Antibacterial activity is the most investigated biological property of honey. The goal of this study was to evaluate the antibacterial activity of 57 Slovak blossom honeys against Staphylococcus aureus and Pseudomonas aeruginosa and investigate the role of several bioactive substances in antibacterial action of honeys. Inhibitory and bactericidal activities of honeys were studied to determine the minimum inhibitory and bactericidal concentrations. The contents of glucose oxidase (GOX) enzyme, hydrogen peroxide (H₂O₂), and total polyphenols (TP) were determined in honeys. We found that honey samples showed different antibacterial efficacy against the tested bacteria as follows: wildflower honeys > acacia honeys > rapeseed honeys. Overall antibacterial activity of the honeys was statistically-significantly correlated with the contents of H₂O₂ and TP in honeys. A strong correlation was found between the H₂O₂ and TP content. On the other hand, no correlation was found between the content of GOX and level of H₂O₂. Antibacterial activity of 12 selected honeys was markedly reduced by treatment with catalase, but it remained relatively stable after inactivation of GOX with proteinase-K digestion. Obtained results suggest that the antibacterial activity of blossom honeys is mainly mediated by H₂O₂ levels present in honeys which are affected mainly by polyphenolic substances and not directly by GOX content.

Honeydew honey is increasingly valued due to its pronounced antibacterial potential; however, the underlying mechanism and compounds responsible for the strong antibacterial activity of honeydew honey are still unknown. The aim of this study was to investigate the inhibition of bacterial growth of 23 honeydew honey samples. Activity of bee-derived glucose oxidase (GOX) enzyme, the content of defensin-1 (Def-1) and hydrogen peroxide (H₂O₂), and total polyphenol content were determined in the 23 honey samples. Our results demonstrated that antibacterial activity of honeydew honey was equivalent to medical-grade manuka and kanuka honey and was abolished by catalase. Although H₂O₂ is an important factor in the inhibition of bacterial growth, polyphenolic compounds and their interaction with H₂O₂ are the key factors responsible for high antibacterial activity of honeydew honey. In addition, our results indicated that the antibacterial activity of honeydew honey is not dependent on GOX-mediated production of H₂O₂ or the presence of Def-1.

Royal jelly (RJ) has successfully been used as a remedy in wound healing. RJ has multiple effects, including antibacterial, anti-inflammatory and immunomodulatory activities, in various cell types. However, no component(s) (other than antibacterial) have been identified in RJ-accelerated wound healing. In this study, we demonstrate that keratinocytes are responsible for the elevated production of matrix metalloproteinase-9 (MMP-9) after incubation with a water extract of RJ. Furthermore, the keratinocyte migration and wound closure rates were significantly increased in the presence of RJ extract. MMP-9 production was reduced significantly following proteinase K treatment but remained stable after heat treatment, indicating that active component(s) have a proteinous character. To identify the component responsible for inducing MMP-9 production, RJ extract was fractionated using C18 RP-HPLC. In fractions exhibiting stimulatory activity, we immunochemically detected the bee-derived antibacterial peptide, defensin-1. Defensin-1 was cloned, and recombinant peptide was produced in a baculoviral expression system. Defensin-1 stimulated MMP-9 secretion from keratinocytes and increased keratinocyte migration and wound closurein vitro. In addition, defensin-1 promoted re-epithelisation and wound closure in uninfected excision wounds. These data indisputably demonstrate that defensin-1, a regular but concentration variable factor found in honey and RJ, contributes to cutaneous wound closure by enhancing keratinocyte migration and MMP-9 secretion.

Many clinically relevant biofilms are polymicrobial. Examining the effect of antimicrobials in a multi-species biofilm consortium is of great clinical importance. The goal of this study was to investigate the effect of different honey types against bacterial wound pathogens grown in multi-species biofilm and to test the antibiofilm activity of honey defensin-1 in its recombinant form. Modified Lubbock chronic wound biofilm formed by four bacterial species (Staphylococcus aureus, Streptococcus agalactiae, Pseudomonas aeruginosa, Enterococcus faecalis) was used for evaluation of honey and recombinant bee-derived defensin-1 antibiofilm efficacy. Defensin-1 was prepared by heterologous expression in Escherichia coli. We showed that different types of honey (manuka and honeydew) were able to significantly reduced cell viability of wound pathogens (S. aureus, S. agalactiae and P. aeruginosa) in polymicrobial biofilm. None of the tested honeys showed the ability to eradicate E. faecalis in biofilm. In addition, recombinant defensin-1 successfully reduced the viability of S. aureus and P. aeruginosa cells within established polymicrobial biofilm after 24 and 48 hours of treatment. Interestingly, recombinant defensin-1 did not affect the viability of S. agalactiae cells within the biofilm whereas both natural honeys significantly reduced the viable bacteria. Although E. faecalis was highly resistant to defensin-1, defensin-1 significantly affected biofilm formation of E. faecalis and S. agalactiae after 24 hours of treatment, most likely through the inhibiting its extracellular polymeric substances production. In conclusion, our study reveals that honey and defensin-1 are effective against established multi-species biofilm; however, E. faecalis grown in multi-species biofilm was resistant to both antimicrobials.