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. Using the sensitive peroxide/peroxidase assay, broth microdilution assay and DNA degradation assays, the quantitative relationships between the content of H₂O₂ and honey’s antibacterial activity was established_. The results showed that: (A) the average H₂O₂ content in honey was over 900-fold lower than that observed in disinfectants that kills bacteria on contact. (B) A supplementation of bacterial cultures with H₂O₂ inhibited E. coli and B. subtilis growth in a concentration-dependent manner, with minimal inhibitory concentrations (MIC₉₀) values of 1.25 mM/10⁷ cfu/ml and 2.5 mM/10⁷ cfu/ml for E. coli and B. subtilis, respectively. In contrast, the MIC₉₀ of honey against E. coli correlated with honey H₂O₂ content of 2.5 mM, and growth inhibition of B. subtilis by honey did not correlate with honey H₂O₂ levels at all. (C) A supplementation of bacterial cultures with H₂O₂ caused a concentration-dependent degradation of bacterial DNA, with the minimum DNA degrading concentration occurring at 2.5 mM H₂O₂. DNA degradation by honey occurred at lower than ≤2.5 mM concentration of honey H₂O₂ suggested an enhancing effect of other honey components. (D) Honeys with low H₂O₂ content were unable to cleave DNA but the addition of H₂O₂ restored this activity. The DNase-like activity was heat-resistant but catalase-sensitive indicating that H₂O₂ participated in the oxidative DNA damage. We concluded that the honey H₂O₂ was involved in oxidative damage causing bacterial growth inhibition and DNA degradation, but these effects were modulated by other honey components.

Honey is recognized as an efficacious topical antimicrobial agent in the treatment of burns and wounds. The antimicrobial activity in some honeys depends on the endogenous hydrogen peroxide content. This study was aimed to determine whether honey's hydrogen peroxide level could serve as a honey-specific, activity-associated biomarker that would allow predicting and assessing the therapeutic effects of honey. Using a broth microdilution assay, I analyzed antibacterial activities of 42 Canadian honeys against two bacterial strains: Escherichia coli (ATCC 14948) and Bacillus subtilis (ATCC 6633). The MIC₉₀ and MIC₅₀ were established from the dose-response relationship between antibacterial activities and honey concentrations. The impact of H₂O₂ on antibacterial activity was determined (i) by measuring the levels of H₂O₂ before and after its removal by catalase and (ii) by correlating the results with levels of antibacterial activities. Canadian honeys demonstrated moderate to high antibacterial activity against both bacterial species. Both MIC₉₀ and MIC₅₀ revealed that the honeys exhibited a selective growth inhibitory activity against E. coli, and this activity was strongly influenced by endogenous H₂O₂ concentrations. Bacillus subtilis activity was marginally significantly correlated with H₂O₂ content. The removal of H₂O₂ by catalase reduced the honeys' antibacterial activity, but the enzyme was unable to completely decompose endogenous H₂O₂. The 25%-30% H₂O₂ "leftover" was significantly correlated with the honeys' residual antibacterial activity against E. coli. These data indicate that all Canadian honeys exhibited antibacterial activity, with higher selectivity against E. coli than B. subtilis, and that these antibacterial activities were correlated with hydrogen peroxide production in honeys. Hydrogen peroxide levels in honey, therefore, is a strong predictor of the honey's antibacterial activity.Key words: honey, antibacterial activity, hydrogen peroxide, catalase, Escherichia coli, Bacillus subtilis.

The fundamental feature of “active honeys” is the presence and concentration of antibacterial compounds. Currently identified compounds and factors have been described in several review papers without broader interpretation or links to the processes for their formation. In this review, we indicate that the dynamic, antagonistic/competitive microbe–microbe and microbe–host interactions are the main source of antibacterial compounds in honey. The microbial colonization of nectar, bees and honey is at the center of these interactions that in consequence produce a range of defence molecules in each of these niches. The products of the microbial interference and exploitive competitions include antimicrobial peptides, antibiotics, surfactants, inhibitors of biofilm formation and quorum sensing. Their accumulation in honey by horizontal transfer might explain honey broad-spectrum, pleiotropic, antibacterial activity. We conclude that honey is an ecological reservoir of antibacterial compounds produced by antagonistic microbial interactions in plant nectars, honey and honey bee. Thus, refocusing research on secondary metabolites resulting from these microbial interactions might lead to discovery of new antibacterial compounds in honey that are target-specific, i.e., acting on specific cellular components or inhibiting the essential cellular function.

We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A- affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log₁₀ reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey’s active principle responsible for this activity.

It has been recently reported that honey hydrogen peroxide in conjunction with unknown honey components produced cytotoxic effects resulting in bacterial growth inhibition and DNA degradation. The objective of this study was twofold: (a) to investigate whether the coupling chemistry involving hydrogen peroxide is responsible for a generation of hydroxyl radicals and (b) whether ^•OH generation affects growth of multi-drug resistant clinical isolates. The susceptibility of five different strains of methicillin-resistant Staphylococcus aureus (MRSA) and four strains of vancomycin-resistant Enterococcus faecium (VRE) isolates from infected wounds to several honeys was evaluated using broth microdilution assay. Isolates were identified to genus and species and their susceptibility to antibiotics was confirmed using an automated system (Vitek^®, Biomérieux^®). The presence of the mec(A) gene, nuc gene and van(A) and (B) genes were confirmed by polymerase chain reaction. Results showed that no clinical isolate was resistant to selected active honeys. The median difference in honeys MICs against these strains ranged between 12.5 and 6.25% v/v and was not different from the MIC against standard Escherichia coli and Bacillus subtilis. Generation of ^•OH during bacteria incubation with honeys was analyzed using 3′-(p-aminophenyl) fluorescein (APF) as the ^•OH trap. The ^•OH participation in growth inhibition was monitored directly by including APF in broth microdilution assay. The growth of MRSA and VRE was inhibited by ^•OH generation in a dose-dependent manner. Exposure of MRSA and VRE to honeys supplemented with Cu(II) augmented production of ^•OH by 30-fold and increased honey bacteriostatic potency from MIC₉₀ 6.25 to MIC₉₀< 0.78% v/v. Pretreatment of honeys with catalase prior to their supplementation with Cu ions fully restored bacterial growth indicating that hydroxyl radicals were produced from H₂O₂ via the Fenton-type reaction. In conclusion, we have demonstrated for the first time that bacteriostatic effect of honeys on MRSA and VRE was dose-dependently related to generation of ^•OH from honey H₂O₂.