management strategies.

15.4.3.3 Nonpredictive variables

The following preoperative variables have not been significantly associated with the prognosis of primary apical periodontitis:

� Age, gender (Sjögren et al. 1990; de Chevigny et al. 2008a; Ng et al. 2011; Ricucci et al. 2011);

� Jaw, specific anatomy (Weiger et al. 2000; de Chevi- gny et al. 2008a; Ng et al. 2011);

� Periodontal support (Sjögren et al. 1990; de Chevi- gny et al. 2008a; Ng et al. 2011). Yet, if present, advanced periodontal disease should be expected to progress over time (Figure 15.8).

The following intraoperative variables have not been significantly associated with the prognosis of primary apical periodontitis.

Root canal instrumentation. The type of instruments used (Sjögren et al. 1990; de Chevigny et al. 2008a), degree of taper (Ng et al. 2011), and apical enlargement size (Sjögren et al. 1990; de Chevigny et al. 2008a). Although extensive apical enlargement has been sug- gested to enhance disinfection in the apical portion of the root canal (Ørstavik et al. 1991; Yared and Dagher 1994; Card et al. 2002), the inability in studies to con- sistently assess the extent of apical enlargement with- out knowing the initial canal dimensions undermines the ability to address this research question.

Restoration type. The type of restoration (temporary, definitive, filling, cast) (Sjögren et al. 1997; de Chevi- gny et al. 2008a; Ng et al. 2011). Yet, one study (Sjögren et al. 1990) reported less healing in teeth restored with crowns and those serving as bridge abut- ments, than in teeth restored with fillings. Posts have not been associated with the outcome either (Sjögren et al. 1990; de Chevigny et al. 2008a).

Number of treatment sessions. (de Chevigny et al. 2008a; Ng et al. 2011) Though intracanal medica- tion applied between treatment sessions has been sug- gested to improve root canal disinfection (Byström and Sundqvist 1981, 1983, 1985; Byström et al. 1985; Molander et al. 1990; Ørstavik et al. 1991; Yared and Dagher 1994; Shuping et al. 2000), a systematic review of studies assessing treatment outcomes after one or

360 Endodontic Microbiology

(a) (b) (c) (d)

Fig. 15.8 Effect of advanced periodontal disease on the prognosis. (a) Mandibular lateral incisor with endodontic infection and advanced marginal periodontitis, resulting in extensive bone loss. (b) Completed treatment. (c) At 8 months, clinical normalcy and drastically decreased radiolucency suggest incomplete healing (tooth is still restored with a temporary filling). (d) At 3 years, recurrent bone loss because of advancing periodontal disease. Source: Friedman (2002). Reproduced with permission of Blackwell Munksgaard.

more treatment sessions concludes that “the biolog- ical benefit of multi-session treatment has not been supported by clinical evidence” (Sathorn et al. 2005).

15.5 Prognosis of posttreatment apical periodontitis after orthograde retreatment

Similarly to the studies in initial treatment, consider- able differences exist in case selection and composi- tion of study materials among the five selected current studies on retreatment (Sjögren et al. 1990; Sundqvist et al. 1998; de Chevigny et al. 2008b; Ng et al. 2011; Ricucci et al. 2011), and their results vary even more than those of initial treatment.

15.5.1 Potential for healing

The proportion of completely healed teeth after orthograde retreatment ranges from 62% (Sjögren et al.

1990) to 84% (in teeth without perforation) (de Chevi- gny et al. 2008b). Falling below the range reported in the other studies, the reported 62% (Sjögren et al. 1990) appears to be an outlier. The variability of the results may be attributed to the same factors as those suggested above for initial treatment. In addition to the healed teeth, progressive healing has been reported in 6% of the teeth (de Chevigny et al. 2008b). Thus, the potential of teeth with persistent apical periodontitis to heal within 2–4 years after orthograde retreatment is 74–84%, while additional 5–6% may still be heal- ing at this time interval. It also is notweworthy that even if complete healing does not occur, 90–93% of the teeth may remain symptom-free and functional (de Chevigny et al. 2008b; Ng et al. 2011).

15.5.2 Time-course of healing

In the same way as healing of primary apical peri- odontitis after initial treatment, healing of persistent apical periodontitis requires considerable time after

Prognosis of Healing in Treated Teeth with Endodontic Infections 361

(a) (b) (c)

Fig. 15.9 Infection associated with perforation healed after orthograde retreatment. (a) Mandibular molar with a distal root perforation and associated bone loss. (b) Completed retreatment and perforation seal with MTA. (c) At 1.5 year, radiographic and clinical normalcy suggest that the tooth has healed. Regrettably, the tooth is not properly restored.

orthograde retreatment. Apparently, as many as 50% of teeth that were not healed 10–17 years after retreat- ment were completely healed a decade later (Fristad et al. 2004). The late healing was mainly characteristic of teeth with surplus root-filling material (Fristad et al. 2004).

15.5.3 Prognostic variables

The similarities between initial treatment and orthograde retreatment justify the consideration of the same outcome predictors. It is noteworthy that the cur- rent best evidence for retreatment is limited to only a few studies that do not allow conclusive assessment of the influence of many variables on the prognosis. Also, specifically for retreatment, characteristics of the previous root canal treatment history have to be consid- ered, including the previous root filling, a perforation that may be present in a minority of retreated teeth, and the time elapsed since initial treatment.

15.5.3.1 Significant outcome predictors

Previous perforation (preoperative). Two studies (de Chevigny et al. 2008b; Ng et al. 2011) have reported a poorer prognosis for retreated teeth with previous perforations sealed with a variety of repair materi- als (about 50% healed), compared to teeth without

perforations (84% healed). It is noteworthy, however, that limited-size case series (Main et al. 2004; Ghod- dusi et al. 2007; Pace et al. 2008; Mente et al. 2010, 2014; Ree and Schwartz 2012; Krupp et al. 2013; Pon- tius et al. 2013) have reported that 73–100% of teeth healed after perforations were sealed with mineral- trioxide aggregate (MTA) (Figure 15.9). Thus, a pre- vious perforation’s impact on the prognosis, or the lack thereof, may depend on the material used for repair.

Apical patency (intraoperative). One study (Ng et al. 2011) reports a better prognosis in teeth where api- cal patency was regained during retreatment (82% healed), compared to no patency regained (70% healed). Note that based on a cohort study, the evi- dence supporting this predictor is weak.

Adjunctive irrigation with ethylenediaminetetraacetic acid (EDTA) (intraoperative). One study (Ng et al. 2011) reports a better prognosis in teeth where canals were irrigated with EDTA in addition to sodium hypochlorite (87% healed), compared to no use of EDTA (78% healed). Note that based on a cohort study, the evidence supporting this predictor is weak.

Number of treatment sessions. Unlike initial treatment (see earlier), one study (de Chevigny et al. 2008b) reports a better prognosis in teeth retreated in one

362 Endodontic Microbiology

session (100% healed), compared with retreatment in two sessions (77% healed). Note that based on a cohort study, the evidence supporting this predictor is weak.

15.5.3.2 Equivocal variables

Radiolucency size (preoperative). One current best evidence study (Ng et al. 2011) reports a better prog- nosis in teeth with small lesions (≤5 mm in diameter; 86% healed) than larger lesions (67% healed), while comparable outcomes have been reported for small and large lesions in two other studies (de Chevigny et al. 2008b; Ricucci et al. 2011).

Sinus tract (preoperative). One study (Ng et al. 2011) reports a poorer prognosis in teeth with a sinus tract (67% healed) than when no tract is present (85% healed), while sinus tracts have not been significantly associated with prognosis in one study (de Chevigny et al. 2008b).

Apparent quality of the previous root filling (preoper- ative). One study (de Chevigny et al. 2008b) reports a better prognosis (86% healed) in teeth with inadequate length or density of the previous root filling, compared with teeth with apparently adequate root fillings (50% healed). Previous root filling quality was not associ- ated with the prognosis in another study (Ng et al. 2011).

15.5.3.3 Nonpredictive variables

The following preoperative variables have not been significantly associated with the prognosis of posttreat- ment apical periodontitis after orthograde retreatment:

� Age, gender (de Chevigny et al. 2008b; Ng et al. 2011);

� Jaw, specific anatomy (de Chevigny et al. 2008b; Ng et al. 2011);

� Periodontal support (de Chevigny et al. 2008b; Ng et al. 2011);

� Number of roots (de Chevigny et al. 2008b; Ng et al. 2011);

� Symptoms (de Chevigny et al. 2008b; Ng et al. 2011);

� Elapsed time after previous treatment (de Chevigny et al. 2008b).

The following intraoperative variables have not been significantly associated with the prognosis of posttreat- ment apical periodontitis after orthograde retreatment:

� Root canal instrumentation (de Chevigny et al. 2008b; Ng et al. 2011);

� Type of root filling (de Chevigny et al. 2008b; Ng et al. 2011);

� Restoration type (de Chevigny et al. 2008b; Ng et al. 2011).

15.6 Prognosis of posttreatment apical periodontitis after apical surgery

Techniques and materials applied in apical surgery have evolved considerably over the past two decades, leading to a surge in the number of studies reporting on prognosis and outcome predictors. Systematic reviews of older and contemporary studies have highlighted the benefits of state-of-the-art apical surgery approaches including the use of magnification and illumination (microscope or endoscope), ultrasonic root-end cavity preparation, and MTA and super ethoxy-benzoic acid (Super-EBA) for root-end filling (Tsesis et al. 2009, 2013; Del Fabbro and Taschieri 2010; Setzer et al. 2010, 2012; Tang et al. 2010; von Arx et al. 2010; Kang et al. 2015; Serrano-Gimenez et al. 2015). Col- lectively, these current techniques, often referred to as “apical microsurgery” (Setzer et al. 2010), have been reported to yield high “success” rates in the range of 89–94% (Tsesis et al. 2009; Del Fabbro and Taschieri 2010; Setzer et al. 2010, 2012; Tang et al. 2010; von Arx et al. 2010; Tsesis et al. 2013; Kang et al. 2015; Serrano-Gimenez et al. 2015). While the recent sys- tematic reviews satisfy the literature searching and sta- tistical analysis criteria, they have consistently fallen short in critically appraising the studies they selected, ignoring flaws in outcome assessment and short-term observation periods. In this regard, those systematic reviews have generally overestimated the prognosis of apical surgery and their conclusions are the subject of controversy (Friedman 2011).

The most contentious methodologic issues are the following.

Short follow-up periods. The 1-year observation in many studies reporting on the outcome of apical micro- surgery (Table 15.3, from Rubinstein and Kim 1999 onwards) does not capture longer-term regression,

Prognosis of Healing in Treated Teeth with Endodontic Infections 363

(a) (b) (c) (d)

Fig. 15.10 Recurrent infection after apical surgery. (a) Maxillary lateral incisor with persistent infection. (b) Completed retrograde retreatment surgery, including a root filling with sealer and injectable gutta-percha. (c) At 6 months, radiographic and clinical normalcy suggest that the tooth has healed. (d) At 2.5 years, renewed radiolucency suggests recurrent infection. Source: Friedman (2005). Reproduced with permission of John Wiley and Sons.

reported in 6–10% of teeth that appeared healed in the short-term after microsurgical treatment (Rubin- stein and Kim 2002; Wesson and Gale 2003; Song et al. 2012, 2014; von Arx et al. 2012, 2014; Tawil et al. 2015) (Figure 15.10). As result of such long- term regression, the healed rates in given populations declined from 84% to 76% at 5 years (von Arx et al. 2007) and from 91% to 88% at 4–8 years (Song et al. 2014), although the decline was occasionally offset by an untypical progression in a few teeth from “not healed” at 1 year to “healed” at 5 years (von Arx et al. 2014; Tawil et al. 2015). By relying primarily on short-term studies, the recent systematic reviews (Tsesis et al. 2009; Del Fabbro and Taschieri 2010; Setzer et al. 2010, 2012; Tang et al. 2010; von Arx et al. 2010; Tsesis et al. 2013; Kang et al. 2015; Serrano- Gimenez et al. 2015) have overestimated the prognosis of apical microsurgery.

Misclassification of “incomplete healing”. The majority of studies on the prognosis of apical micro- surgery appear to classify outcomes based on well- established criteria (Rud et al. 1972a; Molven et al. 1987; Grung et al. 1990), although with critical dis- crepancies. According to the classic criteria, both “complete” and “incomplete” healing represent a favorable outcome or success, while “uncertain” and

“unsatisfactory” healing represent unfavorable out- come or failure. “Incomplete healing” is strictly reserved for fibrous scars, occurring in less than 10% of cases (Molven et al. 1987; Grung et al. 1990; Yazdi et al. 2007; von Arx et al. 2012, 2014; Çalişkan et al. 2016). Instead, it has been erroneously assigned to teeth showing reduced radiolucency (Gagliani et al. 2005; Ortega-Sanchez et al. 2009; Li et al. 2014), which should be classified as “uncertain healing.” The importance of this misclassification cannot be over- stated: it counts many teeth (often 12–32% of the cohort) with an unfavorable outcome as having a favor- able outcome, thus overestimating the prognosis. In specific studies, outcomes can be properly interpreted when both complete and incomplete healing cate- gories are specified (Chong et al. 2003; Maddalone and Gagliani 2003; Lindeboom et al. 2005; Marin-Botero et al. 2006; Yazdi et al. 2007; Walivaara et al. 2009, 2011; Song and Kim 2012; Song et al. 2012; von Arx et al. 2012, 2014); however, some researchers lump both outcome categories together without breakdown, precluding reinterpretation of missclassified outcomes (Taschieri et al. 2005, 2006a,b, 2007a,b 2008, 2010, 2011, 2013).

Misclassification of “uncertain healing”. The prog- nosis has been further inflated by a specific research

364 Endodontic Microbiology

group, frequently reporting on the prognosis of api- cal microsurgery, who include teeth with “uncertain healing” in the favorable outcome or success tally (Taschieri et al. 2013).

Inclusion of previous orthograde retreatment. In sev- eral studies frequently cited for the prognosis of api- cal microsurgery, the entire cohort (Zuolo et al. 2000; Marin-Botero et al. 2006; Li et al. 2014) or a large proportion thereof (Chong et al. 2003; Saunders 2008) was comprised of teeth where infection persisted after previous orthograde retreatment. In those teeth, apical periodontitis may have been sustained by bacteria col- onizing apical root canal ramifications or extraradic- ular infection (see section 15.8), both effectively eliminated by apical surgery, as opposed to teeth with persistent root canal infection where outcome of apical surgery critically relies on the root-end barrier (Fried- man 2005). Accordingly, the outcome of apical surgery is better when it is preceded by orthograde retreatment (Taschieri et al. 2010). Thus, studies that include many teeth with treatment history of previous orthograde retreatment, but that do not characterize their cohorts in this regard, may overestimate the prognosis of apical surgery.

Similarly to the studies on nonsurgical treatment, some differences in case selection and composition of study materials exist among the three selected current studies on apical surgery (Barone et al. 2010; von Arx et al. 2012, 2014) contributing to some inconsistencies among the reported outcomes.

15.6.1 Potential for healing

The proportion of healed teeth (including scars) after first-time apical microsurgery ranges from 74% (Barone et al. 2010) to 84% (von Arx et al. 2014). The reported outcomes may have varied somewhat because of differences in proportions of previously retreated teeth and in treatment procedures. Beyond the assess- ment of healing, some 85–95% of the teeth may remain symptom-free and functional (Barone et al. 2010; von Arx et al. 2012, 2014).

15.6.2 Time-course of healing

Healing after apical surgery progresses rapidly within the first year (Halse et al. 1991; Kvist and Reit 1999). Of all the teeth that heal eventually, 35–60% appear

completely healed by 1 year (Grung et al. 1990; Halse et al. 1991; Molven et al. 1996; Maddalone and Gagliani 2003; Wesson and Gale 2003), while approximately 85% appear healed by 3 years (Grung et al. 1990). Healing by a fibrous scar occurs infre- quently (Rud et al. 1972a; Molven et al. 1996) (Fig- ure 15.4), when both the buccal and lingual bone plates are perforated at the conclusion of the surgical proce- dure (Molven et al. 1991). Postsurgery apical scars usually remain stable over time (Molven et al. 1996) and are considered at par with a healed site (Rud et al. 1972a; Molven et al. 1996).

Unlike the very low occurrence of healing regres- sion after nonsurgical treatment, recurrent infection in the long term after apical microsurgery has been reported in 6–10% of healed teeth (Rubinstein and Kim 2002; Wesson and Gale 2003; Song et al. 2012, 2014; von Arx et al. 2012, 2014; Tawil et al. 2015) (Figure 15.10). To address this risk of regression, it is advisable to re-examine teeth 3 years or longer after apical microsurgery.

15.6.3 Prognostic variables

Many variables associated with the prognosis of apical microsurgery differ from those that have been exam- ined in relation to nonsurgical treatment.

15.6.3.1 Significant outcome predictors

Interproximal bone level (preoperative). One study consistent with the current best evidence reported bet- ter prognosis in teeth where the measured distance between the interproximal bone level and the cemen- toenamel junction was ≤3 mm (78% healed) compared to >3 mm (53% healed) (von Arx et al. 2012). This finding supported earlier observations in a rather large number of nonselected studies (Rud et al. 1972b; Finne et al. 1977; Hirsch et al. 1979; Skoglund and Persson 1985; Forssell et al. 1988; Kim et al. 2008; Saunders 2008) of poor outcomes in teeth with considerable vertical or marginal bone loss, which can compromise periodontal reattachment. Taken together, both higher and lower level evidence suggests that the prognosis may be compromised by considerable attachment loss of the treated tooth.

Type of root-end filling material (intraoperative). Studies based on an in vivo model developed to sim- ulate clinical conditions (Friedman et al. 1991b) have

Prognosis of Healing in Treated Teeth with Endodontic Infections 365

(a) (b) (c)

Fig. 15.11 Root-end management with bonded Retroplast. (a) Clinical view of maxillary first molar with Retroplast “caps” bonded to the three roots. (b) Maxillary central incisor with persistent infection and extruded root filling. (c) At 9 years after surgery and bonding Retroplast at the root end, radiographic and clinical normalcy suggest that the tooth has healed. Courtesy of Dr. Vibe Rud. Source: Friedman (2005). Reproduced with permission of John Wiley and Sons.

highlighted intermediate restorative material (IRM) (Andreasen and Pitt Ford 1994; Pitt Ford et al. 1994; Tawil et al. 2009), Super-EBA (Pitt Ford et al. 1995; Trope et al. 1996), MTA (Torabinejad et al. 1995; Tawil et al. 2009), and Diaket (Witherspoon and Gutmann 2000) as potentially superior root-end filling materi- als. However, animal studies do not rank as evidence for effectiveness of clinical interventions. While sev- eral RCTs (Jensen et al. 2002; Chong et al. 2003; Lindeboom et al. 2005), a nonrandomized trial (von Arx et al. 2007), and a systematic review (Tang et al. 2010) have suggested equivalence of IRM, Super- EBA, MTA, and Retroplast (Figure 15.11) in the short- term, recent 5-year studies consistent with the cur- rent best evidence reported a better prognosis in teeth root-end filled with MTA (86–93% healed) compared to Retroplast (77–79% heaed) and Super-EBA (67% healed) (von Arx et al. 2012, 2014). Note that based on cohort studies, the evidence supporting this predictor is weak.

Surgical crypt size (intraoperative). One study (Barone et al. 2010) reports a better prognosis in teeth with smaller crypt size (≤10 mm in diameter; 80%

healed) than larger crypts (>10 mm; 53% healed). Note that this finding was not correlated with pre- operative radiolucency size in the same study and this variable has not been addressed in the other current best evidence studies.

15.6.3.2 Equivocal variables

Patient’s age (preoperative). One current best evi- dence study (Barone et al. 2010) reports a better prog- nosis for older patients (>45 years; 84% healed) than for younger patients (≤45 years; 68% healed), while comparable outcomes have been reported for younger and older patients in two other current best evidence studies (von Arx et al. 2012, 2014).

Length of the existing root filling (preoperative). One current best evidence study (Barone et al. 2010) reports a better prognosis in teeth with root fillings of inade- quate length (≥2 mm short of the root end or extruded; 84% healed) than adequate length (68% healed), while comparable outcomes have been reported for all root filling lenghts in another study (von Arx et al. 2012).

366 Endodontic Microbiology

(a) (b) (c)

Fig. 15.12 Persistent infection healed after repeat (second-time) surgery. (a) Maxillary lateral incisor with persistent infection after previous surgery, and a gutta-percha cone tracing the sinus tract. (b) Completed repeat surgery, comprising retrograde retreament and filling with MTA. (c) At 3 years radiographic and clinical normalcy suggest that the tooth has healed. Source: Friedman (2002). Reproduced with permission of John Wiley and Sons.

15.6.3.3 Nonpredictive variables

The following preoperative variables have not been significantly associated with the prognosis of posttreat- ment apical periodontitis after apical microsurgery:

� Gender (Barone et al. 2010; von Arx et al. 2012, 2014).

� Tooth type, jaw, specific anatomy (Barone et al. 2010; von Arx et al. 2012). Note that incomplete healing by scar tissue occurs more frequently in maxillary lateral incisors than in other teeth (Molven et al. 1991; von Arx et al. 2014).

� Symptoms (Barone et al. 2010; von Arx et al. 2012). � Radiolucency size (Barone et al. 2010; von Arx et al.

2012). Note that healing by scar tissue frequently occurs in very large lesions (>10 mm in diameter) (Molven et al. 1991).

� Material and density of existing root-filling (Barone et al. 2010).

� Restoration type, post (Barone et al. 2010; von Arx et al. 2014).

� Elapsed time after nonsurgical treatment (Barone et al. 2010).

� Second-time surgery (Barone et al. 2010; von Arx et al. 2012, 2014). Note that modified case selec- tion criteria and techniques have been suggested to improve the outcome of second-time surgery (Wang et al. 2004) (Figure 15.12).

� Nature of pathologic lesion as revealed by biopsy (Barone et al. 2010).

The following intraoperative variables have not been significantly associated with the prognosis of posttreat- ment apical periodontitis after apical microsurgery:

� Method of hemostasis (Barone et al. 2010). Note that effective hemostasis is critical for quality root-end filling (Carr 1998) and bonding of Retroplast apical caps (Jensen et al. 2002).

� Depth of root-end filling (Barone et al. 2010). Note that with the use of conventional ultrasonic tips, root-end cavities can vary in depth from 1 to 3 mm

Prognosis of Healing in Treated Teeth with Endodontic Infections 367

(a)

(b)

Fig. 15.13 Root-end cavity preparation with ultrasonic tips. (a) Assortment of ultrasonic tips for root-end cavity preparation. (b) Clinical view of root-end cavity preparation with an ultrasonic tip. Source: Friedman (2005). Reproduced with permission of John Wiley and Sons.

(Figure 15.13); cavities extending further coronally qualify as retrograde retreatment.

� Procedural complications, including perforation of the opposing bone plate or sinus (Barone et al. 2010). Note that sensory deficit, while unrelated to

healing, must be considered as a risk when mandibu- lar molars are treated. Paresthesia was reported in 20% of patients after apical surgery in mandibular molars; it was transient in 19% of patients but lin- gered for 2 years in 1% of patients (Wesson and Gale 2003).

� Antibiotics after treatment (Barone et al. 2010; von Arx et al. 2012).

15.6.3.4 Variables not addressed at the level of current best evidence

The following pre- and postoperative variables have been addressed in different studies but are not consis- tent with the current best evidence.

Root dentin defects (intraoperative). A pioneering but nonselected study (Tawil et al. 2015) reports a better 3-year healed rate (97%) in teeth where no root dentin defects were evident under microscopic and transillu- mination examination, than in roots where root dentin defects were evident (32%). This variable requires fur- ther investigation.

Presence or absence of a root-end filling (intraop- erative). A root-end filling is placed to establish an effective barrier against interaction of intracanal bacte- ria with the periapical tissues (Friedman 1991). Many nonselected studies have reported better outcomes with root-end fillings than without (Friedman et al. 1991a; Rapp et al. 1991). Collectively, these low-evidence studies suggest that placement of a root-end filling to curtail persistent root canal infection improves the prognosis.

Operator’s skill (intraoperative). Three nonselected studies suggest that the prognosis is associated with the individual operator’s skill (Nord 1970; Altonen and Mattila 1976; Lustmann et al. 1991). While it is widely accepted that apical surgery is technique-sensitive, the evidence supporting this predictor is suggestive at best.

Laser irradiation, bone grafts, and barriers (intra- operative). Application of laser irradiation (Bader and Lejeune 1998), guided regeneration barriers, and bone grafting substances (Saad and Abdellatief 1991; Grimes 1994; Pecora et al. 1995; Rankow and Kras- ner 1996; Tobon et al. 2002; Lin et al. 2010) have not been shown to influence the prognosis. Bone regeneration after apical surgery may be improved by

368 Endodontic Microbiology

application of guided regeneration barriers only in teeth with through-and-through defects (Tsesis et al. 2011).

Level of apical resection (intraoperative). A more coronal resection (approximately 3 mm from the apex) may avoid exposing canal ramifications that can allow intracanal bacteria to sustain disease after surgery (Carr and Bentkover 1998), while also facilitating preparation of the root-end cavity and filling.

Concurrent surgical and orthograde management (intraoperative). When surgical and orthograde treat- ment are performed concurrently “infection is elim- inated and reinfection is prevented” (Molven et al. 1991), augmenting the prognosis compared to apical surgery alone (Friedman 1991; Hepworth and Fried- man 1997). While contemporary treatment planning for persistent endodontic infections usually prescribes either orthograde or surgical management, in selected cases both procedures can be performed concurrently to comprehensively manage anatomic and technical complexities.

Retrograde root canal retreatment (intraoperative). Retrograde retreatment comprises instrumentation, irrigation, and filling the root canal as far coro- nally as can be reached from the apical end (Fig- ure 15.14) (Nygaard-Ostby 1971; Storms 1978; Serota and Krakow 1983; Reit and Hirsch 1986; Flath and Hicks 1987; Amagasa et al. 1989; Goldberg et al. 1990; Jonasson et al. 2008), with reported healed rates ranging from 71% to 100% (Reit and Hirsch 1986;

Amagasa et al. 1989; Goldberg et al. 1991; Wang et al. 2004; Jonasson et al. 2008). While the coronally extended barrier offers an advantage over the standard root-end filling, continued bacterial ingress into the canal under restorations and along posts may result in recurrence of disease (Figure 15.10).

Type of magnification and illumination (intraopera- tive). Contemporary systematic reviews of short-term studies (Tsesis et al. 2009, 2013; Del Fabbro and Taschieri 2010; von Arx et al. 2010; Setzer et al. 2012) suggest that the prognosis may be improved when api- cal microsurgery is perfomed with the aid of the oper- ating microscope, compared to the endoscope.

15.7 Prognosis of posttreatment apical periodontitis after intentional replantation

In accordance with current concepts, intentional replantation may be used as an alternative to extrac- tion when both retreatment and apical surgery are not feasible in situ (Guy and Goerig 1984; Dumsha and Gutmann 1985) (Figure 15.15). The expected goal is survival of the replanted tooth, considered as suc- cess (Grossman 1982; Torabinejad et al. 2015) even if pathologic processes persist. Healing of the attachment apparatus without root resorption depends on survival of the periodontal ligament and cementum along the root surface (Andreasen 1985; Andreasen et al. 1995), and prevention of infection (Tronstad 1988; Trope and Friedman 1992). Reattachement without resorption is conditional on controlled trauma associated with

(a) (b) (c) (d) (e)

Fig. 15.14 Persistent infection healed after retrograde retreatment. (a) Maxillary second premolar with persistent infection. (b) Retrograde retreatment is carried out with ultrasonic files. (c) Completed surgery, including root filling with sealer and injectable gutta-percha. (d,e) At 1 and 7 years, respectively, radiographic and clinical normalcy suggest that the tooth has healed. Source: Friedman (2005). Reproduced with permission of John Wiley and Sons.

Prognosis of Healing in Treated Teeth with Endodontic Infections 369

(a) (b) (c) (d)

Fig. 15.15 Persistent infection healed after intentional replantation, where conventional treatment was unfeasible. (a) Maxillary first premolar with persistent infection, associated with palatal root perforation at the distal aspect, untreated palatal canal, buccal post perforation and total loss of the buccal bone plate. (b) Completed intentional replantation that included repair of two perforations and two root-end fillings, all with MTA. (c) At 3 months, the roots of both teeth have become realigned, and the radiolucency considerably decreased. (d) At 1 year and 4 months, radiographic and clinical normalcy suggest that the tooth has healed with two small scars remaining.

extraction, extraoral manipulation and replantation, short extraoral time within suitable storage medium, and splinting being functional and applied for short duration (Hammarström et al. 1986; Oikarinen 1993).

Outcomes of intentional replantation are compared with those of implants in a systematic review and meta-analysis (Torabinejad et al. 2015), with 88% mean survival rate for intentional replantation; how- ever, because the review included older studies dating back to an era before systematic research on replanta- tion of teeth was available, its findings underestimate the realistic outcomes of a state-of-the-art replantation procedure.

Contemporary studies on the prognosis of inten- tional replantation (Table 15.4) are mostly at a low level of evidence. Also, their clinical procedures have varied, including replantation of teeth with primary infections (Koenig et al. 1988) or persistent root canal infections (majority of studies), surgical repositioning (extrusion) of horizontally fractured teeth (Kahnberg et al. 1982; Kahnberg 1985, 1988, 1996; Warfvinge and Kahnberg 1989; Çalişkan et al. 1998), 180◦ rota- tion (Tegsjö et al. 1987), and replantation preceeded by orthodontic extrusion (Choi et al. 2014). According to these studies, the “success”/survival rate of inten- tional replantation ranges from 71% (Kahnberg 1988) to 91% (Keller 1990) to 95% (Çalişkan et al. 1998; Cho et al. 2016), but the assessment criteria may have been vague. Importantly, specific studies listed in Table 15.4 may not have followed the current clinical protocols for replantation of teeth. For example, on occasion roots

were only sealed apically without a root filling, predis- posing them to inflammatory root resorption (Koenig et al. 1988; Keller 1990).

Consideration of prognosis after intentional replan- tation takes into account not just periapical healing, but also reattachment without external root resorption (Hammarström et al. 1986; Torabinejad et al. 2015; Cho et al. 2016). Reported incidence of root resorp- tion in contemporary studies has varied from zero (Kahnberg 1988; Warfinge 1989; Keller 1990; Kahn- berg 1996; Çalişkan 1998) to 6% (Kingsbury and Wiesenbaugh 1971; Will 1974; Koenig et al. 1988; Bender and Rossman 1993; Choi et al. 2014; Cho et al. 2016), likely thanks to optimal extraoral time and con- ditions. Nevertheless, incidence of resorption as high as 14% and 35% has been reported in several studies (Tegsjö et al. 1987; Raghoebar and Vissink 1999). Per- sistent infection ranges from 5% (Cho et al. 2016) to 29% (Kahnberg 1988), because the infected root canal can be effectively sealed with a root-end filling that is easily placed. The predictable healing potential after intentional replantation performed in well-controlled conditions has been further demonstrated, albeit at the lowest level of evidence, in many case reports (Feldman et al. 1971; Rosenberg et al. 1980; Solomon and Abelson 1981; Stroner and Laskin 1981; Kaufman 1982; Lubin 1982; Guy and Goerig 1984; Nosonowitz and Stanley 1984; Ross 1985; Dryden 1986; Linde- berg et al. 1986; Lu 1986; Madison 1986; Messkoub 1991; Kawai and Masaka 2002; Penarrocha et al. 2007; Subay et al. 2014).

370 Endodontic Microbiology

15.7.1 Dynamics of external root resorption

Resorption after intentional replantation is usually dis- cernible within 1 year (Emmertsen and Andreasen 1966). This is definitely true regarding inflamma- tory resorption. Replacement resorption may be first observed radiographically several years after replan- tation (Andreasen et al. 1995; Cho et al. 2016). For example, in a recent long-term study (Cho et al. 2016), occurrence of external root resorption increased from 2/159 teeth (1%) by 1 year to 5/129 teeth (3%) by 3 years. However, it may be indicated clinically much earlier than radiographically, by a specific pitch (or metallic sound) upon percussion.

15.8 Etiology of persistent apical periodontitis after endodontic treatment

While persistence of apical periodontitis after endodontic treatment may occur in the absence of microbial factors, due to foreign materials, cholesterol crystals, and true cysts (Penick 1961; Bhaskar 1966; Byström et al. 1987; Nair et al. 1990b, 1993, 1999; Selden 1999; Nair 2003a,b; Saunders 2008; Lin et al. 2009; Schulz et al. 2009; Barone et al. 2010; Lui et al. 2014; Siqueira et al. 2014; Bornstein et al. 2015), this is not a common occurrence (Sjögren et al. 1990). For the major part, persistent apical periodontitis is sus- tained by persistent or recurrent infection (Siqueira 2001; Friedman 2002a). The sites colonized by bacte- ria and the pathways of bacteria–host interactions may differ after nonsurgical and surgical treatment. Fur- thermore, the reason for nonhealing or delayed heal- ing seen in some cases may be related to the presence of specific virulent microorganisms in these cases that exert significant impact on periapical tissues and elicit a pronounced host response.

15.8.1 Persistent infection after nonsurgical treatment

Microorganisms, mainly bacteria, sustain the infection process by colonizing different sites within or outside the affected tooth.

Root canal system. This is the most frequently colo- nized site in teeth with persistent infection according to numerous studies (Borssen and Sundqvist 1981; Fukushima et al. 1990; Nair et al. 1990a; Baumgartner

and Falkler 1991; Lin et al. 1991, 1992, 2008; Sjögren et al. 1997; Molander et al. 1998; Sundqvist et al. 1998; Nair et al. 1999; Peciuliene et al. 2000, 2001; Cheung and Ho 2001; Hancock et al. 2001; Rolph et al. 2001; Siqueira 2001; Haapasalo et al. 2003; Pin- heiro et al. 2003a,b; Sundqvist and Figdor 2003; Adib et al. 2004; Gomes et al. 2004, 2006, 2008; Hommez et al. 2004; Nair 2004, 2006; Rôças et al. 2004, 2008; Siqueira and Rôças 2004, 2008; Fouad et al. 2005; Kaufman et al. 2005; Zerella et al. 2005; Williams et al. 2006; Schirrmeister et al. 2007; Ricucci and Siqueira 2008; Ricucci et al. 2009; Subramanian and Mickel 2009; Siqueira et al. 2014). The bacteria have either survived the root canal treatment procedures (Sjögren et al. 1997; Siqueira 2001) or invaded the filled canal space after treatment, possibly through a coronal path- way (Friedman et al. 1997, 2000; Mah et al. 2003; Shipper et al. 2005; Duggan et al. 2009; Santos et al. 2014).

Extraradicular sites. Specific bacteria, particularly Actinomyces israelii and Propionibacterium pro- pionicum, can colonize in the periapical tissues (Sundqvist and Reuterving 1980; Weir and Buck 1982; Martin and Harrison 1984; Nair and Shroeder 1984; Happonen et al. 1985; Happonen 1986; Nishimura 1986; Haapasalo et al. 1987; O’Grady and Reade 1988; Sjögren et al. 1988; Iwu et al. 1990; Fig- ures and Douglas 1991;Wayman et al. 1992; Sakellar- iou 1996; Kalfas et al. 2001; Hirshberg et al. 2003; Siqueira 2003; Sundqvist and Figdor 2003; Figdor 2004; Ricucci and Siqueira 2008; Subramanian and Mickel 2009; Signoretti et al. 2011; Siqueira et al. 2014), after penetrating the host tissues during a long- term infection of the root canal system, or when inoc- ulated periapically during treatment. Other bacterial species also have been implicated in extraradicular infection (Gatti et al. 2000; Sunde et al. 2000a,b, 2002, 2003; Tronstad and Sunde 2003). They can colonize the cementum on the root surface (Pitt Ford 1982; Nair 1987; Kiryu et al. 1994) and around the api- cal foramina (Tronstad et al. 1987, 1990a,b; Siqueira and Lopes 2001; Leonardo et al. 2002; Noiri et al. 2002; Tronstad and Sunde 2003; Ricucci et al. 2005; Vera et al. 2012; Wang et al. 2012; Siqueira et al. 2014), or in dentin debris inadvertently extruded dur- ing treatment (Yusuf 1982). Whether these bacteria can exclusively sustain infection is unclear; however, cur- rent knowledge suggests that the predominant cause of persistent apical periodontitis is root canal infection,

Prognosis of Healing in Treated Teeth with Endodontic Infections 371

while exclusive extraradicular infection comprises a small percentage of cases (Friedman 2002a) (see also Chapter 6).

Even after orthograde retreatment, pathogenic bac- terial strains can still survive in the root canal system and sustain persistent infection (Sundqvist et al. 1998; Peciuliene et al. 2001; Zerella et al. 2005; Schirrmeis- ter et al. 2007). Nevertheless, the disease can also be sustained by extraradicular infection (Sundqvist and Reuterving 1980; Pitt Ford 1982; Weir and Buck 1982; Yusuf 1982; Martin and Harrison 1984; Nair and Shroeder 1984; Happonen et al. 1985; Happo- nen 1986; Nishimura 1986; Haapasalo et al. 1987; Nair 1987; Tronstad et al. 1987, 1990a,b; O’Grady and Reade 1988; Sjögren et al. 1988; Iwu et al. 1990; Figures and Douglas 1991; Wayman et al. 1992; Sakellariou 1996; Gatti et al. 2000; Sunde et al. 2000a,b, 2002, 2003; Kalfas et al. 2001; Siqueira and Lopes 2001; Hirshberg et al. 2003; Siqueira 2003; Tronstad and Sunde 2003; Figdor 2004; Ricucci et al. 2005; Subramanian and Mickel 2009; Signoretti et al. 2011; Vera et al. 2012; Wang et al. 2012; Siqueira et al. 2014). As apical surgery can effec- tively eliminate these etiologic factors, it is the treat- ment of choice for persistent infection after orthograde retreatment.

15.8.2 Persistent infection after apical surgery

Persistence of infection after apical surgery usually suggests that root canal bacteria are not effectively enclosed within the canal space by the root-end filling (Friedman 1991). Placement of a root-end filling is a challenging procedure and several pathways may remain that allow continued interaction of root canal bacteria with the host tissues, resulting in persistent or recurrent infection.

Margins of the root-end filling. Compromised place- ment, adaptation to the canal walls, and sealing ability of the filling material, can all compromise the seal of the root-end filling (Friedman 1991).

Accessory canals or isthmuses. Accessory foramina and, in specific teeth, isthmuses are frequently present in the apical portion of root canals. A root-end fill- ing may not seal these pathways, particularly when it is placed without magnification and illumination aids (Hsu and Kim 1997; Carr and Bentkover 1998).

Exposed dentinal tubules. Apical resection is typically performed with a bevel, exposing dentinal tubules at the cut surface (Gilheany et al. 1994). A shal- low root-end filling does not internally seal all the tubules. The exposed tubules provide a pathway for root canal bacteria to interact with the host tissues (Vertucci and Beatty 1986; Tidmarsh and Arrowsmith 1989; Gilheany et al. 1994).

Vertical root defect, crack, or fracture. Oral bacteria can colonize the crack or fracture line and sustain the infection even if a root-end filling effectively seals the root canal. Presence of a root crack or fracture should be ruled out before further treatment. Occasionally, there are dentinal defects that are not extensive enough to be diagnosed as fractures, but may adversely impact the prognosis (Tawil et al. 2015).

Because persistent infection after apical surgery is likely to be sustained by persistent root canal bacte- ria (Friedman 1991), the treatment of choice for its management is orthograde retreatment, perhaps with an MTA plug (Mente et al. 2015). When retreatment is not feasible, apical surgery should be repeated with an emphasis on effective sealing of the infected root canal.

15.9 Conclusions

The projected prognosis of treatment is a key consid- eration in selection of cases for endodontic treatment. Well-informed clinicians should project a specific prognosis for every tooth considered for treatment. As highlighted in this chapter, the chance of teeth with pri- mary and persistent apical periodontitis to heal after appropriate endodontic treatment ranges from good to very good, depending on specific outcome predic- tors. Furthermore, the chance for functional retention of the tooth in the long term is excellent. Therefore, whenever patients consider it feasible and acceptable, nonsurgical or surgical endodontic treatment should be attempted before considering tooth extraction and replacement. This is certainly the most conservative and, frequently, the most economic manner in which to treat endodontic infections. Given that the lack of healing is primarily mediated by persistent infective process, more research is required to define microbial virulence factors that mediate the diasease, as well as the microbial loads necessary to sustain clinically detectable disease. In addition, a better definition of the

372 Endodontic Microbiology

acceptable follow-up periods should be determined to help the practitioner make treatment decisions on cases that do not respond to treatment with complete radio- graphic and clinical healing.

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Chapter 16 Endodontic Infections and Systemic Disease Ashraf F. Fouad

16.1 Introduction 16.2 Systemic pain syndromes that mimic

endodontic pathosis 16.2.1 Myofacial pain 16.2.2 Maxillary sinus mucosal pain 16.2.3 Neurovascular pain 16.2.4 Neuropathic pain 16.2.5 Angina pectoris

16.3 Jawbone radiolucencies that mimic endodontic pathosis

16.4 Systemic diseases or conditions that may influence the pathogenesis or course of endodontic pathosis

16.4.1 Diabetes mellitus 16.4.2 Smoking

16.5 Systemic viral infections 16.5.1 HIV/AIDS 16.5.2 Herpes zoster 16.5.3 Other viral infections

16.6 Sickle cell anemia 16.7 Malignant neoplasms 16.8 Other systemic disease or

abnormalities 16.9 Hormonal variation and pregnancy

16.10 Patients on systemic medications 16.11 Genetic and epigenetic variations 16.12 Can endodontic infections contribute

to the pathogenesis of systemic disease? 16.12.1 Severe spreading endodontic

infections 16.12.2 Bacteremia as a result of

endodontic pathosis and/or treatment

16.12.3 Endodontic pathosis and cardiovascular disease

16.13 References

16.1 Introduction

In the past three decades, there has been an increasing awareness and recognition of the remarkable interac- tion between oral and systemic diseases. Various sys- temic diseases have been found to not only have oral manifestations, but also influence the presentation and healing of oral diseases. Likewise, a number of oral diseases, particularly periodontal disease, have been linked to the pathogenesis of some systemic diseases. Some studies have even suggested that periodontal therapy may contribute to the overall improvement of the systemic condition of the patient.

Endodontic pathosis is the result of the interplay of infectious agents and host response in the dental pulp and periapical tissues. Recent evidence suggests that certain systemic conditions may have an important role in modulating this interaction. Likewise, the root canal system may act as a pathway for, and/or reservoir of, certain unique microbial communities to contribute to, or cause systemic diseases. This chapter outlines the available information on the endodontic–systemic interrelationship, and provides some hypotheses for future exploration.

In the first section of this chapter, a brief presen- tation of several nonendodontic painful conditions or

385

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jawbone radiolucencies are presented. The clinician is frequently confronted with these conditions, and has to decide whether they involve pathosis of the pulp and periapical tissues. In this instance, the endodontic and the systemic or nonendodontic entity are not linked, but may be similar in their presentation such that they present a diagnostic dilemma. Clearly, the recognition of these entities is essential to allow the clinician to perform adequate diagnosis and to manage the patient effectively.

16.2 Systemic pain syndromes that mimic endodontic pathosis

In this section, the most common orofacial pain entities are reviewed. While some of these may have an inflam- matory or infectious origin, the tooth pulp is usually not involved in the pathogenesis of these conditions.

16.2.1 Myofacial pain

Myofacial pain is a type of chronic orofacial pain that is associated with inflammation of muscles in the head and neck area, primarily the muscles of mastication. The accumulation of inflammatory mediators such as cytokines, eicosanoids, and neuropeptides in certain areas of these muscles create a state of chronic pain that may mimic endodontic pain. Careful palpation of the muscles in the region and examination of alteration of function reveals the source of pain.

16.2.2 Maxillary sinus mucosal pain

The maxillary sinus lies in close proximity to maxillary posterior teeth. Sinus mucosa is frequently inflamed as a result of infections, allergies, or other forms of irritation. The inflamed sinus is frequently filled with serous exudates. This results in feeling of fullness, pain with sudden head movement, and headaches, par- ticularly early in the morning. Imaging of the sinuses by extraoral radiographs such as panoramic radiograph or a cone-beam computed tomography (CBCT) reveals the unilateral or bilateral opacity of the maxillary sinus. A CBCT would also reveal thickening of the Schnei- derian membrane which has relevance in cases where endodontic and sinus disease may coexist or the defini- tive diagnosis of the primary source of disease is dif- ficult to discern (Shanbhag et al. 2013; Nunes et al. 2016).

16.2.3 Neurovascular pain

This type of pain involves hemodynamic changes in vasculature within hard unyielding structures, such as the skull, and causes a variety of headaches that are frequently accompanied by referred pain to the dental tissues. Migraines are a form of neurovascular pain, and so are cluster and tension headaches. These forms of pain frequently complicate the diagnosis of dental pain, and may exaggerate the symptoms of postopera- tive pain.

16.2.4 Neuropathic pain

Neuropathic pain involves a pathologic change within the neural elements supplying a particular tissue, particularly sensory neurons. Neuralgia, neuroma, neuropathy, and neuritis are forms of neuropathic pain. These conditions frequently lead to a symptom of toothache, which brings the patient to the dentist’s office. Pulp testing is essential in identifying pulpal from nonpulpal causes of the pain in some of these conditions, but the diagnosis is more complicated in patients with previous endodontic treatment. A common systemic disease that leads to neuropathy is diabetes mellitus. The degree to which diabetic neuropathy contributes to dental pain in patients with endodontic pathosis is not known.

16.2.5 Angina pectoris

Anginal pain is a form of chest pain that arises from ischemia to the cardiac muscles. The pain is ill-defined, associated with exercise, and is frequently attributed to a gastric reflux or indigestion by the patient. It is often referred to the left shoulder, arm, neck, and face (Kreiner and Okeson 1999). Anginal pain may also be manifested as pain in the left mandible (Batchelder et al. 1987) and this may be the first presentation of the disease. In a recent multicenter trial of 186 patients with cardiac ischemia, it was found that 11 (6%) of them had pain exclusively in the craniofacial area and 60 (32%) had craniofacial pain concomitant with other types of pain. Craniofacial pain was most commonly in the throat, left and right mandible, left temporo- mandibular region, and teeth (Kreiner et al. 2007). A follow-up study was performed by the same group on 359 dental patients and 115 cardiac patients to deter- mine the quality of pain that patients described from the two different sources. It was found that “pressure”

Endodontic Infections and Systemic Disease 387

and “burning” were statistically associated with pain from cardiac origin, whereas “throbbing” and “aching” indicated an odontogenic cause (Kreiner et al. 2010).

There are several other pain syndromes whose signs and symptoms may mimic endodontic pain, although to a lesser degree that those described ear- lier: fibromyalgia, primary and metastatic malignan- cies, sickle cell disease, arteritis syndromes, and viral infections such as herpes zoster.

16.3 Jawbone radiolucencies that mimic endodontic pathosis

There are many other (usually nonpainful) jawbone radiolucencies that are similar to endodontic infections in their presentation (Table 16.1). The infections listed in Table 16.1 are likely to be sequelae of endodontic infections, while the remaining lesions are not likely

to be of endodontic origin. In a more recent study of 9723 periapical biopsies, 33% of the cases were peri- apical cysts and 40% were apical granulomas (Koivisto et al. 2012). The remaining 27% had a large number of diverse pathologic lesions (Figure 16.1).

Traditionally, the presence of an intact lamina dura around the root in a periapical radiograph has been a major sign that the radiolucency is nonendodontic in origin. However, the importance of the lamina dura in defining an endodontic lesion has been questioned (Ricucci et al. 2006a). Common examples of nonen- dodontic lesions include periapical cemental dysplasia (Wilcox and Walton 1989), keratocystic odontogenic tumor (Garlock et al. 1998; Koivisto et al. 2012), cen- tral giant cell granuloma (Dahlkemper et al. 2000), and metastatic carcinoma (Nevins et al. 1988). Occasion- ally, these lesions present in conjunction with teeth that have already been endodontically treated (possi- bly because of missed diagnosis) (Nevins et al. 1988;

Table 16.1 Unusual periapical diagnoses

Category Type Number of

cases

Cysts Odontogenic keratocyst Nasopalatine duct cyst Lateral periodontal cyst Residual cyst Globulomaxillary cyst (which is no longer a valid diagnosis)

22 4 4 3 1

Infections Actinomycosis Histoplasmosis Aspergillosis

15 1 3

Benign aggressive lesions Central giant cell granuloma Central ossifying fibroma Myxomas Central odontogenic fibroma Pindborg tumor Osteoblastomas Langerhans cell disease

24 1 2 1 1 2 3

Benign fibro-osseous lesions Periapical cemental dysplasia Other

30 2

Granulomatous inflammation Foreign body Pulse granuloma

40 22

Malignant lesions Carcinoma, including adenocarcimoma and metastatic lesions Sarcoma Lymphoma Multiple myeloma Leukemia

10 4 7 2 1

Source: Peters and Lau (2003). Reproduced with permission of Canadian Dental Association.

388 Endodontic Microbiology

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Fig. 16.1 Percentage occurrence of nonendodontic lesions in biopsied clinical cases where the jawbone radiolucency was similar to a periapical lesion. Source: Adapted from Koivisto et al. (2012). Reproduced with permission of Elsevier.

Wilcox and Walton 1989), thus a biopsy is the only method to determine the definitive diagnosis. In gen- eral, surveys of biopsy analyses following endodontic surgery in large populations show that the incidence of non-endodontic pathosis ranges from 1% to 27% (Spatafore et al. 1990; Nobuhara and del Rio 1993; Kuc et al. 2000; Koivisto et al. 2012). In addition to these lesions, systemic metabolic diseases such as primary or secondary hyperparathyroidism (Loushine et al. 2003) or osteoporosis may lead to an erroneous diagnosis of endodontic pathosis (Figure 16.2).

The astute clinician should therefore obtain a detailed history, perform all the necessary tests, obtain the necessary consultations, formulate a differential diagnosis, and send surgically excised tissues for

histopathologic examination in order to reach an accu- rate diagnosis.

16.4 Systemic diseases or conditions that may influence the pathogenesis or course of endodontic pathosis

16.4.1 Diabetes mellitus

Diabetes represents a group of diseases characterized by increased serum glucose caused by decreased pro- duction or action of insulin. Type 1 diabetes is the result of destruction of the pancreatic islet cells from autoimmune, genetic, or environmental causes, and

Endodontic Infections and Systemic Disease 389

Fig. 16.2 Periapical radiographs of multiple teeth with periapical radiolucencies that mimic endodontic pathosis. All these teeth responded normally to pulp testing. This patient had hyperparathyroidism secondary to kidney failure.

represents 5–10% of all diabetic patients. Type 2 dia- betes, which is the most prevalent form of diabetes, is associated with increased age, obesity, lack of exercise, and race/ethnicity. In the USA, African-American, Hispanic, and Native American populations have about twice the prevalence of type 2 diabetes as Caucasians. The most recent statistical estimates are from 2014 and reveal that about 9.6% of the US population or 29.1 million individuals are diabetic (in the first edition of this book, the number was 20 million). This figure con- sists of 21 million individuals who are diagnosed dia- betics, and the balance (about 28%) are undiagnosed. Diabetes is relatively rare among people younger than 20 years of age; however, it affects 12.3% (1 in 8) of people over 20 years of age, and 26% (1 in 4) of peo- ple over 65 years of age. (For full access to the data see: http://www.cdc.gov/diabetes/pubs/statsreport14/ national-diabetes-report-web.pdf.)

Numerous studies have shown that diabetes mellitus is associated with increased periodontal disease in all age groups, and after controlling for many confound- ing factors; for recent review see Mealey and Rose (2008). Meta-analyses reveal that periodontal treat- ment has a small but statistically significant associa- tion with improved glycemic control (Engebretson and Kocher 2013; Sgolastra et al. 2013). However, one

recent well-designed randomized clinical trial failed to demonstrate a direct causation effect (Engebretson et al. 2013).

Interest in the relationship of endodontic pathosis and its treatment to diabetes mellitus has a long his- tory. Older papers based on subjective findings con- cluded that for well-controlled diabetic patients there does not appear to be a risk involved with respect to postoperative healing (Bender et al. 1960). Another study among endodontic patients showed a reduction in postoperative healing of about 26% after 30 months’ follow-up with increase of about 20 mg/dL in postpran- dial glycemia (Cheraskin and Ringsdorf 1968). This showed the possible relationship of the healing to the glycemic measure, regardless of the diagnosis of dia- betes. More recently, a number of cross-sectional stud- ies have been reported on the prevalence of endodontic pathosis in diabetic and nondiabetic patients. Diabetic patients seemed to have a disproportionately higher percentage of acute odontogenic infections according to one study (Ueta et al. 1993). Compared to nondi- abetics, periapical radiolucencies were more preva- lent in relation to teeth with or without endodon- tic treatment (Britto et al. 2003; Segura-Egea et al. 2005; Lopez-Lopez et al. 2011; Marotta et al. 2012) in patients with type 2 diabetes. Likewise, long duration,

http://www.cdc.gov/diabetes/pubs/statsreport14/national-diabetes-report-web.pdf.

390 Endodontic Microbiology

insulin-dependent diabetes was associated with a higher percentage of nonhealing lesions in relation to endodontically treated teeth (Falk et al. 1989). A recent review has documented the strong association of existing periapical disease with diabetes and with increased glycemia (Segura-Egea et al. 2016).

Clearly, cross-sectional studies provide a limited perspective on endodontic healing because of the long duration necessary for healing of periapical lesions (see Chapter 15). Therefore, prospective cohort stud- ies with adequate follow-up periods and a high recall rate are optimal to describe the relationship of diabetes and endodontic pathosis.

One such study was made possible by the availabil- ity of an electronic patient record for endodontics in a dental school patient population that kept informa- tion on all patients from 1995 till 2001 (Fouad and Burleson 2003). At that time, the type 1 and 2 clas- sifications were not in use, so patients were classified according to whether they were on insulin. A total of 5210 endodontic cases were completed for nondiabetic patients, 70 cases for insulin-dependent (IDDM) and 214 cases from noninsulin-dependent diabetic patients (NIDDM). There was a strong trend for increased peri- apical pain in the IDDM group (p = 0.058). This may be related to the neuoropathy that is reported as one of the sequelae of diabetes, as noted earlier. Despite the presence of an active follow-up program in that institution, in which all patients were contacted and offered the opportunity to return for follow-up, only about one-third of patients returned for follow-up at any time after treatment, and only 544 cases (about 10%) were available for follow-up 2 years or longer after treatment. This is clearly a limitation of this study; however, given the large number there is merit in con- sidering these data from an epidemiologic perspective. As had been determined in numerous other endodon- tic outcome studies and, most relevant to this textbook, the presence of infection in the form of a preoperative lesion was significantly associated with reduced out- comes. However, infections were only present in 189 cases, including 17 from diabetic patients.

In that study, when the entire patient population (with all pulpal and periapical diagnoses) that reported for the 2-year or longer period was considered, there were no differences between diabetic and nondiabetic patients. However, when only patients with infections were considered (and both IDDM and NIDDM had to be pooled because of the low numbers), diabetic patients had a significantly higher risk of lack of

complete healing than nondiabetics. This was true even after controlling for gender, age, presence of a permanent restoration at the time of follow-up, pri- mary treatment versus retreatment, provider category (dental student versus resident), time to last follow-up, and the presence of periodontal disease on the tooth that was treated endodontically. Interestingly, this lat- ter factor by itself showed a significant increase in diabetic patients, consistent with the data that were described earlier.

These data were consistent with earlier animal research data, which had shown that compared with nondiabetics, diabetic animal models developed larger periapical lesions (Kohsaka et al. 1996), and were more prone to have increased morbidity and mortality in response to endodontic infections (Fouad et al. 2002).

Two more recent endodontic treatment outcome studies examined diabetes as an independent variable and found that it had no effect in one study (Ng et al. 2011) and marginal effect in another (Doyle et al. 2007). However, these studies did not examine the relationship between cases with periapical lesions and diabetes separately, and this was the seminal finding from the older study. Finally, the long-term survival of endodontically treated teeth was found to be sig- nificantly reduced in diabetic patients (Mindiola et al. 2006; Wang et al. 2011).

There are many potential mechanisms that can explain these findings about the diabetic host. It has long been known that diabetic patients have increased levels of a glycated form of tissue proteins called advanced glycated end-products (AGE) (Yan et al. 2004; Janket et al. 2008). These molecules interact with receptors (RAGE) to increase the expression of a number of local and systemic inflammatory media- tors, such as interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α). The chronic nature of the release of these cytokines creates a host environment that is susceptible to increased bone resorption and tissue damage. Some studies have also shown that in the diabetic host, immune cells such as mono- cytes develop a form that is hyperexcitable in that they respond to external irritation by releasing a large amount of inflammatory mediators, which would pro- duce the same chronic inflammation described earlier (Salvi et al. 2000). Diabetes may favor an increased reactive oxygen species in the dental pulp and peri- apical lesion, which increases inflammation and bone loss (Wolle et al. 2013). Finally, it has been hypothe- sized that the diabetic host may favor more bacteria,

Endodontic Infections and Systemic Disease 391

particularly virulent species, which may compromise the host responses more than the nondiabetic host. In endodontic infections, Eubacterium infirmum, a anaer- obic Gram-positive organism, was found to be more prevalent in diabetic patients (Fouad et al. 2003).

Complicating the study of diabetes and healing of periapical lesions are two factors. Glycemia may be the operative variable rather than diabetes, espe- cially given that many patients are not aware of their glycemic status. In addition, it has also been shown in animal studies that metformin, a drug commonly used to treat type 2 diabetes, reduces periapical bone resorption (Liu et al. 2012). Recent animal studies have also suggested that periapical lesions may contribute to an increase in glycemia in diabetic rats (Cintra et al. 2014), and that periapical lesions increased plasma TNF-α and insulin resistance but not glycemia in rats (Astolphi et al. 2013).

16.4.2 Smoking

Smoking as a general social habit is on the decline in western countries. Major tobacco manufacturers have been successfully prosecuted in recent years on the premise that significant health information was with- held or misrepresented to consumers. There is clear evidence of the contribution of smoking to cardiovas- cular disease, neoplasia, chronic bronchopulmonary disease, and periodontal disease. The latter associa- tion has been shown in numerous well-designed stud- ies in recent years, to the extent that a study assess- ing the extent of periodontal disease or evaluating the effectiveness of periodontal therapy performed at the present time would be lacking significant information if it did not control for smoking.

It is only recently that the association of endodon- tic pathosis, and its treatment, with smoking has been explored. In a cross-sectional Swedish study of 247 individuals, a significant association was found between smoking and the presence of apical periodon- titis or root canal treatment (Bergstrom et al. 2004). However, this relationship disappeared after control- ling for the age of the patient. More recently, a study was reported from Spain in which apical periodontitis in at least one tooth was found in 74% of smokers and in 41% of nonsmokers (p < 0.01) (Segura-Egea et al. 2008). Amongst smokers 5% of the teeth had apical periodontitis, whereas in nonsmokers 3% of teeth were affected (p = 0.008). The same group later showed that the prevalence of periapical lesions is even higher

in smokers who are hypertensive (Segura-Egea et al. 2011). Finally, they showed that this association is still statistically significant after adjusting for age, gender, number of teeth, endodontic status, quality of root fill- ing, and diabetic status (Lopez-Lopez et al. 2012).

The results of a larger longitudinal study provided even more compelling data (Krall et al. 2006). In this study, known as the Longitudinal VA Dental Study, 811 men were recruited from 1968 to 1973 and followed till the present time. The age-adjusted incidence of root canal treatment—which would be a surrogate for pulp pathosis—was greater in current cigarette smok- ers relative to never-smokers and in men who stopped smoking less than 9 years compared to more than 9 years before. Incidence of root canal treatment was also greater in men who smoked for 12 years than in those who smoked for 5–12 years, which was more than in those who smoked for less than 4 years. These findings clearly show a dose–effect relationship.

Smoking increased postoperative pain following endodontic surgery in a prospective Swiss cohort of 102 patients (Garcia et al. 2007). In this study, the number of cigarettes smoked was not significant, but patients who also had poor oral hygiene had significant postoperative pain and swelling. Therefore, taken together, it appears that smoking may have an influence on the pathogenesis of pulpal disease, the prevalence of apical periodontitis, and postoperative discomfort. It remains to be determined in longitudinal studies if smoking affects long-term endodontic treatment outcomes.

16.5 Systemic viral infections

16.5.1 HIV/AIDS

Human immunodeficiency virus (HIV) infects CD4 cells, resulting in significant deficiency in specific immunity. It is generally accepted that patients whose CD4 count is below 200 per cubic millimeter have more severe clinical manifestations of the disease, and frequently present with a number of comorbid bacterial and fungal infections. When HIV was first identified, and the mechanisms first described, there was a concern that patients with endodontic infec- tions would have significant perioperative symptoms because they are immunocompromised. Earlier case reports of endodontic postoperative symptoms and flareups in HIV-infected patients raised some concerns

392 Endodontic Microbiology

about this issue (Hillman 1986; Gerner et al. 1988). It became clear that the virus can easily be detected in pulpal (Glick et al. 1989) and periapical (Elkins et al. 1994) tissues, as would be expected, and therefore these tissues are likely to be compromised as well.

A series of animal studies examined the influence of specific immune responses on the pathogenesis of periapical lesions. Pulp necrosis and the development of periapical lesions was shown to occur at compa- rable rates in normal and severe combined immune deficiency (scid) mice when pulp exposures were left open to the oral cavity (Fouad 1997). Scid mice lack all types of T and B cells, and therefore have no form of specific immunity. It was also shown that RAG-2 scid mice that are exposed to large amounts of viru- lent endodontic pathogens develop disseminating oral infections, although the lesion sizes were not larger than those in control animals (Teles et al. 1997). To further isolate the specific immunodeficiency associ- ated with the spreading acute infections, a later study revealed that T-cell-deficient animals were equivalent in their response to virulent endodontic pathogens, and that B-cell-deficient animals did have the significant morbidity identified in the earlier study (Hou et al. 2000). This clearly shows that T-cell deficiency, as is the case in HIV infection, does not seem to com- promise the ability of the host to mount an effective immune response to endodontic infections.

Consistent with these results, a large clinical study of over 330 patients reporting for dental treatment was later published to show that infection with HIV and a CD4 count of <200/mm3 was not a risk factor for more extensive or aggressive endodontic infections (Glick et al. 1994). Another study was reported, in which endodontic treatment of 57 HIV-positive patients (and 17 patients who did not report HIV infection) were endodontically treated. In the 1–3 month postoper- ative period, there were no differences in the com- plications reported between the two groups (Cooper 1993). Finally, an endodontic treatment outcome paper was more recently reported, in which 33 HIV-positive patients were compared with an equivalent number of control subjects using the Periapical Index (PAI) at 1 year postoperatively. The results revealed no differ- ences between the groups (Quesnell et al. 2005).

A recent study examined the immune response dif- ferences of HIV-positive and HIV-negative patients undergoing endodontic treatment in teeth with peri- apical lesions immediately after root canal instrumen- tation and 1 week later (de Brito et al. 2015). There were significantly higher levels of CD4+CD28+ and

CD8+ T cells at the second appointment than the first appointment in both groups. In HIV-negative patients, an increase in IL-10 and CXCR4 and a decrease in proinflammatory cytokines such as receptor activator of NF-κB ligand (RANK-L), IFN-γ, IL-1β, and CCL5 was shown. However, in HIV-positive patients there was an increase in cytokines IFN-γ, IL-1β, TNF-α, and IL-17A, and chemokines CCL-2, CXCR4, and CCR5. These results reveal a prolonged inflammatory response in the HIV-negative patients after control of the root canal irritants.

16.5.2 Herpes zoster

Herpes zoster is caused by varicella zoster virus, which causes chicken pox in childhood, then remains dor- mant with periodic exacerbations in the form of her- pes zoster in adults. The viral infection presents as vesiculo-bullous lesions, which typically follow the innervation pathway of a nerve such as a somatic nerve, or a branch of the trigeminal nerve. The herpes lesions are self-limiting, and the infection subsides within 1– 2 weeks. Occasionally, the viral infection is followed by neuropathic neural pain along the pathway of the affected nerve, causing post-herpetic neuralgia which may be protracted in type and extent.

There have been a few case reports in the endodontic literature in which well-documented herpetic lesions were followed by pulpal pathosis in teeth that are in the path of affection by the viral infection. A case was reported in which a 23-year-old Asian male, who 5 months earlier had herpes zoster infection, developed pulpitis in teeth #9 and 10. These teeth were endodon- tically treated, and the patient was followed up. Later, he presented with pulp necrosis of teeth #11–13 (Goon and Jacobsen 1988) (Figure 16.3). It was surmised from this report that the viral infection had initiated pulpal pathosis in several teeth that were in the path of the affected nerve. However, what is more common is for patients with herpes zoster infection or post- herpetic neuralgia to present with symptoms of pulpal pain, but not to have any documented pulp disease. In these cases, accurate diagnosis and management of the underlying condition remain the appropriate course of action (Sigurdsson and Jacoway 1995).

16.5.3 Other viral infections

As seen in the discussion presented in Chapter 8, other viruses such as cytomegalovirus (CMV) and Epstein–Barr virus, but not herpes simplex virus, may

Endodontic Infections and Systemic Disease 393

Fig. 16.3 Periapical radiograph taken 5 months after the herpes zoster infection. Endodontic treatment can be seen in maxillary left lateral and central incisors. Periapical radiolucent lesions can also be seen at the apices of left canine, first and second premolar. Except for the endodontic access openings, all teeth were intact with negligible carious involvement or minimal restorations. Source: Adapted from Goon and Jacobsen (1988). Reproduced with permission of Elsevier.

394 Endodontic Microbiology

be involved in the pathogenesis of symptomatic peri- apical lesions, and lesions that are larger in size. CMV was shown to be equally prevalent in both periapi- cal cysts as well as odontogenic keratocysts (Andric et al. 2007). Much is still unknown about the relation- ship between viral infections and endodontic pathosis. It remains to be established whether viral infections (herpes zoster and other viruses) can initiate endodon- tic pathosis in the absence of bacterial infection. As noted in Chapter 8, the exact nature of viral involve- ment in pathogenesis of periapical lesion development is also poorly understood and data on the association of viral infections with pathogenesis of endodontic pathosis are equivocal at this time (Jakovljevic and Andric 2014; Jakovljevic et al. 2015).

16.6 Sickle cell anemia

Sickle cell anemia is a very prevalent congenital dis- ease inherited via autosomal recessive transmission. It is a homozygous disease in which both parents are at least carriers. Carrier or heterozygous state is expressed as sickle cell trait and is much milder in its clinical presentation. Sickle cell anemia is character- ized by sickle shape of the red cells due to substitution of a single amino acid—valine for glutamic acid—at the sixth residue of the β chain of hemoglobin (Lit- tle et al. 2008). Interestingly, sickle cell anemia tends to be prevalent in communities and countries where malaria (which results from infection by the parasite Plasmodium falciparum) is endemic, such as central African countries. It has been shown that this type of anemia confers protection against malaria by reducing adhesion of parasite-infected erythrocytes to endothe- lial cells of the microvasculature of the brain and other organs, which causes the life-threatening complica- tions of malaria (Cholera et al. 2008).

Two case series have been reported in which sickle cell anemia seemed to be involved in the pathogene- sis of pulpal and periapical disease. In the first series (Andrews et al. 1983), 22 patients with sickle cell ane- mia were examined. Five of 22 (23%) had jawbone radiolucencies. Three of 22 (14%) had pulp necrosis with periradicular lesions in eight teeth. Five of these eight lesions were in noncarious teeth, indicating a possible role of sickle cell defect in initiating spon- taneous endodontic infections (Figure 16.4). Another study had a case–control design, and included 36 sickle cell patients and an equivalent control group (Demir- bas Kaya et al. 2004). In this study, it was shown

Fig. 16.4 Examples of teeth in which no apparent etiology other than sickle cell anemia was reported for the development of pulp necrosis and periapical lesions. Reproduced with permission from Andrews et al. 1983.

that sickle cell anemia can be involved in initiat- ing pulpal pathosis, endodontic pain, as well as jaw- bone radiolucencies that mimic endodontic pathosis (Table 16.2).

Recent studies have shown that gene expression of proinflammatory cytokines IFN-γ, TNF-α, IL-1β, and

Endodontic Infections and Systemic Disease 395

Table 16.2 The effects of sickle cell anemia (SCA) on orofacial pain, bone radiolucency, and pulpal and periapical lesions

Patient or tooth characteristic SCA

(n = 36) Normal (n =36)

Number of patients with orofacial pain (not during SCA crises)

30 (83%) 5 (14%)

Number of patients with “step ladder” bone pattern

10 (28%) 0

Number of teeth examined 827 1084 Number of noncarious

unrestored teeth with pulp necrosis

51 (6%) 0

Source: Demirbas Kaya et al. (2004). Reproduced with permission of John Wiley and Sons.

IL-17A were significantly higher in apical periodonti- tis from sickle cell anemia patients than in the control group (Ferreira et al. 2015).

16.7 Malignant neoplasms

Primary malignant neoplasms may arise in a num- ber of oral tissues, including keratinized and nonker- atinized oral mucosa, mandibular and maxillary bone, and salivary glands. Locally invasive neoplasms such as ameloblastoma, which arises from odontogenic tis- sues, like other jawbone radiolucencies, may present in close proximity to teeth, thus mimicking the pre- sentation of endodontic infections. In this context, it is of interest that primary neoplasms have rarely if ever been reported in the dental pulp. It is not known why this may be the case.

Reports have been published of metastatic neo- plasms in the maxilla or mandible, causing radiolucent lesions, occasionally with invasive resorption of the root, pain, parasthesia, including numbness. While the tooth root and pulp may be affected in these situations, clearly, proper diagnosis and appropriate corrective therapy, which may include endodontic therapy, must be performed in consultation with the treating physi- cian or surgeon. Examples of these neoplasms include those that arise in the lung, prostate, breast, pancreas, or colon. Primary neoplasms of the immunologic cells that manifest as bone radiolucencies include multiple myeloma, eosinophilic granuloma, Letterer–Siwe dis- ease, and Gaucher’s disease.

16.8 Other systemic disease or abnormalities

Most clinicians appreciate that if a patient’s immune system is compromised, the patient may be more sus- ceptible to perioperative pain and flareups, dissemi- nated infections, and/or reduced postoperative healing. One study showed that patients with systemic aller- gies were more likely to have endodontic flareups than patients without allergies (Torabinejad et al. 1988). In another study, patients undergoing bone marrow transplantation and who had asymptomatic periapical lesions in relation to endodontically treated teeth were divided into two groups. In one group, retreatment, root-end resection, or extraction was performed while the control group had no treatment carried out before the bone marrow transplant. The dependent variables were number of days in the hospital with neutrope- nia (<500/mL) or elevated temperature (>100◦F). The results showed no significant differences between the two groups (Peters et al. 1993).

With respect to long-term outcomes, in one study 66 patients, who had normal or immunocompromised medical status, were followed for a minimum of 30 months (Marending et al. 2005). The medical con- ditions of the patients included type 1 and 2 dia- betes, kidney insufficiency, breast cancer and concomi- tant chemotherapeutic drugs, gout, steroids, ulcerative colitis, gastroesophageal reflux disease (GERD), and rheumatoid arthritis. The study showed that endodon- tic treatment outcome was significantly dependent on whether they were in the normal or immunocompro- mised group. Conversely, another study examined the effects of osteoradionecrosis on endodontic treatment outcomes (Lilly et al. 1998). In this study, endodon- tic treatment of 22 patients was performed on teeth in the area of irradiation with a mean of 5000 cGy within the previous 6 months. The mean postoperative evaluation period was 19 months. The study showed a success rate of 91%, which was consistent with that of other endodontic outcome studies of noncompromised patients.

16.9 Hormonal variation and pregnancy

Animal studies have shown that reduction in estro- gen (by ovariectomy) leads to an increase in periapical lesion production (Gilles et al. 1997). More recently, it was shown that inoculating the pulp space of mice with Porphyromonas gingivalis, isolated from a patient with

396 Endodontic Microbiology

persistent endodontic infection, caused translocation of the organism to the placenta, systemic inflammation, and premature birth (Ao et al. 2015). Another group showed that pregnant rats developed not only sys- temic inflammation, but also insulin resistance when periapical lesions were induced (Bain et al. 2009), and that with periapical infection the pups showed manifestations of brain inflammation (Bain et al. 2013).

16.10 Patients on systemic medications

Patients undergoing radiation therapy do not appear to have compromised healing following endodontic treatment. Another category of patients at risk of osteonecrosis, particularly following surgical proce- dures, are patients on intravenous bisphosphonates. These patients typically receive bisphosphonates for the treatment of multiple myeloma, Paget’s dis- ease, metastatic breast cancer, or oral bisphospho- nates for osteoporosis (Badros et al. 2006; Gutta and Louis 2007). Bisphosphonates interfere with osteo- clast action, thereby reducing bone resorption. Animal studies have shown that bisphosphonates reduce peri- apical lesion size (Xiong et al. 2010; Kang et al. 2013). The risk of osteonecrosis with oral bisphosphonates is significantly reduced, compared to intravenous drugs. One study showed no effects of oral bisphosphonates on the outcome of nonsurgical endodontic treatment (Hsiao et al. 2009).

Another category of drugs that are used to reduce bone resorption are antagonists for RANK-L, such as denosumab (Prolia). In one review it was shown that they cause osteonecrosis following implant placement in about 2% of cases (Boquete-Castro et al. 2016). In a rodent model, these drugs were shown to cause osteonecrosis during the pathogenesis of periapical lesions (Aghaloo et al. 2014).

In addition to their cholesterol-lowering properties, statins have been known for a long time to have osteo- conductive properties. A recent animal study showed that simvastatin reduced the size of induced periapical lesions in a rodent model (Lin et al. 2013). In patients, statins have been linked to pulp canal calcifications, thereby fostering their association with mineralization of hard tissues (Pettiette et al. 2013). Likewise, met- formin (a hypoglycemic agent commonly used for the treatment of type 2 diabetes) reduced periapical lesion size in animal models (Liu et al. 2012).

Finally, selective serotonin reuptake inhibitors, commonly used for the treatment of depression and anxiety, have been shown to mediate bone resorption, and were recently shown to interfere with the heal- ing of implants (Wu et al. 2014). Effects of these common drugs on endodontic lesions have not been reported.

16.11 Genetic and epigenetic variations

Genetic variations generally refer to differences in the phenotype of the host that are related to their gene expression and follow the Mendelian pattern of inher- itance. However, more recently it has been recognized that minor variations in the genome DNA sequence could also cause a different degree or extent to which a particular gene is expressed. This sequence variation is typically in a single nucleotide, thus the term sin- gle nucleotide polymorphism (SNP). These variations may allow an inflammatory mediator, a growth factor, or a receptor to be over- or underexpressed, thus lead- ing to a variation in the pathogenesis of disease or the rate of healing.

Case–control studies have shown significant reduc- tions in periapical healing, in otherwise adequately treated cases, in association with IL-1β allele 2 (Mor- sani et al. 2011), allele H131 of the FcγRIIA gene and its combination with allele NA2 of the FcγRIIIB gene (Siqueira et al. 2009). More recently, it was shown that a genetic variant of the COX-2 gene (a haplo- type composed of rs2383515 G, rs5277 G, rs5275 T, and rs2206593 A) was associated with postop- erative endodontic pain (Applebaum et al. 2015). Several other gene polymorphisms have been stud- ied, and showed less prominent or no associations with postoperative responses to endodontic treatment (Aminoshariae and Kulild 2015).

In addition to the DNA sequences, genetic expres- sion is also controlled by epigenetic variations. These primarily relate to the degree of methylation of a gene, or the presence of small noncoding RNAs in the nucleus, which significantly influence the gene expres- sion. A recent study revealed interesting epigenetic variations, related to the degree of methylation, of the FOXP3 gene promoter which acts as a master regulator of T-regulatory cells in periapical lesions of patients (Campos et al. 2015). In this study, periapical gran- ulomas and cysts had significantly higher methyla- tion of this gene than gingival controls, indicating that

Endodontic Infections and Systemic Disease 397

methylation has an important role in the suppressive activity involved in chronic periapical lesions.

16.12 Can endodontic infections contribute to the pathogenesis of systemic disease?

16.12.1 Severe spreading endodontic infections

As described in Chapter 10, acute endodontic infec- tions can spread to regional lymph nodes, fascial spaces, and distant organs, particularly when left untreated for long durations. In developing countries, and regions in industrialized countries where there is limited access to dental care, these types of infec- tion are seen with some prevalence and contribute to numerous hospital emergency room visits. The litera- ture contains several reports of spreading endodontic infections to cause mediastinitis (Garatea-Crelgo and Gay-Escoda 1991; Bonapart et al. 1995), fatal necro- tizing fasciitis (Stoykewych et al. 1992), and brain abscess (Corson et al. 2001). In 2007, there was a case of a 12-year-old child in the Washington DC area of the USA, who had a dental abscess, and whose treat- ment was delayed for 2 years because of access to care issues. He then developed a brain abscess and was treated for 6 weeks in a hospital emergency room, but eventually died as a result of complications of this condition. A series of epidemiologic studies related to hospital-based endodontic infections in the USA have recently been published which revealed that they account for over 403 000 annual emergency hospital visits (Nalliah et al. 2011), about 8000 annual admis- sions or hospitalizations (Allareddy et al. 2010), and 66 deaths over a period of 8 years (Shah et al. 2013). Therefore, it is clear that endodontic infections can result in spreading, life-threatening, infections.

Routine acute endodontic infections, which are seen frequently in dental and endodontic practices, may result in a number of signs and symptoms of systemic inflammation, albeit with less morbidity. These include fever, malaise, regional lymphadenopathy, anorexia, somnolence, accelerated degradation of skeletal mus- cle proteins, hypotension, and hepatic synthesis of a number of proteins such as complement and coagu- lation proteins (Kumar et al. 2003). Like other types of acute infections, these infections may also result in leukocytosis and increased erythrocyte sedimentation

rate. Systemic inflammation results in an increase in serum cytokines such as TNF-α, IL-1, and IL-6. IL-1 and TNF-α are responsible for temperature elevation because of their action on the thermoregulatory center of the hypothalamus through local prostaglandin pro- duction (thus, the effectiveness of nonsteroidal anti- inflammatory drugs to reduce fever). IL-6 causes an increase in serum acute phase proteins. Acute phase proteins, such as C-reactive protein (CRP), serum amy- loid A (SAA), and others, are produced in the liver and mediate many of the systemic effects of inflamma- tion. CRP has been implicated in the development of atherosclerotic plaque by inducing adhesion molecule expression in human endothelial cells (Pasceri et al. 2000), activating vascular smooth muscle cells causing induction of monocyte chemoattractant peptide (MCP) (Hattori et al. 2003), and the uptake of low-density lipoprotein by macrophages (Zwaka et al. 2001).

In endodontic infections, CRP has been investigated in a number of studies in the past two decades. In an older study, the serum concentration of acute phase proteins, CRP, α2-macroglobulin (AMG), α1-antitripsin (AAT), haptoglobin (HPT), comple- ment component C3, and ceruloplasmin (CER) were measured following endodontic treatment of cases with periapical lesions. The levels of AMG and AAT fell significantly as early as 7 days after treatment. All investigated acute phase proteins decreased signifi- cantly 3 months after treatment (Marton et al. 1988). In another study, CRP increased in the pulp of teeth with pulpitis, but not in serum (Proctor et al. 1991). CRP and SAA did not increase in a dog model of chronic apical periodontitis (Buttke et al. 2005). However, it was more recently shown that the number of patients with acute apical abscess who have a significant increase in CRP decreases significantly within 1 week of treat- ment (Ren and Malmstrom 2007). Interestingly, in this study, the number of patients with periodontal abscess or acute osteitis, who also had elevated CRP, did not change significantly after 1 week. Another study showed that the number of teeth with asymptomatic periapical lesions did not correlate with the levels of CRP (Willershausen et al. 2009). Taken together, these results suggest that acute phase proteins, but mainly CRP, increase systemically in acute periapical infections (but not acute pulpitis) and following endodontic therapy. They decrease gradually within 1–9 weeks following treatment. Chronic apical peri- odontitis, at least in the short term and with a limited number of teeth, does not seem to increase CRP. There

398 Endodontic Microbiology

are several other markers of systemic inflammation such as proinflammatory cytokines. Elevated systemic antibodies (immunoglobulins) indicated exposure to bacteria (oral and otherwise). A recent systematic review and meta-analysis examined the association of 31 systemic molecular markers with apical disease (Gomes et al. 2013). It was revealed that CRP, IL-1, IL-2, IL-6, asymetrical dimethylarginine (ADMA), IgA, IgG, and IgM were significantly increased in patients with apical periodontitis compared to con- trols. Meta-analysis showed that serum levels of IgA, IgG, and IgM were significantly increased in humans with apical periodontitis compared to healthy controls. More recently, it was shown that men and women with periapical lesions may have different expressions of markers of cardiovascular disease (Cotti et al. 2011, 2015). These studies showed that both men and women with apical periodontitis have higher IL-2 and lower endothelial flow reserve (EFR) than controls. However, only men with apical periodontitis have significantly higher ADMA and only women with apical periodontitis have significantly higher reactive oxygen species than controls.

16.12.2 Bacteremia as a result of endodontic pathosis and/or treatment

As noted in Chapter 12, instrumentation of the root canal in a patient with endodontic infection or peri- apical surgery can lead to bacteremia (Table 16.3). It appears that extrusion of bacteria occurs whether or not the instruments in the necrotic pulp cases are

maintained within the canal or extend past the work- ing length (Debelian et al. 1995). A study showed that the instrumentation itself appears to be causative of an increase in the incidence of bacteremia periop- eratively, compared to preoperatively (Savarrio et al. 2005). It is of interest in this study that instrumenta- tion of cases with vital pulp resulted in no bacteremia, and that the incidence of bacteremia was not differ- ent in patients with primary or persistent endodontic infections. Preoperative bacteremia in patients with chronic infections was found in only 7% of all cases (Savarrio et al. 2005). While this incidence is much lower than that which occurs during instrumentation, the duration of lesion induction, until it is discov- ered and treated, can take a long time measured in months or years, compared to the brief period that it takes during instrumentation. Furthermore, it is known that 15–20% of patients with preoperative lesions will not respond to nonsurgical treatment (as discussed in Chapter 15), and about 50% or more of persistent lesions may have bacteria in the periapical lesions (as discussed in Chapter 6). Therefore, the amount of bac- teremia that is caused by chronic endodontic infections preoperatively, and to some degree postoperatively if they do not respond to treatment, remains largely unknown.

The interest in bacteremia in this context is related to the effects that endodontic pathogens can create in distant systemic sites. While it is evident that acute periapical infections can result in transmission of bac- teria to distant locations, the situation is less clear with chronic infections, which are of longer duration and may result in chronic bacteremia. The effect may also

Table 16.3 Bacteremia associated with endodontic treatment

Treatment Incidence

(%) Study

NST 25 Bender et al. (1960) NST 17 Baumgartner et al. (1976) NST 20 Heimdahl et al. (1990) NST (intracanal) 31 Debelian et al. (1995) NST (overinstrumentation) 54 Debelian et al. (1995) NST 23–30a Savarrio et al. (2005) Flap reflection 83 Baumgartner et al. (1977) Periradicular curettage 33 Baumgartner et al. (1977)

NST, nonsurgical treatment. aThirty percent of patients had positive blood culture; but in 23% the same organism was identified in the endodontic and blood cultures.

Endodontic Infections and Systemic Disease 399

Table 16.4 Number of studies in which a particular oral pathogen has been detected in bacteremia specimens

Bacteria Single tooth extraction

Multiple extractions

(or not specified) Brushing or

flossing Periodontal

scaling Endodontic treatment

Gram-positive cocci Viridans streptococci 1 S. oralis group 2 1 1 2 S. sanguis (S. sanguis I) 4 2 1 1b

S. oralis (S. mitior, S. sanguis II) 2 1 1 1b

S. milleri group S. intermedius 5 1 1 1 S. mutans 3 1 S. salivarius group S. salivarius 3 1 1 1 3a

Parvimonas micros, Peptostreptococcus asaccharolyticus, P. evolutus, P. anaerobius

5 2 1 1

Staphylococcus aureus 1 1 1 Staphylococcus epidermidis 1 1 1 1

Gram-negative bacilli/rods 3 Fusobacterium nucleatum, F. fusiforme 5 3 1 Aggregatibacter actinomycetemcomitans 1 Prevotella melaninogenica 5 2 1 1 Bacteroides (Tannerella forsythia,

Eikenella(?) corrodens, Prevotella ruminicola, P. oris, Parabacteroides (?) distasonis, Mitsuokella (?) multiacidus)

1 1 1 1 1b

Porphyromonas gingivalis) 1

?, Taxonomic update is to the best of this author’s knowledge. aBaumgartner et al. (1976); Beighton et al. (1994); Bender and Pressman (1956). bHeimdahl et al. (1990). Source: Adapted from Lockhart and Durack (1999). Reproduced with permission of Elsevier.

be cumulative, if the patient has multiple teeth with endodontic infections. Chronic bacteremia may con- tribute to diseases such as infective endocarditis, in susceptible patients (see Chapter 12), or atheroscle- rosis. A number of oral and endodontic pathogens have been implicated in the etiology of infective endo- carditis (Lockhart and Durack 1999) (Table 16.4). Furthermore, virulence genes that are critical in the pathogenesis of endocarditis, such as those for fibrinogen-binding protein and fibronectin-binding protein, have been identified in endodontic bacteria (Bate et al. 2000). However, the impact of bacteremia resulting from dental procedures or any one oral source on the pathogenesis of chronic systemic diseases is difficult to ascertain. To illustrate this difficulty, one study showed that tooth extraction was associated with a cumulative incidence of bacteremia (measured in

several perioperative specimens) of 60%, which was reduced to 33% if the patient was on amoxicillin, given according to the most recent American Heart Associ- ation prophylaxis guidelines (Lockhart et al. 2008). However, the cumulative incidence of bacteremia fol- lowing tooth brushing in this study was 23%, raising the concern that this daily routine may be more haz- ardous for the susceptible patient in the long term than a single tooth extraction.

16.12.3 Endodontic pathosis and cardiovascular disease

Recent studies have shown with some degree of certainty that cardiovascular disease (CVD) is a result of the interaction of genetic predisposition and environmental risk factors. The pathogenesis of

400 Endodontic Microbiology

Fig. 16.5 Transmission electron micrograph of internalized Porphyromonas endodontalis. Internalized P. endodontalis (arrow) in human coronary artery endothelial cell following a 90-minute infection. Source: Dorn et al. (2002). Reproduced with permission of John Wiley and Sons.

atherosclerosis, which is the underlying disease that leads to CVD mortality, stroke, and myocardial infarc- tion, involves the formation of an atheromatous plaque on the intimal walls of major vessels. The inflamma- tory etiology of this lesion may start with the invasion of vascular wall with pathogenic strains of bacteria, thus inducing an inflammatory response. An early lesion is composed of fatty streaks or plaques, which eventually mature into established lesions that include lymphocytes, macrophages, and bacteria. In an in vitro experiment, it was shown that Porphyromonas gingi- valis strain 381 (an invasive periodontal and endodon- tic pathogen) and Porphyromonas endodontalis ATCC 35406 (an endodontic pathogen) are capable of invad- ing human coronary artery endothelial cells (Dorn et al. 2002) (Figure 16.5).

Acute endodontic infections, endodontic treatment and, presumably, endodontic infections that go through phases of exacerbation and remission without appro- priate treatment, may raise systemic inflammation, and acute phase proteins.

The release of bacteria from the necrotic pulp space into the periapex, and systemically in chronic endodontic infections is less clear. As noted in

Chapter 6, a significant proportion of persistent endodontic infections appear to have bacteria within the periapical lesions (Sunde et al. 2003). In primary endodontic infections, the physical presence of bac- teria in the periapical lesion has been shown in a number of older studies but not with a high degree of consistency, presumably because of the limitations of the older techniques (Nair 1987; Fouad et al. 1992; Ricucci et al. 2006b). More recently, molecular tech- niques have more consistently shown the presence of bacteria periapically in models of chronic periapi- cal lesions (Russo et al. 2008) (Figure 16.6). Clearly, once the bacteria escape to the periapical region, there is no barrier to prevent slow systemic dissemination. This could occur from biofilms located within the root canal, on the root apex (Ricucci et al. 2005), or within the lesion itself such as with periapical actinomycosis (see Chapter 6).

In recent years, a number of investigations have shown the presence of oral bacterial pathogens in atheromatous plaques removed from patients who underwent endarterectomies for the management of carotid artery and other major vessel thrombosis. In one study, Tannerella forsythia was prevalent in 30%, P. gingivalis in 26%, and Prevotella intermedia in 14% of the endarterectomy specimens from 50 patients (Haraszthy et al. 2000). Interestingly, Chlamydia pneumoniae, which is one of the key organisms that are thought to be involved in the pathogenesis of atherosclerosis, was present in only 18% of these patients. In another report, 23% of the same type of specimens in 26 patients had Treponema denticola. This organism was not found in any of the nondiseased aorta specimens that were used as controls (Okuda et al. 2001). More recently, atheromatous specimens from two groups of patients were investigated: a young group that died of cardiovascular disease who were sampled post-mortem (n = 20), and an elderly patient group following endarterectomy (n = 9) (Kozarov et al. 2006). Quantitative real-time polymerase chain reaction (PCR) was used to identify the following organisms: Aggregatibacter actinomycetemcomitans, P. gingivalis, P. intermedia, Streptococcus mutans, T. denticola, Eikenella corrodens, and the nonoral Staphylococcus aureus, Staphylococcus epidermidis, and C. pneumoniae. The prevalence of these organ- isms in both groups is in the range 40–80%, with P. gingivalis, P. intermedia, A. actinimycetemcomi- tans, C. pneumonia, and S. epidermidis being the most prevalent, in this order. These studies have not

Endodontic Infections and Systemic Disease 401

AF = apical foramen

B = bone

C = cementum

D = dentin

PA = periapical lesion

Fig. 16.6 Mouse molar stained with universal bacterial PNA-FISH probe positive fluorescence (red) for bacteria in periapical tissues: (1) H&E stain of lesion (x200); (2) FISH stain of the same region at the same magnification (x200); (3) magnification of (2) (x1000) (unpublished data).

documented the periodontal or endodontic status of these patients; however, most of these organisms have been consistently identified in endodontic infections.

It is also noteworthy that the oral cavity, and indeed the pulp space of teeth with endodontic infections, may act as a reservoir for some bacteria, which are not common to the oral cavity, but are capable of causing significant systemic disease. These bacteria could then escape into systemic organ systems either directly or through a hematogenous route. For example, Strepto- coccus pneumonia and C. pneumoniae are organisms involved in the initiation of pneumonia, bronchitis, and sinusitis, among other respiratory diseases, and have been identified in the oral cavity (Mantyla et al. 2004; Suzuki et al. 2006). However, a recent analysis of 40 endodontic infections failed to identify either of these

organisms from endodontic specimens (Nandakumar et al. 2008).

In the last decade, there have been a number of epidemiologic studies that provided compelling evi- dence for the association between the presence and severity of periodontal disease and cardiovascular dis- ease. A meta-analysis on the subject showed statis- tically significant overall associations in 15 prospec- tive, cross-sectional, or case–control studies that were analyzed (Bahekar et al. 2007). It has been proposed that periodontal disease translates to the exposure of a large area of sulcular surface to pathogenic organ- isms, estimated to be approximately the area of the palm of a hand (Page 1998). The available surface area in endodontic lesions is likely much smaller than that, unless the patient has multiple teeth involved and

402 Endodontic Microbiology

has large periapical lesions. However, there is a high prevalence of periapical lesions in one or more remain- ing or root-filled teeth, as reported in cross-sectional studies. This has been estimated be in the range of 22– 72% of research subjects/patients (Frisk and Hake- berg 2005). Therefore, there may be some contribu- tions to systemic disease that this bacterial load exerts on patients, which has hitherto not been sufficiently studied.

Several epidemiologic studies on the association between endodontic pathosis and cardiovascular dis- ease have been reported. In a study of 1056 older women in Sweden, recruited in 1992–1993, endodon- tic variables were examined in relation to the inci- dence of coronary heart disease (CHD; comprising angina pectoris and myocardial infarction) (Frisk et al. 2003). The independent variables included were num- ber of root-filled teeth, number of teeth with peri- apical radiolucencies, tooth loss, age, marital status, smoking, alcohol habits, body mass index, waist–hip ratio, serum cholesterol and triglyceride concentra- tions, hypertension, and diabetes. A multivariate logis- tic regression analysis did not prove the endodontic variables to be predictive of CHD, but showed that age and tooth loss (of more than 16 teeth) were sig- nificantly associated with CHD. A bivariate logistic regression analysis showed a positive significant asso- ciation between subjects with root fillings = 2 and CHD, but for teeth with periapical radiolucencies the bivariate analysis did not show an association with CHD. The effects of endodontic treatment (as a sur- rogate variable for pulp inflammation) on CHD were evaluated in a large longitudinal study of older male health care professionals (Joshipura et al. 2006). In this study, the subjects were recruited in 1986–2000, and were surveyed about the history and timing of root canal therapy, as well as the presence of caries. Med- ical records were also evaluated, including cause of death for subjects who died during this period. Data for 34 683 participants were analyzed. Multivariate analysis showed that men with root canal treatment had significant (albeit small) CVD risk, were older, and were slightly more likely to be current smokers. There was a stronger association between history of root canal treatment and CVD when men with two or more root canal treatments were included than any root canal treatment, indicating a dose–response effect. There was also a stronger association between history of root canal treatment and CVD when men who were less than 56 years old at baseline, or who were current

smokers (vs. past smokers or had never smoked) were analyzed separately (Joshipura et al. 2006). Interest- ingly, the strongest associations with CVD in this study were seen among dentists who were current smokers or had two or more root canal treatments.

Another epidemiologic study sought to link the inci- dence of CVD more directly to endodontic infections (referred to as lesions of endodontic origin) (Caplan et al. 2006). In this study, 708 men (mean age 47.4) who were a part of the VA Dental Longitudinal Study in the Boston area, and who were not VA patients themselves, were followed for 24 years. Thirty-five percent of the patients had periapical lesions, and 23% were even- tually diagnosed with CVD after being recruited in the study. A multivariate regression analysis showed a significant association between lesion-years and CHD, but only in men ≤40 years of age. Taken together, these findings suggest that endodontic infections may pose a significant risk of CVD, at least among men in younger age groups.

More recently, another longitudinal cohort study was reported, in which 278 individuals were moni- tored for 17.4 ± 11 years in 1962–1995 in the Bal- timore Longitudinal Study of Aging (Gomes et al. 2016). The investigators correlated incidence of car- diovascular events with apical periodontitis, root canal treatment (collectively referred to as endodontic bur- den), and periodontal disease (all three referred to as oral inflammatory burden). Over the length of the study, 22% of individuals developed a cardiovascular event. Multivariate analysis revealed significant asso- ciations of these events with age, hypertension, and the endodontic burden.

Finally, it is noteworthy that a patient history (but not a family history) of CVD seemed to correlate with the presence of pulp stones in a pilot study (Edds et al. 2005). More studies, with control of some of the con- founding factors and in different patient populations, need to be performed to determine if these findings are consistent and could be of diagnostic or prognostic value.

Despite an interest that spanned over a century in the relationship of endodontic pathosis and systemic dis- ease, this area of knowledge is still in its infancy. Very few relationships have been established (Figure 16.7). More epidemiologic data, which are based on large patient and nonpatient populations, are clearly needed to determine if more specific associations can be estab- lished. Furthermore, better models need to be devel- oped in order to identify the mechanisms of disease,

Endodontic Infections and Systemic Disease 403

Fig. 16.7 Most likely relationships between endodontic disease and different systemic conditions according to available data. The question marks indicate doubtful associations or only at the level of animal models.

the pathogenicity of the involved endodontic microor- ganisms, and the contributions of the host.

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Glossary

Abscess. A pus-containing lesion encapsulated by a fibrotic capsule and an inflammatory reaction.

Antibody. A glycoprotein synthesized in a plasma cell, which is derived from a B cell that has interacted with a specific antigen. The antibody molecule can bind specif- ically to this antigen.

Antigen. A molecule that (a) triggers syntheses of antibody and/or a T-cell response and (b) binds specifically to an antibody or a lymphocyte receptor.

Apical periodontitis. Used for the inflammatory lesion in the apical area of the tooth that can be seen on radiographs.

Apoptosis. Programmed cell death. Cell suicide. A process controlled by a cell that results in the death of the cell.

Assembly. The stage (in virology) in the virus replication cycle, when components come together to form virions.

Bacterial artificial chromosome (BAC). A cloning vector able to carry very large inserts of DNA up to 150 kb.

Bacterial competence. The ability of a bacterium to take up DNA from the environment.

Bactericidal. The killing of bacteria. Bacteriostatic. The inhibition of the growth of bacteria with-

out their killing. Biofilm. Adherent microbial community that forms at a

solid–liquid interface. Blastospore. An asexual fungal spore produced by budding. B lymphocyte. Cell with surface receptors that can recognize

a specific antigen. Antigen binding can trigger a B cell to develop into an antibody-secreting plasma cell.

Capsid. The protein coat that encloses the nucleic acid of a virus.

Capsomere. A discrete component of a capsid, constructed from several identical protein molecules.

Chemokine. A cytokine that stimulates the migration and activation of cells in the animal body, especially cells involved in inflammation.

Chlamydospore. The thick-walled, big resting spore of sev- eral kinds of fungi.

Codon. Three base units of DNA that code for amino acids. Commensal. Microorganism living on or in a host but caus-

ing the host no harm. Complement. A series of proteins that are activated when

the body is infected. Activation has a number of antiviral effects, including lysis of infected cells and enhancement of phagocytosis.

Conjugation. The transfer of DNA from one bacterium to another through sex pili.

Cytokine. Protein that is secreted from a cell and has a spe- cific effect on other cells, including cells of the immune system.

Disinfection. A procedure to achieve a state without any pathogenic microorganisms.

DNA-dependent RNA polymerase. An enzyme that syn- thesizes RNA from a DNA template.

Domain. Discrete portion of a protein or a nucleic acid with its own structure and function.

Envelope. A lipid bilayer and associated protein, forming the outer component of an enveloped virion.

Fluorescent in situ hybridization (FISH). A histologic technique in which specific DNA probes hybridize to complementary DNA in tissues and are seen by visualizing a fluorescent marker using a confocal microscope.

Fosmid. A type of plasmid that can carry large inserts of foreign DNA.

Genome. The DNA or RNA that encodes the genes of an organism or virus.

Genotype. The complete set of genes of an organism or virus. Genus. Defined for a microorganism in the first part of the

Latin name, for example, Streptococcus, which cover all streptococcal species group.

409

410 Glossary

Hapten. A specific non-protein substance that does not itself elicit antibody formation, but does elicit the immune response when coupled with a carrier protein.

Horizontal gene transfer. DNA with the potential to trans- fer between bacteria by transformation, transduction, or conjugation, including plasmids, transposons, insertion sequences, bacteriophages’ genomes, and chromosomal DNA.

Host. A cell or an organism in which a virus or a plasmid can replicate.

Hypha (plural, hyphae). Septate or aseptate filament of a fungus. Hyphae are the main mode of vegetative growth and are collectively called a mycelium.

Icosahedral symmetry. A type of symmetry present in viruses where the capsid is constructed from protein molecules arranged to form 20 triangular faces.

Immunoglobulin. The glycoprotein that functions as an anti- body.

Latent infection. Infection of a cell where the replication cycle is not completed, but the virus genome is maintained in the cell.

Lipopolysaccharide. Molecule consisting of lipid and polysaccharide, found in the cell walls of Gram-negative bacteria.

Metagenomics. The study of all of the constituent genomes of a community as a single entity.

Major histocompatibility complex (MHC). A region of the vertebrate genome that encodes major histocompatibility proteins. MHC class I and II molecules are cell surface proteins that have important roles in immune responses.

Microbiome. All of the constituent members of a microbial community, considered as a single entity.

Microflora. All of the constituent members of a microbial community, considered as individual taxa.

Minimal bactericidal concentration (MBC). The antimi- crobial concentration that results in 99.9% reduction of CFU/mL compared with the original inoculum.

Minimal inhibitory concentration (MIC). The lowest antimicrobial concentration that completely inhibits detectable bacterial growth.

Mutation. An alteration in the sequence of DNA or RNA. Nuclear envelope. The structure composed of two mem-

branes that separate the nucleus from the cytoplasm in a eukaryotic cell.

Open reading frame (ORF). A fragment of DNA that encodes a protein, delimited by start and stop codons.

Operon. A group of genes that are able to produce messenger RNA from one or more structural genes within the operon.

Operational taxonomic unit (OTU). In bacteria, a taxo- nomic unit of a bacteria species or genus depending on the sequence similarity threshold. Typically, OTU cluster

are defined by a 97% identity threshold of the 16S gene sequence variants; however, 99% identity may also be used for species separation.

Osteomyelitis. Infection of the bone and bone marrow. Pathogen. A microorganism that produces disease in another

organism. Peptidoglycan. Polymer making up part of the cell wall in

bacteria. Periapical osteitis. An older term that was used synonymous

to apical periodontitis; however, it strictly means that the inflammation is within the bone tissue and that it cannot be evaluated on radiographs.

Phylotype. A term that indicates species or taxon (plural, taxa) particularly for bacteria that have not been culti- vated in vitro, and for which the phenotype has not been studied.

Plasmid. A self-replicating, extrachromosomal DNA molecule that is generally circular though can be linear and can replicate autonomously. Plasmids are common in prokaryotes and rare in eukaryotes. Artificial plasmids can be created to clone DNA sequence. Plasmids can carry genes for antibiotic resistance and virulence factors.

Polymerase. An enzyme that synthesizes RNA from a DNA template.

Polymerase chain reaction (PCR). An in vitro technique for amplifying specific DNA sequences.

Primer. A molecule (often short strand RNA or DNA) that provides a template for formation of another (usually longer) molecule such as in a PCR reaction.

Probe. A short, single strand DNA sequence attached to a marker and designed to hybridize to complementary DNA.

Proteomics. The collective study of expressed proteins in a host tissue or microbial community.

Pseudohypha (plural, pseudohyphae). Not true septate hyphae. Pseudohyphae are most often found in yeasts as the result of a sort of incomplete budding where the cells remain attached after division.

Quorum sensing. The ability of a bacterial community to determine the total number of organisms in the community, and to express certain genes when the total number reaches a threshold level.

Regeneration. In endodontics, regeneration refers to re- establishing a vital pulp tissue in a previously devitalized root canal space.

Repair. In endodontics, repair refers to re-establishing a non- native vital hard and/or soft tissue in a previously devital- ized root canal space.

Revascularization. In endodontics, revascularization refers to re-establishing a vascularized vital tissue in a previously devitalized root canal space.

Glossary 411

Revitalization. In endodontics, revitalization refers to re- establishing a vital tissue in a previously devitalized root canal space.

Reverse transcriptase PCR (RT-PCR). An in vitro tech- nique for converting data in RNA to complementary DNA (cDNA), using a reverse transcriptase. The DNA is then amplified by PCR.

Reverse transcription. Syntheses of RNA from DNA. Real-time PCR. Quantitative PCR in which every PCR cycle

is calibrated to known concentrations of DNA to calculate the number of amplified copies.

Resistance transfer factor (R factor). A plasmid carrying bacterial chromosomal DNA that is passed on to daughter cells during replication.

Resuscitation-promoting factors (Rpf). Proteins produced by bacteria that stimulate the growth of other bacteria and can revive dormant bacteria.

Retrovirus. A member of the family Retroviridae, so named because these viruses carry out reverse transcription.

Ribonuclease RNase. An enzyme that hydrolyses RNA. (RNase H is a ribonuclease that specifically digests the RNA in an RNA–DNA duplex.)

Sinus tract. A spontaneous drainage of an abscess, some- times designated fistula.

Species. Defined for a microorganism in the last part of the Latin name, for example, S. intermedius, where S. stands for Streptococcus and is usually abbreviated once it has been given in full in the text. Undefined species are given as spp. (plural) or sp. (singular), for exam- ple, Streptococcus spp. for several undefined streptococcal species.

Sterilization. A procedure used for obtaining a state with absence of any living (viable) microorganism. Microbio- logically, this includes the very resistant bacterial spores. In practice it also includes virus even if its absence cannot be controlled. In this book, sterilization is used for absence of culturable microorganisms.

Strain. Used for all isolates of a particular microorganism obtained from a sample. Different strains may belong to the same species.

Superinfection. A new infection complicating the course of antimicrobial therapy of an existing infection, caused by invasion by bacteria or fungi resistant to the drug(s) in use.

T cell (T lymphocyte). A cell with surface receptors that can recognize a specific antigen. Antigen binding can trigger a T cell to perform one of several roles, including helper T cell or cytotoxic T cell.

Tegument. A layer of protein and RNA between the capsid and the envelope of a herpes virus particle.

Transcriptase. An enzyme that carries out transcription. Transcription. Synthesis of RNA from complementary

DNA or an RNA template. Transcriptomics. The study of the transcriptome—the com-

plete set of RNA transcripts that are produced by the genome, under specific circumstances or in a specific cell—using high-throughput methods, such as microarray analysis.

Transduction. Transfer of genetic material (and its pheno- typic expression) from one cell to another by viral infection or the injection of genetic material by a virus (bacterio- phage) into a bacterium.

Transformation. The uptake of extracellular DNA and incorporation of the DNA into the recipient’s chromosome where there is a similar base sequence.

Translation. Synthesis of protein from the genetic informa- tion in mRNA.

Transposition. The insertion of DNA transposons into a chromosome without the need for homology between the donor and the recipient cells.

Transposon. A type of mobile genetic element, a section of DNA that can move within the genome of an organism, or between organisms. A transposable element encoding a function such as antibiotic resistance.

Virion. Virus particle. Virulence. A measure of the severity of disease or

pathogenicity that a microorganism is capable of causing. Virulence factors. Factors enabling a microorganism to

establish itself on or within a host, thereby contributing to the disease process.

Yeast. Any of various unicellular fungi.

Index

454 pyrosequencing NGS 95–7

A-δnociceptor fibers, pain 252–62 AAT (α1-antitripsin) 397–8 Abiotrophia 33–7 Abiotrophia defectiva 33–7 abscesses 13, 55, 64–72, 92,

106–19, 133, 134–44, 164, 167–9, 170, 180–1, 190–1, 213–14, 231–49, 270, 276, 277, 280, 281, 323–7, 397–8

see also cysts; sinus tracts definition 68–9, 133, 134–5,

234, 241 diagnosis 13, 135, 241, 245,

276 fistula formation 68–9 head and neck infections 69,

231–49, 270, 280, 397–8 incompletely developed teeth

323–7 pus 61–3, 64–72, 134–5,

234–49, 277 recommendations 280, 281 statistics 65–72, 106, 111,

134–5 treatments 135–6, 241, 244–9,

270, 280, 281, 323–7 accessory genetic elements

see also bacteriophages; insertion sequences; pathogenicity islands; plasmids; transposons

genetic aspects of bacterial virulence 153–7

Ace 37, 166–7 acetaminophen, pain management

261 acids 27–45

see also pH dental caries 27–45 tolerant caries species 32–7

acnes vulgaris 167 Actinobacteria 3–4, 29–37, 92–3,

104–18, 131 Actinomyces 7, 27–45, 54–75, 77,

85, 92, 104–18, 134, 137–9, 140–2, 153, 159, 161, 167–8, 187, 200, 292

definition 140–1, 167–8 stability effects 34–7

Actinomyces georgiae 33–7 Actinomyces gerencseriae 33–7 Actinomyces gerensceriae 30–7 Actinomyces israelii 28–37, 92,

104–18, 159, 167–8, 295, 370

Actinomyces meyeri 137–9 Actinomyces naeslundii 7, 30–7,

38–45, 137–9, 159, 167–8, 292, 316

Actinomyces odontolyticus 30–7 Actinomyces radicidentis 141 Actinomyces timonensis 30–7 actinomycosis 130–44, 169,

387–8, 400 see also extraradicular

endodontic infections acute necrotizing ulcerative

gingivitis (ANUG) 240

acute respiratory distress syndrome 241

see also breathing difficulties adaptive immune system 159, 164,

189–91, 256–60, 270–1, 392–4