Abstract
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.
Highlights
Honey has well established function as an effective antibacterial agent with a broad spectrum of activity against Gram-positive and Gram-negative bacteria
Since H2O2 is a common component in honeys, a byproduct of glucose oxidation by honeybee glucose oxidase, we explored a possibility that generation of hydroxyl radicals from honey hydrogen peroxide represents a general mechanism by which honey affects bacterial growth
BACTERIOSTATIC EFFECT OF HONEY AGAINST ANTIBIOTIC-RESISTANT CLINICAL ISOLATES From a large pool of over 200 honeys screened for their bacteriostatic activity against E. coli and B. subtilis (Brudzynski and Kim, 2011), we selected eleven honeys that showed MIC90 exceeding those of sugar solution
Summary
Honey has well established function as an effective antibacterial agent with a broad spectrum of activity against Gram-positive and Gram-negative bacteria (for review, Lusby et al, 2002; Irish et al, 2011). Despite a progress in identification of compounds that are involved in growth inhibitory and bactericidal actions of honey, the mechanism underlying these activities remained unknown. Our data indicate that H2O2 content in honeys (0.4–2.6 mM), is much below its biocidal levels. Even at these low H2O2 concentrations, honeys effectively inhibited bacterial growth and caused DNA strand breaks (Brudzynski et al, 2011). In our accompanying paper, we provided the first evidence that honeys of high bacteriostatic activity (MIC90 12.5–6.25% v/v) possessed significantly higher levels of phenolic compounds of higher radical scavenging activities than honey of the average bacteriostatic activity (MIC90 25% v/v), suggesting the involvement of phenolics and H2O2 in bacterial growth inhibition (Brudzynski et al, 2012)
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