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Aims: To determine the sensitivity to honey of Gram‐positive cocci of clinical significance in wounds and demonstrate that inhibition is not exclusively due to osmotic effects. Methods and Results: Eighteen strains of methicillin‐resistant Staphylococcus aureus and seven strains of vancomycin‐sensitive enterococci were isolated from infected wounds and 20 strains of vancomycin‐resistant enterococci were isolated from hospital environmental surfaces. Using an agar incorporation technique to determine the minimum inhibitory concentration (MIC), their sensitivity to two natural honeys of median levels of antibacterial activity was established and compared with an artificial honey solution. For all of the strains tested, the MIC values against manuka and pasture honey were below 10% (v/v), but concentrations of artificial honey at least three times higher were required to achieve equivalent inhibition in vitro. Comparison of the MIC values of antibiotic‐sensitive strains with their respective antibiotic‐resistant strains demonstrated no marked differences in their susceptibilities to honey. Conclusions: The inhibition of bacteria by honey is not exclusively due to osmolarity. For the Gram‐positive cocci tested, antibiotic‐sensitive and ‐resistant strains showed similar sensitivity to honey. Significance and Impact of the Study: A possible role for honey in the treatment of wounds colonized by antibiotic‐resistant bacteria is indicated.

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SUMMARYThe antibacterial action of honey in infected wounds does not depend wholly on its high osmolarity. We tested the sensitivity of 58 strains of coagulase-positive Staphylococcus aureus, isolated from infected wounds, to a pasture honey and a manuka honey. There was little variation between the isolates in their sensitivity to honey: minimum inhibitory concentrations were all between 2 and 3% (v/v) for the manuka honey and between 3 and 4% for the pasture honey. Thus, these honeys would prevent growth of S. aureus if diluted by body fluids a further seven-fold to fourteen-fold beyond the point where their osmolarity ceased to be completely inhibitory. The antibacterial action of the pasture honey relied on release of hydrogen peroxide, which in vivo might be reduced by catalase activity in tissues or blood. The action of manuka honey stems partly from a phytochemical component, so this type of honey might be more effective in vivo. Comparative clinical trials with standardized honeys are needed.

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were exposed to sub-lethal concentrations of manuka honey in continuous and stepwise training experiments to determine whether susceptibility to honey diminished. Reduced susceptibilities to manuka honey in the test organisms during long-term stepwise resistance training were found, but these changes were not permanent and honey-resistant mutants were not detected. The risk of bacteria acquiring resistance to honey will be low if high concentrations are maintained clinically.Response to Reviewers: Dear Editor, Thank you for the reviewers' comments on our paper entitled "Absence of bacterial resistance to medical-grade manuka honey". We were pleased that they liked the design and execution of our study and we understood the reservations that reviewer had about our conclusions. We have made the following changes in response to each specific observation, which we feel strengthen our paper: Reviewer 1. We have inserted line numbers into the paper. In order to rebut the reservations about our conclusions we have recently performed further tests on the four clinical isolates that were collected at the end of the recovery period and stored at -80⁰C. For each of the thawed cultures, MICs and MBCs were determined in duplicate on three separate occasions. We found that three cultures (P.aeruginosa, S. epidermidis and MRSA) had returned to pre-training levels of susceptibility and that the MIC of E. coli was only 1.4 times higher than at time 0. That information is included as Table 2 (line 370) and described in lines 201 to 209. We respectfully maintain that honey-resistant mutants were not recovered. To support this conclusion as advised we noted (lines 241-243) that antibiotic-resistance training led to MICs that increased by factors of either 32 or 64 (citation 17-Blair et al, 2009), while honey resistance training led to an increase of 1.4. We also used the EUCAST definition of clinical resistance to show that honey susceptibility which had increased by a factor of 1.4 is unlikely to lead to therapeutic failure when wound care products normally contain at least 80% manuka honey and normally 95%(w/v) (lines 258 to 260). We have removed the statement "gradually increased towards pre-treatment levels". We have made clear the proportional changes in MICs (lines 195 to 197 and 208 and 251). We have commented on the need to maintain high concentrations of manuka honey in wounds to avoid the selection of resistant strains (lines 278 to 284). We have modified the Abstract (lines 42 to 45) to explain that reduced susceptibility was found during long-term training and we have commented on the need to maintain high concentrations of manuka honey during clinical use (lines 45 to 46). In the concluding paragraph we also note that prolonged exposure to antimicrobial agents should be avoided. We have deleted statements about the development of antibiotic resistance from the Introduction and the Discussion. We have inserted "preceding days' culture" into line 169, and "day" into line 194. We have removed the reference to high sugar c...

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Because there is no ideal therapy for burns infected with Pseudomonas aeruginosa, there is sufficient need to investigate the efficacy of alternative antipseudomonal interventions. Honey is an ancient wound remedy for which there is modern evidence of efficacy in the treatment of burn wounds, but limited evidence for the effectiveness of its antibacterial activity against Pseudomonas. We tested the sensitivity of 17 strains of P. aeruginosa isolated from infected burns to two honeys with different types of antibacterial activity, a pasture honey and a manuka honey, both with median levels of activity. All strains showed similar sensitivity to honey with minimum inhibitory concentrations below 10% (vol/vol); both honeys maintained bactericidal activity when diluted more than 10-fold. Honey with proven antibacterial activity has the potential to be an effective treatment option for burns infected or at risk of infection with P. aeruginosa.