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Re-Examining the Role of Hydrogen Peroxide in Bacteriostatic and Bactericidal Activities of Honey

Re-Examining the Role of Hydrogen Peroxide in Bacteriostatic and Bactericidal Activities of Honey ORIGINAL RESEARCH ARTICLE published: 25 October 2011 doi: 10.3389/fmicb.2011.00213 Re-examining the role of hydrogen peroxide in bacteriostatic and bactericidal activities of honey 1,2 2 2 2 Katrina Brudzynski *, Kamal Abubaker , Laurent St-Martin and Alan Castle Bee-Biomedicals Inc., St. Catharines, ON, Canada Department of Biological Sciences, Brock University, St. Catharines, ON, Canada Edited by: The aim of this study was to critically analyze the effects of hydrogen peroxide on growth Mirian A. F. Hayashi, Universidade and survival of bacterial cells in order to prove or disprove its purported role as a main Federal de São Paulo, Brazil component responsible for the antibacterial activity of honey. Using the sensitive perox- Reviewed by: ide/peroxidase assay, broth microdilution assay and DNA degradation assays, the quanti- Jun Liu, Mount Sinai School of Medicine, USA tative relationships between the content of H O and honey’s antibacterial activity was 2 2 Dmitri Debabov, NovaBay established The results showed that: (A) the average H O content in honey was over . 2 2 Pharmaceuticals, USA 900-fold lower than that observed in disinfectants that kills bacteria on contact. (B) A sup- *Correspondence: plementation of bacterial cultures with H O inhibited E. coli and B. subtilis growth in a 2 2 Katrina Brudzynski , Department of concentration-dependent manner, with minimal inhibitory concentrations (MIC ) values of Biological Sciences, Brock University, 7 7 500 Glenridge Avenue, St. Catharines, 1.25 mM/10 cfu/ml and 2.5 mM/10 cfu/ml for E. coli and B. subtilis, respectively. In con- ON, Canada L2S 3A1. trast, the MIC of honey against E. coli correlated with honey H O content of 2.5 mM, 90 2 2 e-mail: beebio@sympatico.ca and growth inhibition of B. subtilis by honey did not correlate with honey H O levels at all. 2 2 (C) A supplementation of bacterial cultures with H O caused a concentration-dependent 2 2 degradation of bacterial DNA, with the minimum DNA degrading concentration occurring at 2.5 mM H O . DNA degradation by honey occurred at lower than ≤2.5 mM concentration 2 2 of honey H O suggested an enhancing effect of other honey components. (D) Honeys 2 2 with low H O content were unable to cleave DNA but the addition of H O restored this 2 2 2 2 activity. The DNase-like activity was heat-resistant but catalase-sensitive indicating that H O participated in the oxidative DNA damage. We concluded that the honey H O was 2 2 2 2 involved in oxidative damage causing bacterial growth inhibition and DNA degradation, but these effects were modulated by other honey components. Keywords: oxidative stress, hydrogen peroxide, bacteriostatic activity, honey, DNA degradation INTRODUCTION bacterial growth by honey (White et al., 1963; Brudzynski, 2006). Hydrogen peroxide is generally thought to be the main compound We have observed that in honeys with a high content of this responsible for the antibacterial action of honey (White et al., oxidizing compound, bacteria cannot respond normally to pro- 1963; Weston, 2000; Brudzynski, 2006). Hydrogen peroxide in liferative signals and their growth remains arrested even at high honey dilutions. Pre-treatment of honey with catalase restored, to honey is produced mainly during glucose oxidation catalyzed by the bee enzyme, glucose oxidase (FAD-oxidoreductase, EC 1.1.3.4; a certain extent, the bacterial growth, thus suggesting that endoge- White et al., 1963).Thelevelsof hydrogenperoxideinhoneyare nous H O was implicated in the growth inhibition (Brudzynski, 2 2 determined by the difference between the rate of its production 2006). and its destruction by catalases. Glucose oxidase is introduced to Most of the conclusions on the H O oxidizing action on bac- 2 2 honey during nectar harvesting by bees. This enzyme is found in teria are drawn from the simplified in vitro models, where direct all honeys but its concentration may differ from honey to honey effects of hydrogen peroxide on bacterial cells were analyzed. In depending on the age and health status of the foraging bees (Per- contrast, honey represent complex chemical milieu consisting of nal and Currie, 2000) as well as the richness and diversity of the over 100 different compounds (including antioxidants and traces foraged diet (Alaux et al., 2010). Catalases on the other hand, are of transition metals), where the interaction between these compo- of pollen origin. Catalase efficiently hydrolyzes hydrogen peroxide nents and hydrogen peroxide may influence its oxidative action. to oxygen and water due to its high turnover numbers. The total We have recently unraveled that honey is a dynamic reaction concentration of catalase depends on the amount of pollen grains mixture which facilitates and propagates the Maillard reaction in honey (Weston, 2000), and consequently, the hydrogen perox- (Brudzynski and Miotto, 2011b). The Maillard reaction which ide levels in different honeys may vary considerably (Brudzynski, initially involves reaction between amino groups of amino acids 2006). or proteins with carbonyl groups of reducing sugars leads to a A substantial correlation has been found between the level of cascade of redox reactions in which several bioactive molecules endogenous hydrogen peroxide and the extent of inhibition of are continuously formed and lost due to their cross-linking to www.frontiersin.org October 2011 | Volume 2 | Article 213 | 1 Brudzynski et al. Re-examining the role of hydrogen peroxide other molecules (gain or loss of function; Brudzynski and Miotto, During last decades, several honey compounds were identified 2011b). We have shown that polyphenol-based melanoidins are as those implicated in honey antibacterial activity (for review, Irish a major group of Maillard reaction products possessing radical- et al., 2011). Despite this knowledge, the mechanisms by which scavenging activity (Brudzynski and Miotto, 2011a,b). These com- these compounds lead to bacterial growth inhibition and bacterial pounds are likely to interact with hydrogen peroxide and, depend- death have never been explained or proven in biochemical terms. ing of their concentration and redox capacity, either enhanced or Since there is a persistent view that hydrogen peroxide is a main diminished the oxidative activity of honey’s H O . In view of these player in these events, the aim of this study was to critically analyze 2 2 facts, we hypothesized that the oxidizing action of honey’s hydro- the effects of hydrogen peroxide on growth and survival of bacte- gen peroxide on bacterial cells may be modulated by the presence rial cells in order to prove or disprove its purported role as a main of other bioactive molecules in honey and therefore, may differ component responsible for the antibacterial activity of honey. from the action of hydrogen peroxide alone. Hydrogen peroxide is commonly used to disinfect and sanitize MATERIALS AND METHODS medical equipment in hospitals. For this purpose, the high concen- HONEY SAMPLES trations of H O in these disinfectants have to be maintained to Honey samples included raw, unpasteurized honeys donated by 2 2 overwhelmed defense systems of bacteria. At high concentrations, Canadian beekeepers and two samples of commercial Active ranging from 3 to 30% (0.8 to 8 M), its bactericidal effectiveness Manuka honey (Honey New Zealand Ltd., New Zealand, UMF has been demonstrated against several microorganisms including 20+, and 25+; M and M2, respectively; Table 1) that were used Staphylococcus -, Streptococcus -, Pseudomonas-species, and Bacillus as a reference in this study. During the study, honey samples were spores (Rutala et al., 2008).Under these conditions, the bacterial kept in the original packaging, at room temperature (22 ± 2˚C) cell death results from the accumulation of irreversible oxidative and in the dark. damages to the membrane layers, proteins, enzymes, and DNA A stock solution of 50% (w/v) honey was prepared by dis- (?Davies, 2000; Rutala et al., 2008; Finnegan et al., 2010). solving 1.35 g honey (average density 1.35 g/ml) in 1 ml of sterile, However, the hydrogen peroxide content in honey is about distilled water warmed at 37˚C. The stock solution was prepared 900-fold lower (Brudzynski, 2006). Moreover, the literature data immediately before conducting the antibacterial assays. indicate that the cell death of cultured mammalian, yeast, and bac- terial cells required H O concentrations higher than 50 mM and PREPARATION OF ARTIFICIAL HONEY 2 2 was associated with chromosomal DNA degradation (Imlay and Artificial honey was prepared by dissolving 76.8 g of fructose and Linn, 1987a,b; Brandi et al., 1989; Davies, 1999; Bai and Konat, 60.6 g of glucose separately in 100 ml of sterile, deionized water, 2003; Ribeiro et al., 2006), which is still five to 10-fold higher and by mixing these two solutions in a 1:1 ratio. The osmolarity then that observed in honeys. Therefore, we have undertaken this of the artificial honey was adjusted to that of the honey samples study to re-examined the role of hydrogen peroxide in antibacterial (BRIX) using refractometric measurements. activity of honeys. The hydrogen peroxide efficacy as an oxidative biocide is related BACTERIAL STRAINS to the bacterial sensitivity to peroxide stress. Defense mecha- Standard strains of Bacillus subtilis (ATCC 6633) and Escherichia nisms to oxidative stress varies between bacterial species such coli (ATCC 14948; Thermo Fisher Scientific Remel Products, as Gram-negative E. coli and Gram-positive B. subtilis used in Lenexa, KS 66215) were grown in Mueller–Hinton broth (MHB; this study and depend on the growth phase (exponential- ver- Difco Laboratories) overnight in a shaking water-bath at 37˚C. sus stationary-phase of growth), and on the adaptive and survival Overnight cultures were diluted with broth to the equivalent mechanisms (non-spore forming versus spore-forming bacteria; of the 0.5 McFarland Standard (approx.10 cfu/ml) which was Dowds et al., 1987; Chen et al., 1995; Storz and Imlay, 1999; Cabis- measured spectrophotometrically at A . 600 nm col et al., 2000). In honey, the effects of H O on the growth 2 2 and survival of microorganisms may be mitigated or enhanced ANTIBACTERIAL ASSAY due to the presence of honey compounds. On one hand, a high The antibacterial activity of honeys was determined using a broth content of sugars in honey that abstracts free water molecules microdilution assay using a 96-well microplate format. Serial from milieu inhibits bacterial growth and proliferation, but honey twofold dilutions of honey were prepared by mixing and transfer- dilutions may create growth-supportive conditions due to the ring 110 μl of honey with 110 μl of inoculated broth (10 cfu/ml abundance of sugars as a carbon source for the growing cells. final concentrations for each microorganism) from row A to row Hydrogen peroxide has deleterious effects on the growth and sur- H of a microplate. Row G contained only inoculum and served as vival of bacterial cells but honey antioxidants such as catalases, a positive control and row H contained sterile MHB and served as polyphenols, Maillard reaction products, and ascorbic acid may a blank. lower the oxidative stress to cells and may have a protective effect After overnight incubation of plates at 37˚C in a shaking water- against endogenous H O (Brudzynski, 2006). Even less informa- bath, bacterial growth was measured at A using the Syn- 2 2 595 nm tion exists on the mechanism of bactericidal action of honey’s ergy HT multi-detection microplate reader (Synergy HT, Bio-Tek hydrogen peroxide. The most fundamental and unsolved ques- Instruments, Winooski, VT, USA). tions concerns the molecular targets of H O cytotoxicity: does The contribution of color of honeys to the absorption was cor- 2 2 molecular hydrogen peroxide at concentrations present in honey rected by subtracting the absorbance values before (zero time) cause DNA degradation? incubation from the values obtained after overnight incubation. Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy October 2011 | Volume 2 | Article 213 | 2 Brudzynski et al. Re-examining the role of hydrogen peroxide Table1|Hydrogenperoxide concentrations in different honeys. Relationship between antibacterial activities of honey and hydrogen peroxide concentrations. Honey sample Plant source Hydrogen peroxide E. coli MIC dilution B. subtilis MIC dilution concentration (mM/l)* (concentration) (concentration) M2 Manuka (UMF 25) 1.04 ± 0.17 16 (6.25%) 16 (6.25%) H58 Buckwheat 2.68 ± 0.04 16 (6.25%) 8 (12.5%) H23 Buckwheat 2.12 ± 0.22 8 (12.5%) 4 (25%) H20 Sweet clover 2.37 ± 0.03 8 (12.5%) 4 (25%) H11 Wildflower/clover 2.49 ± 0.03 8 (12.5%) 8 (12.5%) H56 Blueberry 0.52 ± 0.11 4 (25%) 2 (50%) H60 Clover blend 0.67 ± 0.11 4 (25%) 2 (50%) M Manuka (UMF 20) 0.72 ± 0.02 4 (25%) 4 (25%) H200 Buckwheat 0.248 ± 0.02 2 (50%) 2 (50%) H203 Buckwheat 0.744 ± 0.01 4 (25%) 4 (25%) H204 Buckwheat 1.168 ± 0.05 4 (25%) 4 (25%) H205 Buckwheat/alfalfa 1.112 ± 0.02 4 (25%) 4 (25%) *Hydrogen peroxide concentration was measured at honey dilution of 8× (25% v/v) and represent an average of three experimental trials, where each honey was tested in triplicate. The absorbance readings obtained from the dose–response solution in a 1:1 ratio (v/v), an artificial honey solution, or with curves were used to construct growth inhibition profiles (GIPs). hydrogen peroxide solutions containing 5, 2.5, 1.2, 0.62, and The minimal inhibitory concentrations (MIC) were determined 0.3125 mM (final concentrations) H O prepared from the 20 mM 2 2 from the GIPs and represented the lowest concentration of honey stock solution. After overnight incubation at 37˚C with continu- that inhibited the bacterial growth. The MIC end point in our ous shaking, the cells were harvested by centrifugation at 3,000×g experiments was honey concentration at which 90% bacterial (Eppendorf ) for 30 s and then their DNA was isolated. growth reduction was observed as measured by the absorbance DNA ISOLATION at A . 595 nm The total genomic bacterial DNA was isolated from the untreated, Statistical analysis and dose response curves were obtained control cells and from the honey- or hydrogen peroxide-treated using KC4 software (Synergy HT, Bio-Tek Instruments, Winooski, cells using a DNA isolation kit (Norgen Biotek Corporation, VT, USA). St. Catharines, ON., Canada), according to the manufacturer’s HYDROGEN PEROXIDE ASSAY instructions. Hydrogen peroxide concentration in honeys was determined using AGAROSE GEL ELECTROPHORESIS the hydrogen peroxide/peroxidase assay kit (Amplex Red, Mol- Agarose gel (1.3%) electrophoresis was carried out in 1× TAE ecular Probes, Invitrogen, Burlington, ON, Canada). The assay buffer containing ethidium bromide (0.1 μg/ml w/v). Ten micro- was conducted in the 96-well microplates according to the man- liters of DNA isolated from the untreated and treated bacterial ufacturer’s instruction. The fluorescence of the formed product, cells was mixed with 5X loading dye (0.25% bromophenol blue, resorufin, was measured at 530 nm excitation and a 590 nm emis- 0.25% xylene xyanol, 40% sucrose) and loaded into the gel. The sion using the Synergy HT (Molecular Devices, BioTek Instru- DNA molecular weight markers selected were the HighRanger ments, Winooski, VT, USA) multi-detection microplate reader, 1 kb DNA Ladder, MidRanger 1 kb DNA Ladder, and PCRSizer and the dose–response curves were generated using the KC4™data 100 bp DNA Ladder from Norgen Biotek (Thorold, Ontario). The reduction software. gels were run at 85 V for 1 h and then visualized and photographed To calculate the hydrogen peroxide concentrations of the hon- using the Gel Doc 1000 system and the Quantity One 1-D Analysis eys, a standard curve was run alongside the honey serial dilutions. software (version 4.6.2 Basic) from Bio-Rad. The standard curve was prepared from the 200 μMH O stock 2 2 solution. Each of the honey samples, and the standard curve, were RESULTS tested in triplicate. DETERMINATION OF THE HYDROGEN PEROXIDE CONCENTRATIONS IN HONEYS CATALASE-TREATMENT OF HONEYS Formation of H O depends on the honey dilution since glu- 2 2 Honey were treated with catalase (13 800 U/mg solid; Sigma- cose oxidase is inactive in undiluted honey (White et al., 1963; Aldrich, Canada) at ratio of 1000 units per 1 ml of 50% honey Brudzynski, 2006). Honeys used in this study required a four to solution in sterile water for 2 h at room temperature. 16-fold dilution for the maximal production of hydrogen per- INCUBATION OF BACTERIAL CULTURES WITH HONEY OR HYDROGEN oxide to be observed (Figure 1). At the peak, H O concentra- 2 2 PEROXIDE tions ranged from 2.68 ± 0.04 to 0.248 ± 0.02 mM in the different Overnight cultures of E. coli and B. subtilis (1.5 ml, adjusted to honeys (Table 1), as measured by a sensitive, high-throughput 10 cfu/ml in MHB) were treated with either the 50% honey hydrogen peroxide/peroxidase assay (Amplex Red assay). www.frontiersin.org October 2011 | Volume 2 | Article 213 | 3 Brudzynski et al. Re-examining the role of hydrogen peroxide FIGURE1|Effect of honey dilutions on the production of hydrogen FIGURE2|Effect of increasing the concentration of exogenous peroxide. Honeys of buckwheat origin, H58 and H23, together with sweet hydrogen peroxide on the growth of E. coli (blue line) andB. subtilis clover (H20), and wildflower/clover (H11) produced distinctively higher (red line). Each point represents the mean and SD of three separate amounts of H O than Manuka (M2) or honey blends (H56 and H60). The experiments conducted in triplicate. 2 2 H O content was measured in twofold serially diluted honeys, the x axis 2 2 represents a log2 values. of honeys against E. coli, it appeared that almost all of the bac- teriostatic activity of honeys could be assigned to the effects of this compound (Figure 3A). In honeys, the endogenous H O of CONCENTRATION-DEPENDENT EFFECT OF HYDROGEN PEROXIDE ON 2 2 2.5 mM was of critical importance for the growth inhibition of E. BACTERIAL GROWTH INHIBITION coli ; the dilutions that reduced H O concentrations below this Throughout this study, we used terms: endogenous hydrogen per- 2 2 value showed a loss of honey potency to inhibit bacterial growth oxide to describe H O produced in honey by glucose oxidase 2 2 at the MIC level (Figure 3A). These data suggest that upon and exogenous hydrogen peroxide, which has been added as a 90 honey dilution, endogenous H O mediates growth inhibition of supplement to the bacterial cultures. These terms were intro- 2 2 E. coli. However, the concentrations required to reach MIC were duced in order to differentiate between the effects of honey’s 90 twofold higher than that found for exogenous hydrogen peroxide endogenous H O whose action on bacterial cells could be modu- 2 2 (2.5 versus 1.25 mM, respectively; Figure 3A). lated/obscured by other honey components as opposed to true, In contrast to E. coli, the inhibition of growth of B. subtilis well-defined action of exogenous hydrogen peroxide directly seemed not to be due to the effect of the levels of honey H O added to bacterial culture. 2 2 (Figure 3B). A rapid increase of B. subtilis growth with honey dilu- In agreement with previous reports (Brudzynski, 2006), we tions occurred despite the presence of high levels of H O (honeys found a strong correlation between the content of honey hydrogen 2 2 H58, H23, H20, and H11, Figure 3B). While exposure of the B. peroxide and the growth inhibitory action of Canadian honeys; subtilis culture to exogenous H O resulted in a concentration- honeys with high MIC values (6.25 to 12.5% v/v) correspond- 2 2 dependent growth inhibition with MIC at 2.5 mM (Figure 2), ing to16 to 8× dilution) also possessed a high content of H O 90 2 2 comparable concentrations of H O in honeys were ineffective. (Table 1). Since the minimum inhibitory concentration values 2 2 This indicated that other honey compounds/physical features were and the hydrogen peroxide peak were both observed at the 4 to responsible for the growth inhibition, such as honey’s high osmo- 16× honey dilutions, we hypothesized that the maximal hydro- larity. Moreover, higher honey dilutions, beyond 16-fold, had a gen peroxide production is required to achieve the bacteriostatic stimulatory effect on B. subtilis growth (Figure 3B). activity of honey at the MIC level. To test this assumption, we Thus, our results demonstrated for the first time that bacterio- first examined the dose–response relationship between the con- static effects of endogenous versus exogenous hydrogen peroxide centration of exogenous hydrogen peroxide, ranging from 10 to are markedly different due to the presence of other honey compo- 0.312 mM, and its growth inhibitory activity against E. coli and B. nents and, more importantly, that the effects of honey H O on subtilis. The dose–response curves and growth inhibitory profiles 2 2 bacterial growth are markedly different in E. coli and B. subtilis. revealed very reproducibly that H O concentrations of 1.25 mM 2 2 7 7 (1.25 μmoles/10 cfu/ml) and 2.5 mM (2.5 μmoles/10 cfu/ml) were required to inhibit the growth of E. coli and B. subtilis by COMPARISON OF EFFECTS OF HONEY AND HYDROGEN PEROXIDE ON 90%, respectively (Figure 2). DNA DEGRADATION IN BACTERIAL CELLS To exert effectively its oxidative biocide action, the concentrations of hydrogen peroxide in various disinfectants are high ranging RELATIONSHIP BETWEEN THE ENDOGENOUS H O CONTENT AND THE from 3 to 30% (0.8 to 8 M). In contrast, we have established that 2 2 GROWTH INHIBITORY ACTIVITY OF HONEYS the average content of H O in tested honeys ranged from 0.5 2 2 To investigate whether the content of honey H O influences to 2.7 mM (Table 1). The concentrations of H O measured in 2 2 2 2 honey’s bacteriostatic potency in a similar manner to that of honeys, therefore was about 260–1600-fold lower than the effec- exogenous H O , each honey was analyzed for growth inhibitory tive bactericidal dose of H O in disinfectants. Therefore, we asked 2 2 2 2 activity and the production of hydrogen peroxide in the same the question whether hydrogen peroxide at concentrations present range of honey dilutions. When the profiles of hydrogen peroxide in honey can cause DNA degradation and ultimately bacterial production were superimposed on the growth inhibitory profiles cell death. Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy October 2011 | Volume 2 | Article 213 | 4 Brudzynski et al. Re-examining the role of hydrogen peroxide FIGURE 3 | The relationship between bacteriostatic effect of described in the Section “Materials and Methods.” Of note: growth inhibition honey and the content of en-H2O2 on E. coli (A) or B. subtilis cultures (B) profiles of artificial honey of osmolarity equal to that of natural honey provided Growth inhibition profiles were determined for different honeys using MIC values of 25% (v/v) against both E. coli and B. subtilis. Each point or the broth microdilution assay (columns). The content of honey H O at column represents the mean values of three separate experiments run in 2 2 each honey dilution was determined using the peroxide/peroxidase assay, as triplicate. To examine the effects of honey and hydrogen peroxide on DNA DEGRADATION IN E. COLI CELLS EXPOSED TO the integrity of bacterial DNA, E. coli cultures (10 cfu/ml) were CATALASE-TREATED OR HEAT-TREATED HONEYS exposed to increasing concentrations of exogenous H O (5– To gain more insight into the role of H O in chromosomal DNA 2 2 2 2 0.3125 mM) or to honeys containing known amounts of H O . degradation, E. coli cultures were exposed to honeys which were 2 2 After 24 h incubation at 37˚C, bacterial DNA was isolated and its treated with catalase. Removal of H O by catalase abolished DNA 2 2 integrity examined on agarose gels. Figure 4 shows that the expo- degrading activity of honey H205 and had a protective effect on sure of E. coli cultures to hydrogen peroxide at concentrations of 5 bacterial DNA (Figure 5). The short incubation of catalase-treated and 2.5 mM caused DNA degradation, while H O concentrations honey H204 with DNA (8 h instead of 24 h) also prevented DNA 2 2 lower than 2.5 mM were ineffective. degradation (Figure 5). Inactive honey H200 remained unable to In contrast, honeys of relatively high H O concentrations degrade DNA after catalase-treatment (Figure 5). However, when 2 2 but below 2.5 mM (H203, 204, 205; Table 1) exerted DNA honey H200 was supplemented with 2 mM H O , and then incu- 2 2 degrading activity (Figure 4). The ability of honeys H O to bated with E. coli culture at 37˚C for 8 h, it became active in degrad- 2 2 degrade DNA appeared to be concentration-dependent. Honey ing DNA and the extent of DNA degradation was comparable H200 containing 0.25 mM H O was unable to cleave DNA to that of honey H204 (Figure 5). On the other hand, catalase– 2 2 (Figure 5). The differences in the concentrations of H O treatment of manuka honey did not prevent DNA degradation, 2 2 between exogenous and honey’s hydrogen peroxide that were consistent with the notion, that manuka honey antibacterial activ- required to effectively degrade chromosomal DNA may indi- ity is hydrogen peroxide-independent (Molan and Russell, 1988; cate that the action of honey H O is enhanced by other honey Allen et al., 1991). 2 2 components. To investigate the potential involvement of DNases in DNA Manuka honey also possessed low concentration of H O degradation, honeys were heat-treated under conditions which 2 2 (0.72 mM), but efficiently degraded DNA (Figures 4 and 5). The inactivate DNase activity (75˚C for 10 min). Unheated and heat- antibacterial activity of manuka honey however is not regulated treated honeys were then incubated with E. coli cultures at 37˚C by the honey H O content (Molan and Russell, 1988; Allen et al., for 8 h, followed by DNA isolation and its analysis on agarose gels. 2 2 1991). Heat-treatment of active honeys H205 and H23 did not prevent www.frontiersin.org October 2011 | Volume 2 | Article 213 | 5 Brudzynski et al. Re-examining the role of hydrogen peroxide FIGURE4|Effect of exposure ofE. coli cultures to honey or exogenous with increasing concentrations of exogenous H O (5–0.312 mM). Untreated 2 2 hydrogen peroxide on the integrity of bacterial DNA. The cells were cells and cells treated with the sugar solution (artificial honey, AH) served as treated with honeys (manuka, buckwheat honeys H203, H204, and H205) or the controls. The integrity of DNA was analyzed on agarose gels. FIGURE5|Effect of exposure of E. coli cultures to honeys untreated E. coli cells, AH-cell treated with artificial honey, or buckwheat untreated and treated with catalase (cat) on the integrity of honeys H200, H205, and M-manuka honey after 24 h incubation and H204 chromosomal DNA. “Cont” represents DNA isolated from after 8 h incubation. DNA degradation suggesting against the involvement of DNase in Firstly, we found that the exponentially growing E. coli and this process (Figures 6 and 7). Moreover, the fact that some honeys B. subtilis cells were inhibited in a concentration-dependent displayed DNA degrading activity (H23 or H205) in bacterial cul- manner by exogenous H O reaching MIC at 1.25 mM 2 2 90 7 7 ture while others did not (H200 and H60) makes it unlikely that (1.25 μmoles/10 cfu/ml) and 2.5 mM (2.5 μmoles/10 cfu/ml), this process was mainly due to the contamination of honeys with respectively. The bacteriostatic efficacy of H O however differed 2 2 DNases. On the other hand, inability of honeys H200 and H60 to significantly from that of honey H O . The main factors that 2 2 degrade DNA was closely related to the very low concentration of contributed to these differences were (a) bacterial susceptibil- H O in these honeys. ity/resistance to the oxidative action of hydrogen peroxide and 2 2 Together, these results provided a strong support for the role of (b) interference from other honey components. H O in DNA degradation. Endogenous H O inhibited the growth of E. coli in a 2 2 2 2 concentration-dependent manner, but its MIC was twofold DISCUSSION higher than those of exogenous H O (2.5 versus 1.25 mM, 2 2 The findings described in this study revise the old views and pro- respectively). The honeys MIC levels against E. coli coincided vide novel information on the role of hydrogen peroxide in the with the dilutions at which a peak of hydrogen peroxide pro- regulation of bacteriostatic and bactericidal activities of honey. duction occurred. Treatment of honeys with catalase led to a Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy October 2011 | Volume 2 | Article 213 | 6 Brudzynski et al. Re-examining the role of hydrogen peroxide H O resulted in a concentration-dependent growth inhibition, 2 2 the comparable concentrations of honey H O in honey were 2 2 ineffective in arresting B. subtilis growth. The rapid decrease in bacteriostatic activity of honey upon dilution was observed even in the presence of high concentrations of honey H O . These results 2 2 suggest that other honey compounds were responsible for the inhi- bition of B. subtilis growth. As a consequence of growth arrest, the change in sensitivity of B. subtilis to honey H O occurred. Instead 2 2 of growth inhibition, we observed growth stimulation of B. sub- tilis at high honey dilutions (16-fold and over) and in the presence of highlevelsof H O . Literature data provides compelling evi- 2 2 dence that transition from the exponential-phase growth to the stationary-phase growth evokes B. subiltis sporulation and with it, increased resistance to hydrogen peroxide. The transition to stationary-phase growth activates RNA polymerase σ transcrip- tional factor which regulates a stationary-phase gene expression of rpoS regulon. The expression of σ factor in B. subtilis evokes spore-formation to enhance bacterial survival (Dowds et al., 1987; FIGURE6|Effect of exposure of E. coli cultures to heat-treated (h), Dowds, 1994; Loewen et al., 1998; Zheng et al., 1999; Chen and catalase-treated (cat), and untreated (un) honeys on the integrity of Schellhorn, 2003). Dowds et al. (1987) have shown that stationary- chromosomal DNA. AH-cell treated with artificial honey, or buckwheat honeys H200, and H205. Lane “H200 2 mM H O ” represents the effect of 2 2 phase cultures of B. subtilis displayed viability even at the 10 mM honey H200 supplementation with hydrogen peroxide on the E. coli DNA concentration of H O . These data may explain, at least in part, 2 2 integrity. an apparent insensitivity of B. subtilis to highlevelsof hydrogen peroxideinhoney. These results revealed significant differences in the sensitivi- ties of E. coli and B. subtilis to oxidative stress caused by honey H O . As aerobic bacteria, both E. coli and B. subtilis are equipped 2 2 with molecular machinery to cope with oxidative stress by acti- vating several stress genes under oxyR- or perR-regulons, in E. coli and B. subtilis respectively (Dowds et al., 1987; Christman et al., 1989; Dowds, 1994; Bsat et al., 1998; Storz and Imlay, 1999). The oxyR and perR genes control expression of inducible forms of katG (catalase hydroperoxidase I, HP1), ahpCF (alkylhydroperox- ide reductase) that function to reduce hydrogen peroxide to levels that are not harmful to growing cells (Hassan and Fridovich, 1978; Loewen and Switala, 1987; Storz et al., 1990; Seaver and Imlay, 2001). While these responses are similar in both bacteria, the main difference concerns their adaptive and survival mechanisms to oxidative stress. Relatively little is known about the contribution of honey’s hydrogen peroxide to bacterial cell death. The most important result obtained in this work is the demonstration that honey H O 2 2 participated in bacterial DNA degradation. Several lines of evi- dence support this finding. Firstly, the treatment of exponential- phase E. coli cultures with increasing concentrations of exoge- nous hydrogen peroxide (5–0.3125 mM) or honeys of different content of endogenous H O led to a concentration-dependent 2 2 DNA degradation. While the minimum DNA degrading activity of exogenous H O occurred at 2.5 mM (2.5 μmoles/10 cfu/ml), 2 2 FIGURE7|Effect of exposure of E. coli naked DNA to unheated and in contrast, honeys possessing H O concentrations lower than 2 2 heat-treated honeys H60 and H23. 2.5 mM were still active in this process. Secondly, DNA degra- dation by active honeys was abolished by removal of H O by 2 2 significant reduction in their bacteriostatic activity (Brudzynski, catalase. Thirdly, honeys with the low content of H O were unable 2 2 2006). Together, these data provide direct evidence that E. coli to degrade DNA but the supplementation with 2 mM of hydrogen growth is sensitive to oxidative action of honey H O . peroxide caused the appearance of this activity. The extent of DNA 2 2 In contrast, growth inhibition of B. subtilis was not due to the degradation by honey, which was supplemented with H O , was 2 2 action of honey H O . While exposure of B. subtilis cultures to comparable to that of active honeys. 2 2 www.frontiersin.org October 2011 | Volume 2 | Article 213 | 7 Brudzynski et al. Re-examining the role of hydrogen peroxide Heat-treatment of active honeys prior to incubation with E. coli the minimum DNA degrading activity of honey H O was below 2 2 cultures did not prevent DNA degradation, suggesting against the 2.5 mM. The lower concentrations of honeys H O required to 2 2 involvement of DNase in this process. Moreover, not all tested hon- effectively degrade chromosomal DNA strongly suggest that the eys displayed DNA degrading activity on E. coli cells. Given that oxidizing effect of H O was augmented by other honey com- 2 2 bacterial cells are impermeable to DNase, the DNA degradation ponents such as transition metals (Fe, Cu) commonly present in by honeys observed in this study could not be simply explained by honeys. In support of this notion, the recent literature evidence the DNase contaminations. Rather, the close relationship between indicates that it is the hydroxyl radical (HO) that is produced in the DNA degradation and H O content in honeys advocates for the metal-catalyzed Fenton reaction from H O rather than molecular 2 2 2 2 role of H O in the mechanism of DNA cleavage. hydrogen peroxide that causes the oxidative damage to membrane 2 2 DNA degradation is a lethal event which ultimately kills the structures, proteins, and DNA (Imlay et al., 1988; Storz and Imlay, cell. Literature data indicate that the concentration of hydrogen 1999; Cabiscol et al., 2000; Imlay, 2003). peroxide plays a decisive role in the type of cell death that fol- In conclusion, our study demonstrated that honey H O 2 2 lows H O exposure. In simplified in vitro models, where direct exerted bacteriostatic and DNA degrading activities to bacter- 2 2 effects of hydrogen peroxide on bacterial cells were analyzed, two ial cells. The extent of damaging effects of honey H O was 2 2 separate modes of killing were observed for E. coli. At low con- strongly influenced by the bacterial sensitivity to oxidative stress, centrations of H O (≤2.5 mM), E. coli cells were dying because the growth phase and their survival strategy (non-spore forming 2 2 of DNA damage inflicted on the metabolically active cells (Imlay versus spore forming species) as well as by the modulation of other and Linn, 1986; Imlay and Linn, 1987a,b; Brandi et al., 1989). honey compounds. At H O concentrations of 10–50 mM, cell death resulted from 2 2 cytotoxic effects due to hydroxyl radicals formed from hydro- ACKNOWLEDGMENTS genperoxide(Imlay and Linn, 1987a,b; Brandi et al., 1989). In This research was supported by funds from the Agricultural Adap- a full agreement with these data, we established that the minimum tation Council, Agriculture and Agri-Food Canada (ADV-380), DNA degrading activity of exogenous H O on E. coli cells was and the Ontario Centres of Excellence (BM50849) awarded to 2 2 2.5 mM (2.5 μmoles/10 cfu/ml). In contrast to exogenous H O , Katrina Brudzynski. 2 2 REFERENCES multiple Fur homologues: identifi- subtilis. FEMS Microbiol. 124, Imlay, J. A., and Linn, S. (1987b). DNA Alaux, C., Ducloz, F., Crauser, D., and cation of the iron uptake (Fur) and 155–264. damage and oxygen radical toxicity. Le Conte, Y. (2010). Diet effects peroxide regulon (PerR) repressors. Dowds, B. 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Oxidative gen peroxide. Bacteriology 166, tices Advisory Committee. (2008). weight melanoidin fractions exhibit- stress, antioxidant defences, and 519–527. Guideline for Disinfection and Steril- ing radical scavenging capacity. Food damage, removal, repair and replace- Imlay, J. A., and Linn, S. (1987a). ization in Healthcare Facilities, 2008. Chem. 127, 1023–1030. ment systems. IUBMB Life 50, Mutagenesis and stress response Centers for Disease Control and Bsat, N., Herbig, A., Casillas-Martinez, 279–289. induced in Escherichia coli by hydro- Prevention (CDC), Department of L., Setlow, P., and Helmann, J. Dowds, B. C. (1994). The oxida- gen peroxide. J. Bacteriol. 169, Health and Human Services USA, D. (1998). Bacillus subtilis contains tive stress response in Bacillus 2967–2976. Chapel Hill, NC. Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy October 2011 | Volume 2 | Article 213 | 8 Brudzynski et al. Re-examining the role of hydrogen peroxide Seaver, L. C., and Imlay, J. A. (2001). products to the antibacterial activ- Conflict of Interest Statement: The activities of honey. Front. Microbio. Alkyl hydroperoxide reductase is the ity of honey: a review. Food Chem. authors declare that the research was 2:213. doi: 10.3389/fmicb.2011.00213 primary scavenger of endogenous 71, 235–239. conducted in the absence of any com- This article was submitted to Fron- hydrogen peroxide in Escherichia White, J. H., Subers, M. H., and Schep- mercial or financial relationships that tiers in Antimicrobials, Resistance and coli. J. Bacteriol. 183, 7173–7181. artz, A. I. (1963). The identification could be construed as a potential con- Chemotherapy, a specialty of Frontiers in Storz, G., and Imlay, J. A. (1999). Oxida- of inhibine, the antibacterial factor flict of interest. Microbiology. tive stress. Curr. Opin. Microbiol. 2, in honey, as hydrogen peroxide and Copyright © 2011 Brudzynski, Abubaker , 188–194. its origin in a honey glucose-oxidase St-Martin and Castle. This is an open- Storz, G., Tartaglia, L. A., and Ames, system. Biochem. Biophys. Acta 73, Received: 01 September 2011; accepted: access article subject to a non-exclusive B. N. (1990). Transcriptional reg- 57–70. 03 October 2011; published online: 25 license between the authors and Frontiers ulator of oxidative stress-inducible Zheng, M., Doan, B., Schneider, October 2011. Media SA, which permits use, distribu- genes: direct activation by oxidation. T. D., and Storz, G. (1999). Citation: Brudzynski K, Abubaker K, tion and reproduction in other forums, Science 248, 189–194. OxyR and SoxRS regula- St-Martin L and Castle A (2011) Re- provided the original authors and source Weston, R. J. (2000). The contribu- tion of fur. J. Bacteriol. 181, examining the role of hydrogen per- are credited and other Frontiers condi- tion of catalase and other natural 4639–4643. oxide in bacteriostatic and bactericidal tions are complied with. www.frontiersin.org October 2011 | Volume 2 | Article 213 | 9 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Microbiology Pubmed Central

Re-Examining the Role of Hydrogen Peroxide in Bacteriostatic and Bactericidal Activities of Honey

Frontiers in Microbiology , Volume 2 – Oct 25, 2011

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Abstract

ORIGINAL RESEARCH ARTICLE published: 25 October 2011 doi: 10.3389/fmicb.2011.00213 Re-examining the role of hydrogen peroxide in bacteriostatic and bactericidal activities of honey 1,2 2 2 2 Katrina Brudzynski *, Kamal Abubaker , Laurent St-Martin and Alan Castle Bee-Biomedicals Inc., St. Catharines, ON, Canada Department of Biological Sciences, Brock University, St. Catharines, ON, Canada Edited by: The aim of this study was to critically analyze the effects of hydrogen peroxide on growth Mirian A. F. Hayashi, Universidade and survival of bacterial cells in order to prove or disprove its purported role as a main Federal de São Paulo, Brazil component responsible for the antibacterial activity of honey. Using the sensitive perox- Reviewed by: ide/peroxidase assay, broth microdilution assay and DNA degradation assays, the quanti- Jun Liu, Mount Sinai School of Medicine, USA tative relationships between the content of H O and honey’s antibacterial activity was 2 2 Dmitri Debabov, NovaBay established The results showed that: (A) the average H O content in honey was over . 2 2 Pharmaceuticals, USA 900-fold lower than that observed in disinfectants that kills bacteria on contact. (B) A sup- *Correspondence: plementation of bacterial cultures with H O inhibited E. coli and B. subtilis growth in a 2 2 Katrina Brudzynski , Department of concentration-dependent manner, with minimal inhibitory concentrations (MIC ) values of Biological Sciences, Brock University, 7 7 500 Glenridge Avenue, St. Catharines, 1.25 mM/10 cfu/ml and 2.5 mM/10 cfu/ml for E. coli and B. subtilis, respectively. In con- ON, Canada L2S 3A1. trast, the MIC of honey against E. coli correlated with honey H O content of 2.5 mM, 90 2 2 e-mail: beebio@sympatico.ca and growth inhibition of B. subtilis by honey did not correlate with honey H O levels at all. 2 2 (C) A supplementation of bacterial cultures with H O caused a concentration-dependent 2 2 degradation of bacterial DNA, with the minimum DNA degrading concentration occurring at 2.5 mM H O . DNA degradation by honey occurred at lower than ≤2.5 mM concentration 2 2 of honey H O suggested an enhancing effect of other honey components. (D) Honeys 2 2 with low H O content were unable to cleave DNA but the addition of H O restored this 2 2 2 2 activity. The DNase-like activity was heat-resistant but catalase-sensitive indicating that H O participated in the oxidative DNA damage. We concluded that the honey H O was 2 2 2 2 involved in oxidative damage causing bacterial growth inhibition and DNA degradation, but these effects were modulated by other honey components. Keywords: oxidative stress, hydrogen peroxide, bacteriostatic activity, honey, DNA degradation INTRODUCTION bacterial growth by honey (White et al., 1963; Brudzynski, 2006). Hydrogen peroxide is generally thought to be the main compound We have observed that in honeys with a high content of this responsible for the antibacterial action of honey (White et al., oxidizing compound, bacteria cannot respond normally to pro- 1963; Weston, 2000; Brudzynski, 2006). Hydrogen peroxide in liferative signals and their growth remains arrested even at high honey dilutions. Pre-treatment of honey with catalase restored, to honey is produced mainly during glucose oxidation catalyzed by the bee enzyme, glucose oxidase (FAD-oxidoreductase, EC 1.1.3.4; a certain extent, the bacterial growth, thus suggesting that endoge- White et al., 1963).Thelevelsof hydrogenperoxideinhoneyare nous H O was implicated in the growth inhibition (Brudzynski, 2 2 determined by the difference between the rate of its production 2006). and its destruction by catalases. Glucose oxidase is introduced to Most of the conclusions on the H O oxidizing action on bac- 2 2 honey during nectar harvesting by bees. This enzyme is found in teria are drawn from the simplified in vitro models, where direct all honeys but its concentration may differ from honey to honey effects of hydrogen peroxide on bacterial cells were analyzed. In depending on the age and health status of the foraging bees (Per- contrast, honey represent complex chemical milieu consisting of nal and Currie, 2000) as well as the richness and diversity of the over 100 different compounds (including antioxidants and traces foraged diet (Alaux et al., 2010). Catalases on the other hand, are of transition metals), where the interaction between these compo- of pollen origin. Catalase efficiently hydrolyzes hydrogen peroxide nents and hydrogen peroxide may influence its oxidative action. to oxygen and water due to its high turnover numbers. The total We have recently unraveled that honey is a dynamic reaction concentration of catalase depends on the amount of pollen grains mixture which facilitates and propagates the Maillard reaction in honey (Weston, 2000), and consequently, the hydrogen perox- (Brudzynski and Miotto, 2011b). The Maillard reaction which ide levels in different honeys may vary considerably (Brudzynski, initially involves reaction between amino groups of amino acids 2006). or proteins with carbonyl groups of reducing sugars leads to a A substantial correlation has been found between the level of cascade of redox reactions in which several bioactive molecules endogenous hydrogen peroxide and the extent of inhibition of are continuously formed and lost due to their cross-linking to www.frontiersin.org October 2011 | Volume 2 | Article 213 | 1 Brudzynski et al. Re-examining the role of hydrogen peroxide other molecules (gain or loss of function; Brudzynski and Miotto, During last decades, several honey compounds were identified 2011b). We have shown that polyphenol-based melanoidins are as those implicated in honey antibacterial activity (for review, Irish a major group of Maillard reaction products possessing radical- et al., 2011). Despite this knowledge, the mechanisms by which scavenging activity (Brudzynski and Miotto, 2011a,b). These com- these compounds lead to bacterial growth inhibition and bacterial pounds are likely to interact with hydrogen peroxide and, depend- death have never been explained or proven in biochemical terms. ing of their concentration and redox capacity, either enhanced or Since there is a persistent view that hydrogen peroxide is a main diminished the oxidative activity of honey’s H O . In view of these player in these events, the aim of this study was to critically analyze 2 2 facts, we hypothesized that the oxidizing action of honey’s hydro- the effects of hydrogen peroxide on growth and survival of bacte- gen peroxide on bacterial cells may be modulated by the presence rial cells in order to prove or disprove its purported role as a main of other bioactive molecules in honey and therefore, may differ component responsible for the antibacterial activity of honey. from the action of hydrogen peroxide alone. Hydrogen peroxide is commonly used to disinfect and sanitize MATERIALS AND METHODS medical equipment in hospitals. For this purpose, the high concen- HONEY SAMPLES trations of H O in these disinfectants have to be maintained to Honey samples included raw, unpasteurized honeys donated by 2 2 overwhelmed defense systems of bacteria. At high concentrations, Canadian beekeepers and two samples of commercial Active ranging from 3 to 30% (0.8 to 8 M), its bactericidal effectiveness Manuka honey (Honey New Zealand Ltd., New Zealand, UMF has been demonstrated against several microorganisms including 20+, and 25+; M and M2, respectively; Table 1) that were used Staphylococcus -, Streptococcus -, Pseudomonas-species, and Bacillus as a reference in this study. During the study, honey samples were spores (Rutala et al., 2008).Under these conditions, the bacterial kept in the original packaging, at room temperature (22 ± 2˚C) cell death results from the accumulation of irreversible oxidative and in the dark. damages to the membrane layers, proteins, enzymes, and DNA A stock solution of 50% (w/v) honey was prepared by dis- (?Davies, 2000; Rutala et al., 2008; Finnegan et al., 2010). solving 1.35 g honey (average density 1.35 g/ml) in 1 ml of sterile, However, the hydrogen peroxide content in honey is about distilled water warmed at 37˚C. The stock solution was prepared 900-fold lower (Brudzynski, 2006). Moreover, the literature data immediately before conducting the antibacterial assays. indicate that the cell death of cultured mammalian, yeast, and bac- terial cells required H O concentrations higher than 50 mM and PREPARATION OF ARTIFICIAL HONEY 2 2 was associated with chromosomal DNA degradation (Imlay and Artificial honey was prepared by dissolving 76.8 g of fructose and Linn, 1987a,b; Brandi et al., 1989; Davies, 1999; Bai and Konat, 60.6 g of glucose separately in 100 ml of sterile, deionized water, 2003; Ribeiro et al., 2006), which is still five to 10-fold higher and by mixing these two solutions in a 1:1 ratio. The osmolarity then that observed in honeys. Therefore, we have undertaken this of the artificial honey was adjusted to that of the honey samples study to re-examined the role of hydrogen peroxide in antibacterial (BRIX) using refractometric measurements. activity of honeys. The hydrogen peroxide efficacy as an oxidative biocide is related BACTERIAL STRAINS to the bacterial sensitivity to peroxide stress. Defense mecha- Standard strains of Bacillus subtilis (ATCC 6633) and Escherichia nisms to oxidative stress varies between bacterial species such coli (ATCC 14948; Thermo Fisher Scientific Remel Products, as Gram-negative E. coli and Gram-positive B. subtilis used in Lenexa, KS 66215) were grown in Mueller–Hinton broth (MHB; this study and depend on the growth phase (exponential- ver- Difco Laboratories) overnight in a shaking water-bath at 37˚C. sus stationary-phase of growth), and on the adaptive and survival Overnight cultures were diluted with broth to the equivalent mechanisms (non-spore forming versus spore-forming bacteria; of the 0.5 McFarland Standard (approx.10 cfu/ml) which was Dowds et al., 1987; Chen et al., 1995; Storz and Imlay, 1999; Cabis- measured spectrophotometrically at A . 600 nm col et al., 2000). In honey, the effects of H O on the growth 2 2 and survival of microorganisms may be mitigated or enhanced ANTIBACTERIAL ASSAY due to the presence of honey compounds. On one hand, a high The antibacterial activity of honeys was determined using a broth content of sugars in honey that abstracts free water molecules microdilution assay using a 96-well microplate format. Serial from milieu inhibits bacterial growth and proliferation, but honey twofold dilutions of honey were prepared by mixing and transfer- dilutions may create growth-supportive conditions due to the ring 110 μl of honey with 110 μl of inoculated broth (10 cfu/ml abundance of sugars as a carbon source for the growing cells. final concentrations for each microorganism) from row A to row Hydrogen peroxide has deleterious effects on the growth and sur- H of a microplate. Row G contained only inoculum and served as vival of bacterial cells but honey antioxidants such as catalases, a positive control and row H contained sterile MHB and served as polyphenols, Maillard reaction products, and ascorbic acid may a blank. lower the oxidative stress to cells and may have a protective effect After overnight incubation of plates at 37˚C in a shaking water- against endogenous H O (Brudzynski, 2006). Even less informa- bath, bacterial growth was measured at A using the Syn- 2 2 595 nm tion exists on the mechanism of bactericidal action of honey’s ergy HT multi-detection microplate reader (Synergy HT, Bio-Tek hydrogen peroxide. The most fundamental and unsolved ques- Instruments, Winooski, VT, USA). tions concerns the molecular targets of H O cytotoxicity: does The contribution of color of honeys to the absorption was cor- 2 2 molecular hydrogen peroxide at concentrations present in honey rected by subtracting the absorbance values before (zero time) cause DNA degradation? incubation from the values obtained after overnight incubation. Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy October 2011 | Volume 2 | Article 213 | 2 Brudzynski et al. Re-examining the role of hydrogen peroxide Table1|Hydrogenperoxide concentrations in different honeys. Relationship between antibacterial activities of honey and hydrogen peroxide concentrations. Honey sample Plant source Hydrogen peroxide E. coli MIC dilution B. subtilis MIC dilution concentration (mM/l)* (concentration) (concentration) M2 Manuka (UMF 25) 1.04 ± 0.17 16 (6.25%) 16 (6.25%) H58 Buckwheat 2.68 ± 0.04 16 (6.25%) 8 (12.5%) H23 Buckwheat 2.12 ± 0.22 8 (12.5%) 4 (25%) H20 Sweet clover 2.37 ± 0.03 8 (12.5%) 4 (25%) H11 Wildflower/clover 2.49 ± 0.03 8 (12.5%) 8 (12.5%) H56 Blueberry 0.52 ± 0.11 4 (25%) 2 (50%) H60 Clover blend 0.67 ± 0.11 4 (25%) 2 (50%) M Manuka (UMF 20) 0.72 ± 0.02 4 (25%) 4 (25%) H200 Buckwheat 0.248 ± 0.02 2 (50%) 2 (50%) H203 Buckwheat 0.744 ± 0.01 4 (25%) 4 (25%) H204 Buckwheat 1.168 ± 0.05 4 (25%) 4 (25%) H205 Buckwheat/alfalfa 1.112 ± 0.02 4 (25%) 4 (25%) *Hydrogen peroxide concentration was measured at honey dilution of 8× (25% v/v) and represent an average of three experimental trials, where each honey was tested in triplicate. The absorbance readings obtained from the dose–response solution in a 1:1 ratio (v/v), an artificial honey solution, or with curves were used to construct growth inhibition profiles (GIPs). hydrogen peroxide solutions containing 5, 2.5, 1.2, 0.62, and The minimal inhibitory concentrations (MIC) were determined 0.3125 mM (final concentrations) H O prepared from the 20 mM 2 2 from the GIPs and represented the lowest concentration of honey stock solution. After overnight incubation at 37˚C with continu- that inhibited the bacterial growth. The MIC end point in our ous shaking, the cells were harvested by centrifugation at 3,000×g experiments was honey concentration at which 90% bacterial (Eppendorf ) for 30 s and then their DNA was isolated. growth reduction was observed as measured by the absorbance DNA ISOLATION at A . 595 nm The total genomic bacterial DNA was isolated from the untreated, Statistical analysis and dose response curves were obtained control cells and from the honey- or hydrogen peroxide-treated using KC4 software (Synergy HT, Bio-Tek Instruments, Winooski, cells using a DNA isolation kit (Norgen Biotek Corporation, VT, USA). St. Catharines, ON., Canada), according to the manufacturer’s HYDROGEN PEROXIDE ASSAY instructions. Hydrogen peroxide concentration in honeys was determined using AGAROSE GEL ELECTROPHORESIS the hydrogen peroxide/peroxidase assay kit (Amplex Red, Mol- Agarose gel (1.3%) electrophoresis was carried out in 1× TAE ecular Probes, Invitrogen, Burlington, ON, Canada). The assay buffer containing ethidium bromide (0.1 μg/ml w/v). Ten micro- was conducted in the 96-well microplates according to the man- liters of DNA isolated from the untreated and treated bacterial ufacturer’s instruction. The fluorescence of the formed product, cells was mixed with 5X loading dye (0.25% bromophenol blue, resorufin, was measured at 530 nm excitation and a 590 nm emis- 0.25% xylene xyanol, 40% sucrose) and loaded into the gel. The sion using the Synergy HT (Molecular Devices, BioTek Instru- DNA molecular weight markers selected were the HighRanger ments, Winooski, VT, USA) multi-detection microplate reader, 1 kb DNA Ladder, MidRanger 1 kb DNA Ladder, and PCRSizer and the dose–response curves were generated using the KC4™data 100 bp DNA Ladder from Norgen Biotek (Thorold, Ontario). The reduction software. gels were run at 85 V for 1 h and then visualized and photographed To calculate the hydrogen peroxide concentrations of the hon- using the Gel Doc 1000 system and the Quantity One 1-D Analysis eys, a standard curve was run alongside the honey serial dilutions. software (version 4.6.2 Basic) from Bio-Rad. The standard curve was prepared from the 200 μMH O stock 2 2 solution. Each of the honey samples, and the standard curve, were RESULTS tested in triplicate. DETERMINATION OF THE HYDROGEN PEROXIDE CONCENTRATIONS IN HONEYS CATALASE-TREATMENT OF HONEYS Formation of H O depends on the honey dilution since glu- 2 2 Honey were treated with catalase (13 800 U/mg solid; Sigma- cose oxidase is inactive in undiluted honey (White et al., 1963; Aldrich, Canada) at ratio of 1000 units per 1 ml of 50% honey Brudzynski, 2006). Honeys used in this study required a four to solution in sterile water for 2 h at room temperature. 16-fold dilution for the maximal production of hydrogen per- INCUBATION OF BACTERIAL CULTURES WITH HONEY OR HYDROGEN oxide to be observed (Figure 1). At the peak, H O concentra- 2 2 PEROXIDE tions ranged from 2.68 ± 0.04 to 0.248 ± 0.02 mM in the different Overnight cultures of E. coli and B. subtilis (1.5 ml, adjusted to honeys (Table 1), as measured by a sensitive, high-throughput 10 cfu/ml in MHB) were treated with either the 50% honey hydrogen peroxide/peroxidase assay (Amplex Red assay). www.frontiersin.org October 2011 | Volume 2 | Article 213 | 3 Brudzynski et al. Re-examining the role of hydrogen peroxide FIGURE1|Effect of honey dilutions on the production of hydrogen FIGURE2|Effect of increasing the concentration of exogenous peroxide. Honeys of buckwheat origin, H58 and H23, together with sweet hydrogen peroxide on the growth of E. coli (blue line) andB. subtilis clover (H20), and wildflower/clover (H11) produced distinctively higher (red line). Each point represents the mean and SD of three separate amounts of H O than Manuka (M2) or honey blends (H56 and H60). The experiments conducted in triplicate. 2 2 H O content was measured in twofold serially diluted honeys, the x axis 2 2 represents a log2 values. of honeys against E. coli, it appeared that almost all of the bac- teriostatic activity of honeys could be assigned to the effects of this compound (Figure 3A). In honeys, the endogenous H O of CONCENTRATION-DEPENDENT EFFECT OF HYDROGEN PEROXIDE ON 2 2 2.5 mM was of critical importance for the growth inhibition of E. BACTERIAL GROWTH INHIBITION coli ; the dilutions that reduced H O concentrations below this Throughout this study, we used terms: endogenous hydrogen per- 2 2 value showed a loss of honey potency to inhibit bacterial growth oxide to describe H O produced in honey by glucose oxidase 2 2 at the MIC level (Figure 3A). These data suggest that upon and exogenous hydrogen peroxide, which has been added as a 90 honey dilution, endogenous H O mediates growth inhibition of supplement to the bacterial cultures. These terms were intro- 2 2 E. coli. However, the concentrations required to reach MIC were duced in order to differentiate between the effects of honey’s 90 twofold higher than that found for exogenous hydrogen peroxide endogenous H O whose action on bacterial cells could be modu- 2 2 (2.5 versus 1.25 mM, respectively; Figure 3A). lated/obscured by other honey components as opposed to true, In contrast to E. coli, the inhibition of growth of B. subtilis well-defined action of exogenous hydrogen peroxide directly seemed not to be due to the effect of the levels of honey H O added to bacterial culture. 2 2 (Figure 3B). A rapid increase of B. subtilis growth with honey dilu- In agreement with previous reports (Brudzynski, 2006), we tions occurred despite the presence of high levels of H O (honeys found a strong correlation between the content of honey hydrogen 2 2 H58, H23, H20, and H11, Figure 3B). While exposure of the B. peroxide and the growth inhibitory action of Canadian honeys; subtilis culture to exogenous H O resulted in a concentration- honeys with high MIC values (6.25 to 12.5% v/v) correspond- 2 2 dependent growth inhibition with MIC at 2.5 mM (Figure 2), ing to16 to 8× dilution) also possessed a high content of H O 90 2 2 comparable concentrations of H O in honeys were ineffective. (Table 1). Since the minimum inhibitory concentration values 2 2 This indicated that other honey compounds/physical features were and the hydrogen peroxide peak were both observed at the 4 to responsible for the growth inhibition, such as honey’s high osmo- 16× honey dilutions, we hypothesized that the maximal hydro- larity. Moreover, higher honey dilutions, beyond 16-fold, had a gen peroxide production is required to achieve the bacteriostatic stimulatory effect on B. subtilis growth (Figure 3B). activity of honey at the MIC level. To test this assumption, we Thus, our results demonstrated for the first time that bacterio- first examined the dose–response relationship between the con- static effects of endogenous versus exogenous hydrogen peroxide centration of exogenous hydrogen peroxide, ranging from 10 to are markedly different due to the presence of other honey compo- 0.312 mM, and its growth inhibitory activity against E. coli and B. nents and, more importantly, that the effects of honey H O on subtilis. The dose–response curves and growth inhibitory profiles 2 2 bacterial growth are markedly different in E. coli and B. subtilis. revealed very reproducibly that H O concentrations of 1.25 mM 2 2 7 7 (1.25 μmoles/10 cfu/ml) and 2.5 mM (2.5 μmoles/10 cfu/ml) were required to inhibit the growth of E. coli and B. subtilis by COMPARISON OF EFFECTS OF HONEY AND HYDROGEN PEROXIDE ON 90%, respectively (Figure 2). DNA DEGRADATION IN BACTERIAL CELLS To exert effectively its oxidative biocide action, the concentrations of hydrogen peroxide in various disinfectants are high ranging RELATIONSHIP BETWEEN THE ENDOGENOUS H O CONTENT AND THE from 3 to 30% (0.8 to 8 M). In contrast, we have established that 2 2 GROWTH INHIBITORY ACTIVITY OF HONEYS the average content of H O in tested honeys ranged from 0.5 2 2 To investigate whether the content of honey H O influences to 2.7 mM (Table 1). The concentrations of H O measured in 2 2 2 2 honey’s bacteriostatic potency in a similar manner to that of honeys, therefore was about 260–1600-fold lower than the effec- exogenous H O , each honey was analyzed for growth inhibitory tive bactericidal dose of H O in disinfectants. Therefore, we asked 2 2 2 2 activity and the production of hydrogen peroxide in the same the question whether hydrogen peroxide at concentrations present range of honey dilutions. When the profiles of hydrogen peroxide in honey can cause DNA degradation and ultimately bacterial production were superimposed on the growth inhibitory profiles cell death. Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy October 2011 | Volume 2 | Article 213 | 4 Brudzynski et al. Re-examining the role of hydrogen peroxide FIGURE 3 | The relationship between bacteriostatic effect of described in the Section “Materials and Methods.” Of note: growth inhibition honey and the content of en-H2O2 on E. coli (A) or B. subtilis cultures (B) profiles of artificial honey of osmolarity equal to that of natural honey provided Growth inhibition profiles were determined for different honeys using MIC values of 25% (v/v) against both E. coli and B. subtilis. Each point or the broth microdilution assay (columns). The content of honey H O at column represents the mean values of three separate experiments run in 2 2 each honey dilution was determined using the peroxide/peroxidase assay, as triplicate. To examine the effects of honey and hydrogen peroxide on DNA DEGRADATION IN E. COLI CELLS EXPOSED TO the integrity of bacterial DNA, E. coli cultures (10 cfu/ml) were CATALASE-TREATED OR HEAT-TREATED HONEYS exposed to increasing concentrations of exogenous H O (5– To gain more insight into the role of H O in chromosomal DNA 2 2 2 2 0.3125 mM) or to honeys containing known amounts of H O . degradation, E. coli cultures were exposed to honeys which were 2 2 After 24 h incubation at 37˚C, bacterial DNA was isolated and its treated with catalase. Removal of H O by catalase abolished DNA 2 2 integrity examined on agarose gels. Figure 4 shows that the expo- degrading activity of honey H205 and had a protective effect on sure of E. coli cultures to hydrogen peroxide at concentrations of 5 bacterial DNA (Figure 5). The short incubation of catalase-treated and 2.5 mM caused DNA degradation, while H O concentrations honey H204 with DNA (8 h instead of 24 h) also prevented DNA 2 2 lower than 2.5 mM were ineffective. degradation (Figure 5). Inactive honey H200 remained unable to In contrast, honeys of relatively high H O concentrations degrade DNA after catalase-treatment (Figure 5). However, when 2 2 but below 2.5 mM (H203, 204, 205; Table 1) exerted DNA honey H200 was supplemented with 2 mM H O , and then incu- 2 2 degrading activity (Figure 4). The ability of honeys H O to bated with E. coli culture at 37˚C for 8 h, it became active in degrad- 2 2 degrade DNA appeared to be concentration-dependent. Honey ing DNA and the extent of DNA degradation was comparable H200 containing 0.25 mM H O was unable to cleave DNA to that of honey H204 (Figure 5). On the other hand, catalase– 2 2 (Figure 5). The differences in the concentrations of H O treatment of manuka honey did not prevent DNA degradation, 2 2 between exogenous and honey’s hydrogen peroxide that were consistent with the notion, that manuka honey antibacterial activ- required to effectively degrade chromosomal DNA may indi- ity is hydrogen peroxide-independent (Molan and Russell, 1988; cate that the action of honey H O is enhanced by other honey Allen et al., 1991). 2 2 components. To investigate the potential involvement of DNases in DNA Manuka honey also possessed low concentration of H O degradation, honeys were heat-treated under conditions which 2 2 (0.72 mM), but efficiently degraded DNA (Figures 4 and 5). The inactivate DNase activity (75˚C for 10 min). Unheated and heat- antibacterial activity of manuka honey however is not regulated treated honeys were then incubated with E. coli cultures at 37˚C by the honey H O content (Molan and Russell, 1988; Allen et al., for 8 h, followed by DNA isolation and its analysis on agarose gels. 2 2 1991). Heat-treatment of active honeys H205 and H23 did not prevent www.frontiersin.org October 2011 | Volume 2 | Article 213 | 5 Brudzynski et al. Re-examining the role of hydrogen peroxide FIGURE4|Effect of exposure ofE. coli cultures to honey or exogenous with increasing concentrations of exogenous H O (5–0.312 mM). Untreated 2 2 hydrogen peroxide on the integrity of bacterial DNA. The cells were cells and cells treated with the sugar solution (artificial honey, AH) served as treated with honeys (manuka, buckwheat honeys H203, H204, and H205) or the controls. The integrity of DNA was analyzed on agarose gels. FIGURE5|Effect of exposure of E. coli cultures to honeys untreated E. coli cells, AH-cell treated with artificial honey, or buckwheat untreated and treated with catalase (cat) on the integrity of honeys H200, H205, and M-manuka honey after 24 h incubation and H204 chromosomal DNA. “Cont” represents DNA isolated from after 8 h incubation. DNA degradation suggesting against the involvement of DNase in Firstly, we found that the exponentially growing E. coli and this process (Figures 6 and 7). Moreover, the fact that some honeys B. subtilis cells were inhibited in a concentration-dependent displayed DNA degrading activity (H23 or H205) in bacterial cul- manner by exogenous H O reaching MIC at 1.25 mM 2 2 90 7 7 ture while others did not (H200 and H60) makes it unlikely that (1.25 μmoles/10 cfu/ml) and 2.5 mM (2.5 μmoles/10 cfu/ml), this process was mainly due to the contamination of honeys with respectively. The bacteriostatic efficacy of H O however differed 2 2 DNases. On the other hand, inability of honeys H200 and H60 to significantly from that of honey H O . The main factors that 2 2 degrade DNA was closely related to the very low concentration of contributed to these differences were (a) bacterial susceptibil- H O in these honeys. ity/resistance to the oxidative action of hydrogen peroxide and 2 2 Together, these results provided a strong support for the role of (b) interference from other honey components. H O in DNA degradation. Endogenous H O inhibited the growth of E. coli in a 2 2 2 2 concentration-dependent manner, but its MIC was twofold DISCUSSION higher than those of exogenous H O (2.5 versus 1.25 mM, 2 2 The findings described in this study revise the old views and pro- respectively). The honeys MIC levels against E. coli coincided vide novel information on the role of hydrogen peroxide in the with the dilutions at which a peak of hydrogen peroxide pro- regulation of bacteriostatic and bactericidal activities of honey. duction occurred. Treatment of honeys with catalase led to a Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy October 2011 | Volume 2 | Article 213 | 6 Brudzynski et al. Re-examining the role of hydrogen peroxide H O resulted in a concentration-dependent growth inhibition, 2 2 the comparable concentrations of honey H O in honey were 2 2 ineffective in arresting B. subtilis growth. The rapid decrease in bacteriostatic activity of honey upon dilution was observed even in the presence of high concentrations of honey H O . These results 2 2 suggest that other honey compounds were responsible for the inhi- bition of B. subtilis growth. As a consequence of growth arrest, the change in sensitivity of B. subtilis to honey H O occurred. Instead 2 2 of growth inhibition, we observed growth stimulation of B. sub- tilis at high honey dilutions (16-fold and over) and in the presence of highlevelsof H O . Literature data provides compelling evi- 2 2 dence that transition from the exponential-phase growth to the stationary-phase growth evokes B. subiltis sporulation and with it, increased resistance to hydrogen peroxide. The transition to stationary-phase growth activates RNA polymerase σ transcrip- tional factor which regulates a stationary-phase gene expression of rpoS regulon. The expression of σ factor in B. subtilis evokes spore-formation to enhance bacterial survival (Dowds et al., 1987; FIGURE6|Effect of exposure of E. coli cultures to heat-treated (h), Dowds, 1994; Loewen et al., 1998; Zheng et al., 1999; Chen and catalase-treated (cat), and untreated (un) honeys on the integrity of Schellhorn, 2003). Dowds et al. (1987) have shown that stationary- chromosomal DNA. AH-cell treated with artificial honey, or buckwheat honeys H200, and H205. Lane “H200 2 mM H O ” represents the effect of 2 2 phase cultures of B. subtilis displayed viability even at the 10 mM honey H200 supplementation with hydrogen peroxide on the E. coli DNA concentration of H O . These data may explain, at least in part, 2 2 integrity. an apparent insensitivity of B. subtilis to highlevelsof hydrogen peroxideinhoney. These results revealed significant differences in the sensitivi- ties of E. coli and B. subtilis to oxidative stress caused by honey H O . As aerobic bacteria, both E. coli and B. subtilis are equipped 2 2 with molecular machinery to cope with oxidative stress by acti- vating several stress genes under oxyR- or perR-regulons, in E. coli and B. subtilis respectively (Dowds et al., 1987; Christman et al., 1989; Dowds, 1994; Bsat et al., 1998; Storz and Imlay, 1999). The oxyR and perR genes control expression of inducible forms of katG (catalase hydroperoxidase I, HP1), ahpCF (alkylhydroperox- ide reductase) that function to reduce hydrogen peroxide to levels that are not harmful to growing cells (Hassan and Fridovich, 1978; Loewen and Switala, 1987; Storz et al., 1990; Seaver and Imlay, 2001). While these responses are similar in both bacteria, the main difference concerns their adaptive and survival mechanisms to oxidative stress. Relatively little is known about the contribution of honey’s hydrogen peroxide to bacterial cell death. The most important result obtained in this work is the demonstration that honey H O 2 2 participated in bacterial DNA degradation. Several lines of evi- dence support this finding. Firstly, the treatment of exponential- phase E. coli cultures with increasing concentrations of exoge- nous hydrogen peroxide (5–0.3125 mM) or honeys of different content of endogenous H O led to a concentration-dependent 2 2 DNA degradation. While the minimum DNA degrading activity of exogenous H O occurred at 2.5 mM (2.5 μmoles/10 cfu/ml), 2 2 FIGURE7|Effect of exposure of E. coli naked DNA to unheated and in contrast, honeys possessing H O concentrations lower than 2 2 heat-treated honeys H60 and H23. 2.5 mM were still active in this process. Secondly, DNA degra- dation by active honeys was abolished by removal of H O by 2 2 significant reduction in their bacteriostatic activity (Brudzynski, catalase. Thirdly, honeys with the low content of H O were unable 2 2 2006). Together, these data provide direct evidence that E. coli to degrade DNA but the supplementation with 2 mM of hydrogen growth is sensitive to oxidative action of honey H O . peroxide caused the appearance of this activity. The extent of DNA 2 2 In contrast, growth inhibition of B. subtilis was not due to the degradation by honey, which was supplemented with H O , was 2 2 action of honey H O . While exposure of B. subtilis cultures to comparable to that of active honeys. 2 2 www.frontiersin.org October 2011 | Volume 2 | Article 213 | 7 Brudzynski et al. Re-examining the role of hydrogen peroxide Heat-treatment of active honeys prior to incubation with E. coli the minimum DNA degrading activity of honey H O was below 2 2 cultures did not prevent DNA degradation, suggesting against the 2.5 mM. The lower concentrations of honeys H O required to 2 2 involvement of DNase in this process. Moreover, not all tested hon- effectively degrade chromosomal DNA strongly suggest that the eys displayed DNA degrading activity on E. coli cells. Given that oxidizing effect of H O was augmented by other honey com- 2 2 bacterial cells are impermeable to DNase, the DNA degradation ponents such as transition metals (Fe, Cu) commonly present in by honeys observed in this study could not be simply explained by honeys. In support of this notion, the recent literature evidence the DNase contaminations. Rather, the close relationship between indicates that it is the hydroxyl radical (HO) that is produced in the DNA degradation and H O content in honeys advocates for the metal-catalyzed Fenton reaction from H O rather than molecular 2 2 2 2 role of H O in the mechanism of DNA cleavage. hydrogen peroxide that causes the oxidative damage to membrane 2 2 DNA degradation is a lethal event which ultimately kills the structures, proteins, and DNA (Imlay et al., 1988; Storz and Imlay, cell. Literature data indicate that the concentration of hydrogen 1999; Cabiscol et al., 2000; Imlay, 2003). peroxide plays a decisive role in the type of cell death that fol- In conclusion, our study demonstrated that honey H O 2 2 lows H O exposure. In simplified in vitro models, where direct exerted bacteriostatic and DNA degrading activities to bacter- 2 2 effects of hydrogen peroxide on bacterial cells were analyzed, two ial cells. The extent of damaging effects of honey H O was 2 2 separate modes of killing were observed for E. coli. At low con- strongly influenced by the bacterial sensitivity to oxidative stress, centrations of H O (≤2.5 mM), E. coli cells were dying because the growth phase and their survival strategy (non-spore forming 2 2 of DNA damage inflicted on the metabolically active cells (Imlay versus spore forming species) as well as by the modulation of other and Linn, 1986; Imlay and Linn, 1987a,b; Brandi et al., 1989). honey compounds. At H O concentrations of 10–50 mM, cell death resulted from 2 2 cytotoxic effects due to hydroxyl radicals formed from hydro- ACKNOWLEDGMENTS genperoxide(Imlay and Linn, 1987a,b; Brandi et al., 1989). In This research was supported by funds from the Agricultural Adap- a full agreement with these data, we established that the minimum tation Council, Agriculture and Agri-Food Canada (ADV-380), DNA degrading activity of exogenous H O on E. coli cells was and the Ontario Centres of Excellence (BM50849) awarded to 2 2 2.5 mM (2.5 μmoles/10 cfu/ml). In contrast to exogenous H O , Katrina Brudzynski. 2 2 REFERENCES multiple Fur homologues: identifi- subtilis. FEMS Microbiol. 124, Imlay, J. A., and Linn, S. (1987b). DNA Alaux, C., Ducloz, F., Crauser, D., and cation of the iron uptake (Fur) and 155–264. damage and oxygen radical toxicity. Le Conte, Y. (2010). Diet effects peroxide regulon (PerR) repressors. Dowds, B. 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Microbiol. 2, in honey, as hydrogen peroxide and Copyright © 2011 Brudzynski, Abubaker , 188–194. its origin in a honey glucose-oxidase St-Martin and Castle. This is an open- Storz, G., Tartaglia, L. A., and Ames, system. Biochem. Biophys. Acta 73, Received: 01 September 2011; accepted: access article subject to a non-exclusive B. N. (1990). Transcriptional reg- 57–70. 03 October 2011; published online: 25 license between the authors and Frontiers ulator of oxidative stress-inducible Zheng, M., Doan, B., Schneider, October 2011. Media SA, which permits use, distribu- genes: direct activation by oxidation. T. D., and Storz, G. (1999). Citation: Brudzynski K, Abubaker K, tion and reproduction in other forums, Science 248, 189–194. OxyR and SoxRS regula- St-Martin L and Castle A (2011) Re- provided the original authors and source Weston, R. J. (2000). The contribu- tion of fur. J. Bacteriol. 181, examining the role of hydrogen per- are credited and other Frontiers condi- tion of catalase and other natural 4639–4643. oxide in bacteriostatic and bactericidal tions are complied with. www.frontiersin.org October 2011 | Volume 2 | Article 213 | 9

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