answer from the powerpoint

Chapters 04, 06, & 13

Bacteria & Antibiotics

Public Health 4030

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Cell Shapes and Patterns

• Morphology: bacteria are found in several common shapes (and arrangements), which are useful in species identification

• Bacillus, (bacilli) rod shaped

• Coccus, (cocci) spherical

• Curved or spiral, (vibrio, curved; spirilla, rigid helix or wavy; spirochete, flexible helix)

• WHY WOULD THIS BE IMPORTANT FOR CONTROL?

Figure 04.01: Bacterial cell shapes and patterns.

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Naming Bacteria

• Bacteria are named using Linnaeus’s binomial classification system (having two Latinized names)

• genus name, capitalized (Escherichia)

• species name, not capitalized (coli)

• both names are italicized: Escherichia coli

• Why are they named this way? • Bacteria names are frequently instructive

• Escherichia is named for Theodor Escherich

• coli indicates its habitat (large intestine)

• Streptococcus indicates shape and arrangement

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Anatomy of the Bacterial Cell

Figure 04.02: A “composite” bacterial cell.

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Envelope—is external to the cytoplasm

• Plasma membrane, selectively permeable

• Cell wall (gram positive vs. gram negative) • G+: thick layer of rigid polymer, peptidoglycan

• G−: thin layer of peptidoglycan

• Prevents osmotic rupture of cell membrane

• Outer membrane (in gram negatives only) • Contains fever inducing endotoxin

• Capsule not integral to the cell • Is a virulence factor, anti-phagocytic

Do you remember this from an earlier chapter?

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The Gram stain is an important first step in the identification of bacteria pathogens. • Choice of antibiotics is influenced by the Gram stain reaction

• Broad spectrum antibiotics work against g+ and g-

• Narrow spectrum antibiotics work against g+ or g-

Figure 04.03: Gram stain.

Amoxicillin, Tetracycline

Z-Pak - Azithromycin

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Cytoplasm—is all of a cell’s contents enclosed within the plasma membrane.

• Nucleoid, region of cytoplasm containing chromosomal DNA

• Double stranded DNA (dsDNA)

• One or more circular and/or linear chromosomes

• E. coli, one circular

• Vibrio cholerae, two circular

• Borrelia spp., linear and circular

Figure 04.05: Binary fission.

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Cytoplasm (cont.) • Plasmids, small, circular, independently replicating, dsDNA

• Encode limited number of genes (few to many)

• Expand genetic capability of host cell • May encode virulence genes

• R factors: plasmids encoding antibiotic resistance genes

• Infectious nature, transmissible from donor to recipient

Figure 04.06: The infectious nature of plasmids.

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Spores – Endospores formed within cell

• Viable for long periods (perhaps centuries or longer)

• Resistant to heat, boiling, drying, radiation, and various chemical compounds, including alcohol

• Important pathogens • Bacillus anthracis, anthrax,

possible bioweapon

• Clostridium spp., tetanus,

botulism, gas gangrene

(all anaerobes)

Figure 04.07: Bacillus anthracis.

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Appendages

• Flagella, used for motility • Rotates like propeller

• Chemotaxis • Move toward attractant

• Move away from repellant

• Long, hollow, filament

made of subunits of flagellin

• Many arrangements (single,

Polar, bipolar, dispersed, etc.)

• Pili (singular pilus) • Shorter, straighter, thinner than flagella

• Filaments made of subunits of pilin protein

• Function • Adhesin, anchor for colonization of host cells and other surfaces

• pili, forms a bridge between donor and recipient bacterial cells, for transmission of DNA

Figure 04.09: Structure and arrangement of flagella.

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Slight Topic Detour – Genetic Material Discussion

• DNA structure • Deoxyribonucleic Acid is a polymer of repeated

nucleotides

• Nucleotide = a nitrogenous base, deoxyribose, and 1-3 phosphates

• The 4 nitrogenous bases (A, G, C, and T) can be grouped by structure into purines and pyrimidines

Figure 06.01A: Nucleotide.

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DNA Replication

• The “parental” double stranded DNA separates into single strands by breaking hydrogen bonds

• Each single strand acts as a template for the new “daughter” strand, following Chargaff’s rule (A-T; G-C)

• DNA replication is semiconservative

• Chromosome replication is usually bidirectional

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Figure 06.03: DNA replication: analogy of two zippers.

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Transcription: DNA to mRNA

• RNA polymerase uses one strand of DNA as a template for transcribing a mRNA copy

• mRNA is complementary to DNA template strand

• Transcription of mRNA • Begins with promoter

• Ends with terminator

• RNA polymerase uses one strand of DNA as a template for transcribing an RNA copy—a process similar to DNA replication

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Translation: mRNA to Protein • mRNA, in the language of nucleic acid

(i.e., the 4 bases, A, G, C, and U), is translated by ribosomes into the 20 amino acid language of protein

• Amino acid structure: • a central carbon atom

with one of 20 side chains

• amino group (NH2)

• carboxyl group (COOH)

Figure 06.09: Amino acids. (a) A

generalized structure for an amino acid.

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Characteristics of the genetic code • The mRNA is read in blocks of three letter codons

• Each codon hydrogen bonds with the complementary anticodon of a tRNA (the opposite end of the tRNA is bound to the amino acid it specifies)

• There are 64 possible codons in the genetic code:

• Only one start codon (AUG codes for methionine in eucaryotes and formyl-methionine in bacteria)

• There are 3 stop codons (no tRNA exists for stop codons)

• It is redundant, as most amino acids are encoded by two or more codons

• Due to “wobble,” the third nucleotide in a codon may not be significant in specifying an amino acid’s identity

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Table 06.02: The Genetic Code Decoder.

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Gene Expression

• A constitutive gene is always expressed (turned on)

• Regulated genes: • An inducible gene is expressed only when an inducer is

present

• A repressed gene is “turned off” when a repressor is present

• Operons, in bacteria, are a group of functionally related genes that are controlled by the same regulatory sequences (promoter)

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Gene Expression • Chromosomes

• Prokaryotes usually have a single chromosome

Table 06.03: Bacterial Disease, Genomes, and Genes.

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WHY SPEND ALL THIS TIME DISCUSSING DNA AND REPLICATION?

Bacterial Genetics • Mutations cause a change in the nucleotide sequence of

the DNA, there are three consequences for the cell:

• Harmful • Beneficial (rare) • Silent

• Cause of mutations: • Unrepaired error in DNA replication • Mutagens

• Chemical agents (nucleotide analogues, etc.)

• Physical agents (UV and ionizing radiation)

• Transposons or “jumping genes”

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Table 06.05: Mutations in bacteria.

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Recombination

• Vertical, sexual reproduction passes on genetic change from parents to offspring

• Horizontal (lateral), recombination occurs between a donor cell and a recipient (not reproduction)

• Three examples:

• Transduction (generalized or specialized)

• Conjugation

• Transformation

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Generalized Transduction

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Specialized Transduction

Bacterial Conjugation

• Requires cell-to-cell contact • ssDNA is transferred from

donor to recipient

• Donor requires F factor

• Donors are F+

• Have F plasmid

• produce sex pilus

• Recipient is F -

Figure 06.15: Bacterial Conjugation: direct transfer of DNA.

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During mating, a single strand of the F plasmid DNA crosses over from F+ donor into the F— recipient

Figure 06.16: Bacterial Conjugation F+ to F-.

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Bacterial Growth

• Limits to growth/multiplication of bacteria • Abiotic: temperature, the availability of oxygen and water,

etc.

• Biotic: disease, competition, and predation

Figure 04.10: Overnight growth in a broth changes from clear to turbid.

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Bacterial Growth – Four major growth phases • Lag - period of adaptation to new conditions

• Exponential/logarithmic - the cell population doubles with each generation (23 = 2 x 2 x 2 = 8; the exponent 3 equals the number of generations

• Stationary - rate of cell division is about equal to the rate of death (nutrients are depleted and toxins accumulate)

• Death - the number of cells dying exceeds the rate of cell division.

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Microbial Generation Times

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Culturing Bacteria: Diagnostics

• A clinical specimen is obtained to grow in culture to identify the suspected cause of the infection

• throat swab, urine or blood culture, etc.

• Inoculated into growth medium

• Streaked across agar plate media to aid identification

• Colonies may have identifiable morphology or properties

• Characteristic, texture, size, pigment, hemolysis, etc.

Figure 04.13: Isolated colonies on an agar plate.

Figure 04.15: Streptococci on

blood agar plate.

Courtesy of Dr. Richard Facklam/CDC

Author’s photo (RIK)

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Culturing Bacteria: Diagnostics

• Metabolic tests are used to identify bacterial pathogens.

• Rapid Strep antigen tests • Identification of Streptococcus

pyogenes

Figure 04.16: A variety of media in test tubes are used to determine the metabolic properties of

bacteria that are useful in identification.

30Figure 04.19: Rapid strep antigen test results.

Culturing Bacteria: Diagnostics

• A variety of media and diagnostic tests are available • Type selected depends on source of the specimen (bacterial

pathogens of the skin may differ from those typically found in a vaginal swab, etc.)

• Some bacteria can’t be grown, or they grow too slowly, requiring alternative approaches

• Detect specific anti-bacterial antibodies in patient’s blood

• Amplification of pathogen’s DNA

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Determining which antibiotics a bacterial isolate is sensitive to is important

• Antibiotic sensitivities are determined as follows:

• Spread an isolate onto the surface of an agar plate

• Apply antibiotic-containing disks to plate surface

• After incubation, “zones of inhibition” (no growth) form around any disks that inhibit the bacterium’s growth

Figure 04.17: Determination of antibiotic sensitivity.

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Rapid and cost-saving multitest procedures

• API-20E test contains twenty tests to differentiate bacteria belonging to the Enterobacteriaceae family (many cause urinary tract infections or diarrhea).

Figure 04.18: API-20E test strip before and after inoculation and incubation.

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Oddball (Atypical) Bacteria • Mycoplasmas

• Have no cell wall, thus, have no shape

• Very small and require specialized media for growth

• Disease: walking pneumonia

• Chlamydiae • Obligate intracellular parasites

• Disease: urethritis, trachoma, lymphogranuloma venereum

• Rickettsiae • Obligate intracellular parasites

• Transmitted by arthropods (except for Q fever)

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Antibiotics

• Considered the single most important discovery for the treatment of infectious diseases in the history of medicine

• Sulfonamide (sulfa) drugs, were the first “wonder” drugs

• Are antimicrobials, not antibiotics, because they are synthetic

• Inhibitor of enzyme involved in folate synthesis – inhibit grown and multiplication of bacteria (but doesn’t kill)

• Why aren’t humans affected?

• Saved millions of lives in World War II

• Antibiotics are produced, by definition, by microbes

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History of Antibiotics

• Penicillin became first antibiotic used in 1941 • Discovered by Alexander Fleming

• Fleming studied staphylococci – streaked a Petri dish and went on a two week vacation. Upon return he noticed his the plate contaminated with a common (Penicillium) mold

• Penicillin became a prescription drug in the mid-1950’s

• Several semisynthetic penicillin derivatives available (methicillin, ampicillin, and penicillin V, etc.), each with distinctive and beneficial properties

• Many other antibiotics were discovered in the post–World War II period

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Types of Antibiotics • Spectrum of activity varies by type of bacterium

• Broad-spectrum: inhibitory for a variety of gram positive and negative bacteria

• Used when type of bacterium is unknown

• May kill normal flora, allowing non-susceptible organisms to flourish (thrush) and antibiotic resistance

• Examples – Amoxicillin, Carbapenems, Streptomycin, Tetracycline, Chloramphenicol

• Treats bacteremia, sepsis, pneumonia

• Narrow-spectrum: treats specific families of bacteria and causes less disruption – know causative agent

• Does not kill as many of normal flora and less resistance • Examples – Azithromycin, Erythromycin, Vancomycin

• Treats ear infections, throat infections, UTI, typhoid, pneumonia

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Mechanisms of Antimicrobial Activity

How do they work? Antibiotics must show selective toxicity for bacteria.

Antibiotics are either…. • Bactericidal: directly kill cells

• Bacteriostatic: inhibits growth of cells, immune system eliminates cells

Mechanisms of antibiotics

A. Interference With Cell Wall Synthesis • Beta-lactam antibiotics interfere with peptidoglycan synthesis

B. Interference With Protein Synthesis • Bacteria have 70S ribosomes, vs. 80S for eucaryotic cells

C. Interference With Cell Membrane Function • Polymyxin B is used topically, because of toxicity (membranes are similar)

D. Interference With Nucleic Acid Synthesis • Block DNA replication (gyrase) and RNA polymerase (transcription)

E. Interference With Metabolic Activity • Antimetabolites competitively bind with enzymes (molecular mimicry) rendering them inactive; the sulfa drugs

mimic a precursor to folic acid

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Mode of action for antibiotics and other antimicrobial agents

Figure 13.16: Mechanisms of antimicrobial activity.

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Acquisition of Antibiotic Resistance • Antibiotic resistance is a major international public health problem

• Antibiotic resistance results from a genetic change – how they acquire….

• A chromosomal mutation (spontaneous genetic change)

• Usually confers resistance to only a single antibiotic

• Acquisition of R (resistance) plasmid from resistant strains

• Can confer resistance to several antibiotics at one time

• First reported in Japan in 1959 with multi-drug-resistant Shigella bacteria

• Transposons, or “jumping genes,” may carry genes for antibiotic resistance and can integrate into chromosomes or plasmids allowing rapid dissemination of antibiotic resistance

• In the presence of an antibiotic, natural selection will favor the survival of resistant cells until they are dominant in the population

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Mechanisms of Antibiotic Resistance Bacteria counter the effects of antibiotics by several mechanisms –

Methods of resistance…

• Enzymatic inactivation: e.g., beta-lactamase cleaves penicillins

• Alter antibiotic uptake

• Acquire membrane pump that expels antibiotics like tetracycline

• Decrease membrane permeability to certain antibiotics

• Modify target of antibiotic (antibiotic receptor site)

• Examples: penicillin resistance in streptococci and methicillin resistance in staphylococci

• Develop alternate metabolic pathway

• Resistance to sulfonamides is an example

• Bacteria share antibiotic resistance genes by horizontal gene transfer

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Bacteria counter the effects of antibiotics by several known mechanisms

Figure 13.17: Microbes fight back.

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Antibiotic Misuse Types of antibiotic misuse:

• Failure to complete dose (stop taking pills when feeling better)

• Failure to take full dose (trying to save pills for future use)

• Inappropriate use (using antibiotics for viral illness, etc.)

Consequences of misuse:

• Gonorrhea resistance to quinolones, in Hawaii, went from 1.4% to 9.5% in the 3 years ending in 2000

• More than 90% of the strains of Staphylococcus aureus are resistant to penicillin and other antibiotics

• Vancomycin resistance is appearing in staphylococci and enterococci

• Drug-resistant strains of tuberculosis are increasing worldwide

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As only the more susceptible bacteria are eliminated by using an insufficient dose or an inappropriate antibiotic, susceptible bacteria are replaced by strains possessing greater resistance

Figure 13.18: The paradox of antibiotic misuse.

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CDC list of the top drug resistant threats in U.S. (some are not bacteria) • Hazard Level – Urgent

• Clostridium difficile • Carbapenem-resistant Enterobacteriaceae • Neisseria gonorrhoeae

• Hazard Level – Serious • Multidrug-resistant Acinetobacter • Drug-resistant Campylobacter • Fluconazole-resistant Candida • Extended spectrum enterobacteriaceae • Vancomycin-resistant enterococcus • Multidrug-resistant Pseudomonas aeruginosa • Drug-resistant non-typhoidal Salmonella • Drug-resistant Salmonella serotype typhi • Drug-reistant Shigella • Methicillin-resistant Staphylococcus aureus • Drug-resistant Streptococcus pneumoniae • Drug-resistant Tuberculosis

• Hazard Level – Concerning • Vancomycin-resistant Staphylococcus Aureus • Erythromycin-resistant Group A Streptococcus • Clindamycin-resistant Group B Streptococcus

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http://www.cdc.gov/drugresistance/biggest_threats.html