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...

The purpose of this study was to investigate the effect of manuka honey on Staphylococcus aureus in order to identify the intracellular target site. The mode of inhibition of manuka honey against S. aureus NCTC 10017 was investigated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the effect of time on viability. Structural changes were observed by scanning (SEM) and transmission electron microscopy (TEM) of cells suspended for 4 h at 37 degrees C in 0.05 mM Tris buffer containing 10% (w/v) manuka honey and were compared to cells in buffer alone or buffer containing 10% (w/v) artificial honey (to assess osmotic damage). A bactericidal mode of inhibition for manuka honey on S. aureus was established. Marked structural changes in honey-treated cells were seen only with TEM, where a statistically significant increase in the number of whole cells with completed septa compared to untreated cells were observed (P < 0.05). Structural changes found with TEM suggest that honey-treated cells had failed to progress normally through the cell cycle and accumulated with fully formed septa at the point of cell division without separating. Sugars were not implicated in this effect. The staphylococcal target site of manuka honey involves the cell division machinery.

The purpose of this study was to investigate the effects of manuka honey on the structural integrity of Pseudomonas aeruginosa ATCC 27853. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of manuka honey for P. aeruginosa were determined by a microtitre plate method, and the survival of bacteria exposed to a bactericidal concentration of manuka honey was monitored. The effect of manuka honey on the structure of the bacteria was investigated using scanning and transmission electron microscopy (SEM and TEM, respectively). The MIC and MBC values of manuka honey against P. aeruginosa were 9.5% (w/v) and 12% (w/v) respectively; a time-kill curve demonstrated a bactericidal rather than a bacteriostatic effect, with a 5 log reduction estimated within 257 min. Using SEM, loss of structural integrity and marked changes in cell shape and surface were observed in honey-treated cultures. With TEM, these changes were confirmed, and evidence of extensive cell disruption and lysis was found. Manuka honey does not induce the same structural changes in P. aeruginosa as those observed in staphylococci. Our results indicate that manuka honey has the potential to be an effective inhibitor of P. aeruginosa.

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Staphylococcus aureus is an important pathogen that can cause many problems, from impetigo to endocarditis. With its continued resistance to multiple antibiotics, S. aureus remains a serious health threat. Honey has been used to eradicate meticillin-resistant S. aureus (MRSA) strains from wounds, but its mode of action is not yet understood. Proteomics provides a potent group of techniques that can be used to analyse differences in protein expression between untreated bacterial cells and those treated with inhibitory concentrations of manuka honey. In this study, two-dimensional (2D) electrophoresis was combined with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to determine the identities of proteins whose levels of expression were changed at least two-fold following treatment with manuka honey. Protein extracts were obtained from cells grown in tryptone soy broth (with or without manuka honey) by mechanical disruption and were separated on 2D polyacrylamide gels. A protein was isolated in gels prepared from untreated cell extract that was absent from gels made using honey-treated cell extract. Using MALDI-TOF MS, the protein was identified as universal stress protein A (UspA). Downregulation of this protein was confirmed by real-time polymerase chain reaction (PCR), which showed a 16-fold downregulation in honey-treated cells compared with untreated samples. This protein is involved in the stress stamina response and its downregulation could help to explain the inhibition of MRSA by manuka honey.