Use of a model to understand the synergies underlying the antibacterial mechanism of H2O2-producing honeys

Honey has been valued as a powerful antimicrobial since ancient times. However, the understanding of the underlying antibacterial mechanism is incomplete. The complexity and variability of honey composition represent a challenge to this scope. In this study, a simple model system was used to investigate the antibacterial effect of, and possible synergies between, the three main stressors present in honey: sugars, gluconic acid, and hydrogen peroxide (H2O2), which result from the enzymatic conversion of glucose on honey dilution. Our results demonstrated that the synergy of H2O2 and gluconic acid is essential for the antibacterial activity of honey. This synergy caused membrane depolarization, destruction of the cell wall, and eventually growth inhibition of E. coli K-12. The presence of H2O2 stimulated the generation of other long-lived ROS in a dose-dependent manner. Sugars caused osmosis-related morphological changes, however, decreased the toxicity of the H2O2/gluconic acid. The susceptibility of catalase and general stress response sigma factor mutants confirmed the synergy of the three stressors, which is enhanced at higher H2O2 concentrations. By monitoring cellular phenotypic changes caused by model honey, we explained how this can be bactericidal even though the antimicrobial compounds which it contains are at non-inhibitory concentrations.