Hydrogen peroxide is generally thought to be the main compound responsible for the antibacterial action of honey (White et al., 1963; Weston, 2000; Brudzynski, 2006). Hydrogen peroxide in honey is produced mainly during glucose oxidation catalyzed by the bee enzyme, glucose oxidase (FAD-oxidoreductase, EC 1.1.3.4; White et al., 1963). The levels of hydrogen peroxide in honey are determined by the difference between the rate of its production and its destruction by catalases. Glucose oxidase is introduced to honey during nectar harvesting by bees. This enzyme is found in all honeys but its concentration may differ from honey to honey depending on the age and health status of the foraging bees (Pernal and Currie, 2000) as well as the richness and diversity of the foraged diet (Alaux et al., 2010). Catalases on the other hand, are of pollen origin. Catalase efficiently hydrolyzes hydrogen peroxide to oxygen and water due to its high turnover numbers. The total concentration of catalase depends on the amount of pollen grains in honey (Weston, 2000), and consequently, the hydrogen peroxide levels in different honeys may vary considerably (Brudzynski, 2006).

A substantial correlation has been found between the level of endogenous hydrogen peroxide and the extent of inhibition of bacterial growth by honey (White et al., 1963; Brudzynski, 2006). We have observed that in honeys with a high content of this oxidizing compound, bacteria cannot respond normally to proliferative signals and their growth remains arrested even at high honey dilutions. Pre-treatment of honey with catalase restored, to a certain extent, the bacterial growth, thus suggesting that endogenous H₂O₂ was implicated in the growth inhibition (Brudzynski, 2006).

Most of the conclusions on the H₂O₂ oxidizing action on bacteria are drawn from the simplified in vitro models, where direct effects of hydrogen peroxide on bacterial cells were analyzed. In contrast, honey represent complex chemical milieu consisting of over 100 different compounds (including antioxidants and traces of transition metals), where the interaction between these components and hydrogen peroxide may influence its oxidative action. We have recently unraveled that honey is a dynamic reaction mixture which facilitates and propagates the Maillard reaction (Brudzynski and Miotto, 2011b). The Maillard reaction which initially involves reaction between amino groups of amino acids or proteins with carbonyl groups of reducing sugars leads to a cascade of redox reactions in which several bioactive molecules are continuously formed and lost due to their cross-linking to other molecules (gain or loss of function; Brudzynski and Miotto, 2011b). We have shown that polyphenol-based melanoidins are a major group of Maillard reaction products possessing radical-scavenging activity (Brudzynski and Miotto, 2011a,b). These compounds are likely to interact with hydrogen peroxide and, depending of their concentration and redox capacity, either enhanced or diminished the oxidative activity of honey’s H₂O₂. In view of these facts, we hypothesized that the oxidizing action of honey’s hydrogen peroxide on bacterial cells may be modulated by the presence of other bioactive molecules in honey and therefore, may differ from the action of hydrogen peroxide alone.

Hydrogen peroxide is commonly used to disinfect and sanitize medical equipment in hospitals. For this purpose, the high concentrations of H₂O₂ in these disinfectants have to be maintained to overwhelmed defense systems of bacteria. At high concentrations, ranging from 3 to 30% (0.8 to 8 M), its bactericidal effectiveness has been demonstrated against several microorganisms including Staphylococcus-, Streptococcus-, Pseudomonas-species, and Bacillus spores (Rutala et al., 2008).Under these conditions, the bacterial cell death results from the accumulation of irreversible oxidative damages to the membrane layers, proteins, enzymes, and DNA (Davies, 1999, 2000; Rutala et al., 2008; Finnegan et al., 2010).

However, the hydrogen peroxide content in honey is about 900-fold lower (Brudzynski, 2006). Moreover, the literature data indicate that the cell death of cultured mammalian, yeast, and bacterial cells required H₂O₂ concentrations higher than 50 mM and was associated with chromosomal DNA degradation (Imlay and Linn, 1987a,b; Brandi et al., 1989; Davies, 1999; Bai and Konat, 2003; Ribeiro et al., 2006), which is still five to 10-fold higher then that observed in honeys. Therefore, we have undertaken this study to re-examined the role of hydrogen peroxide in antibacterial activity of honeys.

The hydrogen peroxide efficacy as an oxidative biocide is related to the bacterial sensitivity to peroxide stress. Defense mechanisms to oxidative stress varies between bacterial species such as Gram-negative E. coli and Gram-positive B. subtilis used in this study and depend on the growth phase (exponential- versus stationary-phase of growth), and on the adaptive and survival mechanisms (non-spore forming versus spore-forming bacteria; Dowds et al., 1987; Chen et al., 1995; Storz and Imlay, 1999; Cabiscol et al., 2000). In honey, the effects of H₂O₂ on the growth and survival of microorganisms may be mitigated or enhanced due to the presence of honey compounds. On one hand, a high content of sugars in honey that abstracts free water molecules from milieu inhibits bacterial growth and proliferation, but honey dilutions may create growth-supportive conditions due to the abundance of sugars as a carbon source for the growing cells. Hydrogen peroxide has deleterious effects on the growth and survival of bacterial cells but honey antioxidants such as catalases, polyphenols, Maillard reaction products, and ascorbic acid may lower the oxidative stress to cells and may have a protective effect against endogenous H₂O₂ (Brudzynski, 2006). Even less information exists on the mechanism of bactericidal action of honey’s hydrogen peroxide. The most fundamental and unsolved questions concerns the molecular targets of H₂O₂ cytotoxicity: does molecular hydrogen peroxide at concentrations present in honey cause DNA degradation?

During last decades, several honey compounds were identified as those implicated in honey antibacterial activity (for review, Irish et al., 2011). Despite this knowledge, the mechanisms by which these compounds lead to bacterial growth inhibition and bacterial death have never been explained or proven in biochemical terms. Since there is a persistent view that hydrogen peroxide is a main player in these events, the aim of this study was to critically analyze the effects of hydrogen peroxide on growth and survival of bacterial cells in order to prove or disprove its purported role as a main component responsible for the antibacterial activity of honey.

The findings described in this study revise the old views and provide novel information on the role of hydrogen peroxide in the regulation of bacteriostatic and bactericidal activities of honey.

Firstly, we found that the exponentially growing E. coli and B. subtilis cells were inhibited in a concentration-dependent manner by exogenous H₂O₂ reaching MIC₉₀ at 1.25 mM (1.25 μmoles/10⁷ cfu/ml) and 2.5 mM (2.5 μmoles/10⁷ cfu/ml), respectively. The bacteriostatic efficacy of H₂O₂ however differed significantly from that of honey H₂O₂. The main factors that contributed to these differences were (a) bacterial susceptibility/resistance to the oxidative action of hydrogen peroxide and (b) interference from other honey components.

Endogenous H₂O₂ inhibited the growth of E. coli in a concentration-dependent manner, but its MIC₉₀ was twofold higher than those of exogenous H₂O₂ (2.5 versus 1.25 mM, respectively). The honeys MIC₉₀ levels against E. coli coincided with the dilutions at which a peak of hydrogen peroxide production occurred. Treatment of honeys with catalase led to a significant reduction in their bacteriostatic activity (Brudzynski, 2006). Together, these data provide direct evidence that E. coli growth is sensitive to oxidative action of honey H₂O₂.

In contrast, growth inhibition of B. subtilis was not due to the action of honey H₂O₂. While exposure of B. subtilis cultures to H₂O₂ resulted in a concentration-dependent growth inhibition, the comparable concentrations of honey H₂O₂ in honey were 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 H₂O₂. These results suggest that other honey compounds were responsible for the inhibition of B. subtilis growth. As a consequence of growth arrest, the change in sensitivity of B. subtilis to honey H₂O₂ occurred. Instead of growth inhibition, we observed growth stimulation of B. subtilis at high honey dilutions (16-fold and over) and in the presence of high levels of H₂O₂. Literature data provides compelling evidence 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^s transcriptional factor which regulates a stationary-phase gene expression of rpoS regulon. The expression of σ^s factor in B. subtilis evokes spore-formation to enhance bacterial survival (Dowds et al., 1987; Dowds, 1994; Loewen et al., 1998; Zheng et al., 1999; Chen and Schellhorn, 2003). Dowds et al. 1987 have shown that stationary-phase cultures of B. subtilis displayed viability even at the 10 mM concentration of H₂O₂. These data may explain, at least in part, an apparent insensitivity of B. subtilis to high levels of hydrogen peroxide in honey.

These results revealed significant differences in the sensitivities of E. coli and B. subtilis to oxidative stress caused by honey H₂O₂. As aerobic bacteria, both E. coli and B. subtilis are equipped with molecular machinery to cope with oxidative stress by activating 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 (alkylhydroperoxide 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 H₂O₂ participated in bacterial DNA degradation. Several lines of evidence support this finding. Firstly, the treatment of exponential-phase E. coli cultures with increasing concentrations of exogenous hydrogen peroxide (5–0.3125 mM) or honeys of different content of endogenous H₂O₂ led to a concentration-dependent DNA degradation. While the minimum DNA degrading activity of exogenous H₂O₂ occurred at 2.5 mM (2.5 μmoles/10⁷ cfu/ml), in contrast, honeys possessing H₂O₂ concentrations lower than 2.5 mM were still active in this process. Secondly, DNA degradation by active honeys was abolished by removal of H₂O₂ by catalase. Thirdly, honeys with the low content of H₂O₂ were unable to degrade DNA but the supplementation with 2 mM of hydrogen peroxide caused the appearance of this activity. The extent of DNA degradation by honey, which was supplemented with H₂O₂, was comparable to that of active honeys.

Heat-treatment of active honeys prior to incubation with E. coli cultures did not prevent DNA degradation, suggesting against the involvement of DNase in this process. Moreover, not all tested honeys displayed DNA degrading activity on E. coli cells. Given that bacterial cells are impermeable to DNase, the DNA degradation by honeys observed in this study could not be simply explained by the DNase contaminations. Rather, the close relationship between DNA degradation and H₂O₂ content in honeys advocates for the role of H₂O₂ in the mechanism of DNA cleavage.

DNA degradation is a lethal event which ultimately kills the cell. Literature data indicate that the concentration of hydrogen peroxide plays a decisive role in the type of cell death that follows H₂O₂ exposure. In simplified in vitro models, where direct effects of hydrogen peroxide on bacterial cells were analyzed, two separate modes of killing were observed for E. coli. At low concentrations of H₂O₂ (≤2.5 mM), E. coli cells were dying because of DNA damage inflicted on the metabolically active cells (Imlay and Linn, 1986; Imlay and Linn, 1987a,b; Brandi et al., 1989). At H₂O₂ concentrations of 10–50 mM, cell death resulted from cytotoxic effects due to hydroxyl radicals formed from hydrogen peroxide (Imlay and Linn, 1987a,b; Brandi et al., 1989). In a full agreement with these data, we established that the minimum DNA degrading activity of exogenous H₂O₂ on E. coli cells was 2.5 mM (2.5 μmoles/10⁷cfu/ml). In contrast to exogenous H₂O₂, the minimum DNA degrading activity of honey H₂O₂ was below 2.5 mM. The lower concentrations of honeys H₂O₂ required to effectively degrade chromosomal DNA strongly suggest that the oxidizing effect of H₂O₂ was augmented by other honey components such as transition metals (Fe, Cu) commonly present in honeys. In support of this notion, the recent literature evidence indicates that it is the hydroxyl radical (HO) that is produced in the metal-catalyzed Fenton reaction from H₂O₂ rather than molecular hydrogen peroxide that causes the oxidative damage to membrane structures, proteins, and DNA (Imlay et al., 1988; Storz and Imlay, 1999; Cabiscol et al., 2000; Imlay, 2003).

In conclusion, our study demonstrated that honey H₂O₂ exerted bacteriostatic and DNA degrading activities to bacterial cells. The extent of damaging effects of honey H₂O₂ was strongly influenced by the bacterial sensitivity to oxidative stress, the growth phase and their survival strategy (non-spore forming versus spore forming species) as well as by the modulation of other honey compounds.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.