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ORIGINAL ARTICLE

Manuka honey inhibits siderophore production in Pseudomonas aeruginosa J.M. Kronda, R.A. Cooper and S.E. Maddocks

Cardiff School of Health Sciences, Cardiff Metropolitan University, Cardiff, UK

Keywords

Chrome azurol S, pyochelin, pyoverdin.

Correspondence

Sarah Maddocks, Cardiff School of Health

Sciences, Cardiff Metropolitan University,

Western Avenue, Llandaff, Cardiff CF5 2YB,

UK.

E-mail: [email protected].

2013/0089: received 14 January 2013,

revised 4 April 2013 and accepted 14 April

2013

doi:10.1111/jam.12222

Abstract

Aims: The aim of this study was to determine whether manuka honey affected

siderophore production by three strains of Pseudomonas aeruginosa.

Methods and results: The minimum inhibitory concentration (MIC) of

manuka honey against each of the test bacteria was determined. The effect of

manuka honey on siderophore production by three strains of Ps. aeruginosa was

investigated using the Chrome azurol S assay (CAS) and CAS-agar plates.

Manuka honey at ½ and ¼ of the MIC for each strain led to reduced production of siderophores (1�3–2�2-fold less) which was found to be statistically significant when compared to the untreated control.

Conclusions: Manuka honey effectively inhibited siderophore production by

all three strains of Ps. aeruginosa used in this study. This suggests that manuka

honey may impact on bacterial iron homoeostasis and identified a new target

for manuka honey in Ps. aeruginosa.

Significance and impact of study: Pseudomonas aeruginosa is an opportunistic

human pathogen that can cause acute, life-threatening or persistent wound

infections. Part of the virulence repertoire of this micro-organism includes the

ability to sequester iron from the host during infection by the synthesis and

secretion of siderophores. Manuka honey may limit wound infection by

Ps. aeruginosa by limiting its ability to capture iron. This is the first time this

mechanism has been investigated.

Introduction

Infections are a considerable risk for patients with

wounds and can result in increased morbidity and mor-

tality. Gram negative bacteria cause the most severe infec-

tions, especially in burns patients, and Pseudomonas

aeruginosa is most commonly encountered (McManus

et al. 1985; Tredget et al. 2004). Systemic antibiotics rep-

resent the current treatment of choice despite problems

sometimes associated with side effects and insufficient tis-

sue penetration as a consequence of impaired blood sup-

ply to wound tissues. Increasingly, multidrug-resistant

strains of Ps. aeruginosa are found associated with wound

infections, and untreated or untreatable infections can

result in impaired wound healing, bacteraemia or sepsis

(Aloush et al. 2006; Strateva and Yordanov 2009). Topi-

cal antimicrobial agents are attractive forms of treatment

that are applied directly to the wound, negating the need

for an intact blood supply to the damaged tissues.

Manuka honey is an example of a versatile topical anti-

microbial agent that is effective against over 80 different

species of bacteria, but its precise mode of antibacterial

action is only just beginning to be understood. So far the

bacterial ‘target sites’ identified include genes involved in

stress response, cell division and adhesion to human

proteins (Henriques et al. 2011; Jenkins et al. 2011;

Maddocks et al. 2012; Roberts et al. 2012).

Bacteria colonizing the human host are subject to iron

restriction with the majority of iron bound tightly to host

proteins (such as transferrin, lactoferrin and ferritins),

which form part of the first line of defence against patho-

gens. To obtain sufficient iron, bacteria must compete with

the host and many rely on secreted siderophores to seques-

ter available iron. Pseudomonas aeruginosa produces two

extensively characterized siderophores, pyochelin and

pyoverdin. Pyoverdin is known to have a high affinity for

Journal of Applied Microbiology ISSN 1364-5072

iron, whereas pyochelin is a lower affinity siderophore

(Cox and Adams 1985). Siderophores have long been

regarded as virulence factors and siderophores in Ps. aeru-

ginosa have been shown to function as signalling molecules

(Beare et al. 2003). Ordinarily, siderophores are produced

only under iron limitation, and therefore interfering with

bacterial iron homoeostasis could be one of the ways in

which manuka honey limits wound infections caused by

Ps. aeruginosa. In this study, the universal chrome azurol S

assay was used to determine whether manuka honey has

the capacity to inhibit siderophore production, and

whether a combination of iron limitation and manuka

honey impacts on the survival of Ps. aeruginosa.

Materials and methods

Bacterial strains

Pseudomonas aeruginosa reference strain ATCC 9027

(NCIMB 8626) and clinical isolates 867 and LE08 were

used throughout the study. The clinical isolates were

from wound swabs collected from patients with chronic

wounds who attended an outpatient clinic at the Univer-

sity Hospital of Wales, Cardiff. All strains were cultured

aerobically at 37°C in nutrient broth. To attain iron restriction, 2,2-dipyridyl was added to the media to

achieve a concentration of 2 mmol l �1 .

Manuka honey

Sterile (gamma irradiated) medical grade manuka honey

(MedihoneyTM) was provided by Comvita in 50-g tubes

and is available as a licensed, commercial medical device

(Comvita, Berkshire, UK). It is supplied as a standard-

ized, 100% pure honey derived from the Leptospermum

scoparium plant in New Zealand.

Minimum inhibitory concentration

The minimum inhibitory concentration for manuka honey

against the test bacteria was determined by serial dilution

(0 – 50% w/v) in a total volume of 5-ml nutrient broth (Oxoid, Cambridge, UK) (according to British Society for

Antimicrobial Chemotherapy methodology for determin-

ing minimum inhibitory concentration (MIC); Andrews

2011; Roberts et al. 2012). Cultures were incubated for

16 h at 37°C in aerobic conditions. Assays were carried out in triplicate on each of three separate occasions. Where

combinations of manuka honey and 2,2-dipyridyl were

tested, cultures were incubated with 2,2-dipyridyl at con-

centrations of 0, 1, 2 and 3 mmol l�1 in media containing manuka honey equivalent to ¼ and ½ MIC (for each strain tested). Cultures were incubated as described above. MIC

readings were taken using a Spectrostar Nano spectropho-

tometer at a wavelength of 620 nm.

Chrome azurol S assay

The method to detect siderophore production was based on

that described by Schwyn and Neilands (Schwyn and Nei-

lands 1987), cultures were grown under conditions of iron

restriction by the addition of 2,2-dipyridyl (2 mmol l�1

was found to be the optimum concentration for sidero-

phore production; data not shown) as described above.

Chrome azurol S assay (CAS) solution was used for quanti-

fication of siderophores in culture supernatants. Cultures

were centrifuged for 10 000 g for 10 min and mixed with

the CAS reagent at a ratio of 1 : 1. After reaching equilibra-

tion, absorbance readings were measured at 620 nm. CAS-

agar plates were prepared as described previously (Deng

et al. 2006). Standardized suspensions (OD 0�5 at 620 nm) of Ps. aeruginosa were inoculated onto separate CAS-agar

plates. This procedure was performed in triplicate, and

plates were incubated at 37°C for 48 h. Callipers were used to measure the diameter (mm) of observable zones.

Results

A combination of iron restriction and manuka honey

treatment impairs growth of Pseudomonas aeruginosa

The MIC for the reference strain (ATCC 9027) was found

to be 30% (w/v) for manuka honey, and 10% (w/v) for

each of the clinical isolates (LE08 and 867) (data not

shown). For ATCC, 9027 growth was inhibited as the con-

centration of 2,2-dipyridyl increased, and the reduction

was statistically significant with each increment (P < 0�05, using Student’s t-test) (Fig 1a). With 1 mmol l�1 2,2-di- pyridyl, ½ MIC manuka honey completely inhibited growth; with 2 and 3 mmol l

�1 2,2-dipyridyl, both ¼ and

½ MIC completely inhibited growth (Fig. 1a). The same pattern of inhibition was observed for clinical isolate 867

(Fig. 1b). However, LE08 required 2 mmol l�1 2,2-dipyr- idyl and above combined with ¼ and ½ MIC to com- pletely inhibit growth (Fig. 1c). When supplemented with

200 lmol l�1 ferric citrate, MICs were restored to that equivalent to honey treatment alone (data not shown).

This suggests that manuka honey treatment in combina-

tion with 2,2-dipyridyl was more deleterious for microbial

growth than either condition individually.

Manuka honey at sublethal concentrations inhibits

siderophore production

Each strain of Ps. aeruginosa was grown under iron-

limited conditions and assessed for siderophore production

J.M. Kronda et al. Pseudomonas siderophores and honey

using the Chrome azurol S (CAS) assay in liquid

culture as well as on solid media. Untreated cells were

compared to honey-treated cells using either ½ or ¼ MIC of manuka honey for each of the three Ps.

aeruginosa strains studied. Cultures were equilibrated to

OD 0�5 (A620 nm) prior to assay. Quantitative CAS assay showed 1�8- and 2�2-fold reductions in sidero- phore production by strain ATCC 9027 treated with ¼ and ½ MIC manuka honey, respectively (Fig. 2). The clinical strains 867 and LE08 showed similar results of

1�6- and 1�3-fold (¼ MIC) and 2�2- and 1�8-fold (½ MIC) reductions in siderophore production, respec-

tively, following honey treatment. In each case, the

reduction in siderophore production compared to the

untreated control, at either ¼ or ½ MIC, was found to be statistically significant (P < 0�05). However, there was no statistically significant reduction in siderophore

production between ¼ and ½ MIC. CAS-agar plate assays supported this data, showing a marked reduction

in siderophore production evident as a smaller zone of

yellow colouration on the blue CAS-agar, following

honey treatment at both ¼ and ½ MIC for each strain studied (Table 1). Again a statistically significant reduc-

tion in zone size was evident for both ¼ and ½ MIC as compared to the untreated control, but not between

¼ and ½ MIC.

Discussion

During the 1960s the relationship between iron and bac-

terial virulence was recognized (Bullen et al. 1967). Due

to the presence of numerous iron-containing proteins in

the mammalian host, free iron is maintained at a concen-

tration of 10 �18

mol l �1

(Rogers 1973; Fischer et al.

1990). This is far below levels of iron necessary to sustain

bacterial growth, most species require between 10�9 and

0 0 mmol l–1 1 mmol l–1 2 mmol l–1

DIP (mmol l–1)

3 mmol l–1

0 mmol l–1 1 mmol l–1 2 mmol l–1

DIP (mmol l–1)

3 mmol l–1

0 mmol l–1 1 mmol l–1 2 mmol l–1

DIP (mmol l–1)

3 mmol l–1

0·5

O D

( 6

2 0

n m

)

1·5

2·5

0·5

O D

( A

6 2

0 n

m )

1·5

2·5

0·5

O D

( A

6 2

0 n

m )

1·5

2·5

* *

* * * *

* *

** **

* *

(a)

(b)

(c)

Figure 1 The combined effect of iron limitation and manuka honey

on growth of Pseudomonas aeruginosa; MIC values are for manuka

honey to which 2,2-dipyridyl has been added. (a) strain ATCC 9027;

(b) strain 867; (c) strain LE08. Dark grey bars, no manuka honey; light

grey bars, ¼ MIC; mid-grey bars, ½ MIC. DIP = 2,2-dipyridyl. Error

bars are the standard error of triplicate biological replicates calculated

using Minitab (v13). * indicates a statistical difference between the

test condition and the control (P < 0�05); ** indicates a statistical dif- ference between both the control and consecutive test condition

(P < 0�05).

100

* * *

% S

id e ro

p h o re

p ro

d u ct

io n

0 0% Honey 1/4 MIC 1/2 MIC

Figure 2 Chrome azurol S assay assay (in liquid) to quantify sidero-

phore production in response to manuka honey treatment. Dark grey,

strain ATCC 9027 (¼ MIC = 7�5%; ½ MIC = 15%); Light grey, strain 8626 (¼ MIC = 2�5%; ½ MIC = 5%); mid-grey, strain LE08 (¼ MIC = 2�5%; ½ MIC = 5%). Error bars are the standard error of trip- licate biological replicates calculated using Minitab (v13). *indicates a

statistical difference between the test condition and the control

(P < 0�05).

Pseudomonas siderophores and honey J.M. Kronda et al.

10�6 mol l�1 (Pradel et al. 2000). It is the lack of avail- able iron in the mammalian host that is believed to act as

a signal to up-regulate both iron acquisition pathways

and virulence components in bacteria.

It is evident from the data presented in this study that

manuka honey, at sublethal doses, reduced siderophore

production in both reference and clinical strains of

Ps. aeruginosa. The loss of ability to scavenge iron would

be highly detrimental to Ps. aeruginosa in the host envi-

ronment. In Ps. aeruginosa, the production of pyoverdin

is regulated in part by PvdQ, LasR (involved in the

regulation of quorum sensing), PtxR (a LysR-type tran-

scriptional regulator) and Fur (Ochsner et al., 1995;

Nadal-Jimenez et al. 2010; Jimenez et al. 2012). Despite

the mechanism remaining unknown, it is possible that

reduced siderophore production was the consequence of

altered responses by these transcriptional regulators.

Therefore potentially, honey treatment could also impact

upon quorum sensing; it is known that chestnut honey

can inhibit quorum sensing in several bacteria including

Yersinia enterocolitica, Erwinia carotovora and Aeromonas

hydrophila (Truchado et al. 2009).

In addition to the observed effect of manuka honey

on siderophore production, this study has revealed that

under conditions of iron limitation lower concentra-

tions of manuka honey effectively inhibit bacterial

growth and to levels equivalent to the MIC. Iron limi-

tation is known to have an inhibitory effect on the

growth of many micro-organisms and is regarded as

being part of the innate immune defence mechanism to

prevent colonization by pathogenic bacteria – the com- bined effect of manuka honey and low intracellular iron

would incur a significant strain on the bacterial cell.

However for some pathogens, iron limitation promotes

growth and makes the population more stable, this has

been noted for mucoid strains of Ps. aeruginosa isolated

from cystic fibrosis sufferers and is often coincident

with increased antimicrobial resistance (Anwar et al.

1989). Conversely, this study indicated that manuka

honey was more efficacious under iron-limiting condi-

tions; however, this may not necessarily be as a conse-

quence of improved antimicrobial activity. Therefore,

the arrest of growth observed in this study might be in

part a consequence of the inhibition of siderophore

production rather than the direct antimicrobial effects

of manuka honey.

Under the conditions of iron limitation used in this

study, Ps. aeruginosa was hypothesized to rely on sid-

erophore production to sequester any available iron; if

siderophore production was inhibited by manuka

honey, fewer siderophores would be available to seques-

ter and deliver iron to the micro-organisms, thus

impeding growth. Manuka honey contains a relatively

low concentration of iron, following dilution of honey

in this study; the iron concentration would have been

between 8�5 9 10�11 and 2�55 9 10�9 mol l�1 (Crane 1975), which is at the very threshold required for bac-

terial growth and would likely have been sequestered

by 2,2-dipyridyl. Consequently, this was unlikely to

contribute significantly to the overall iron availability,

thus negating the possibility that additional iron pres-

ent in the honey would have impacted on siderophore

production.

During infection, bacteria are exposed to iron restric-

tion and the application of manuka honey may further

compound bacterial stress potentially leading to greater

inhibition of growth as observed in this study. Sidero-

phores are classed as virulence factors and their reduced

production in response to manuka honey indicates that

it acts as an ‘antivirulence’ therapy. The growing prob-

lem of antimicrobial resistance means that identifying

new, efficacious treatments are imperative. Compounds

that exhibit antivirulence properties are attractive

because they don’t afford the same evolutionary pres-

sures associated with medications that result in bacterial

death. Previous studies have clarified that manuka honey

is indeed bactericidal, and this study has shown that it

is also an antivirulence agent. The apparent multifaceted

action of manuka honey therefore makes it both versa-

tile and effective as an antimicrobial treatment which is

paramount in an age where antibiotics are becoming

increasingly inadequate.

Acknowledgements

The authors would like to thank the Society for Applied

Microbiology for funding this project (JK; Students

into Work Scheme) and Richard Rowlands for technical

assistance.

Table 1 Chrome azurol S (CAS)-agar plate assay

CAS-agar plate assay: zone sizes in mm

Honey

treatment

Bacterial strains tested

ATCC 9027 LE08 827

0% 7�67 (SE: 0�23) 8�26 (SE: 0�1) 9�77 (SE: 0�1) ¼ MIC 4�59* (SE: 0�18) 5�56* (SE: 0�16) 5�04* (SE: 0�01) ½ MIC 4�26* (SE: 0�03) 4�58* (SE: 0�13) 4�38* (SE: 0�02)

Showing zone sizes (indicating siderophore production) in mm for

Pseudomonas aeruginosa ATCC 9027, LE08 and 867 in response to

manuka honey at the appropriate MIC, ½ MIC and ¼ MIC for each

strain studied. Statistically significant (P < 0�05) changes as compared to the untreated control are marked with an asterisk (*). The differ-

ence between ¼ and ½ MIC was not statistically significant

(P > 0�05).

J.M. Kronda et al. Pseudomonas siderophores and honey

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