With the rise in prevalence of antibiotic-resistant bacteria, honey is increasingly valued for its antibacterial activity. To characterize all bactericidal factors in a medical-grade honey, we used a novel approach of successive neutralization of individual honey bactericidal factors. All bacteria tested, including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase producing Escherichia coli, ciprofloxacin-resistant Pseudomonas aeruginosa, and vancomycin-resistant Enterococcus faecium, were killed by 10-20 (v/v) honey, whereas > or = 40 (v/v) of a honey-equivalent sugar solution was required for similar activity. Honey accumulated up to 5.62 +/- 0.54 mM H(2)O(2) and contained 0.25 +/- 0.01 mM methylglyoxal (MGO). After enzymatic neutralization of these two compounds, honey retained substantial activity. Using B. subtilis for activity-guided isolation of the additional antimicrobial factors, we discovered bee defensin-1 in honey. After combined neutralization of H(2)O(2), MGO, and bee defensin-1, 20 honey had only minimal activity left, and subsequent adjustment of the pH of this honey from 3.3 to 7.0 reduced the activity to that of sugar alone. Activity against all other bacteria tested depended on sugar, H(2)O(2), MGO, and bee defensin-1. Thus, we fully characterized the antibacterial activity of medical-grade honey.
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