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 (H 2 O 2 ), which result from the enzymatic conversion of glucose on honey dilution. Our results demonstrated that the synergy of H 2 O 2 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 H 2 O 2 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 H 2 O 2 /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 H 2 O 2 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.