Manuka honey treatment of biofilms of Pseudomonas aeruginosa results in the emergence of isolates with increased honey resistance

 Background Medical grade manuka honeys are well known to be efficacious against Pseudomonas aeruginosa being bactericidal and inhibiting the development of biofilms; moreover manuka honey effectively kills P. aeruginosa embedded within an established biofilm. Sustained honey resistance has not been previously documented for planktonic or biofilm P. aeruginosa. Methods Minimum inhibitory concentrations for manuka honey and antibiotics were determined using broth micro-dilution methods. Minimum biofilm eliminating concentrations (MBEC) and biofilm biomass were determined using the crystal violet method. Sub-culture used non-selective media and the grid-plate method. Results When honey treated biofilm biomass of two strains of P. aeruginosa (reference strain ATCC 9027 and the clinical isolate 867) were sub-cultured onto non-selective media isolates emerged that exhibited reduced susceptibility to manuka honey. Significantly, this characteristic was sustained with repeated sub-culture onto non-selective media resulting in increased minimum inhibitory concentrations (MIC) of between 5-7% (w/v) and increased minimum biofilm eliminating concentrations (MBEC) of up to 15% (w/v). Interestingly the resistant isolates showed reduced susceptibility to antibiotic treatment with rifampicin and imipenem as well as being more prolific biofilm-formers than the progenitor strains. Conclusions P. aeruginosa biofilms treated with manuka honey equivalent to the MBEC harbour slow growing, viable persistor organisms that exhibit sustained, increased resistance to manuka honey and antibiotic treatment, suggesting a shared mechanism of resistance. This sheds new light on the propensity for biofilm embedded organisms to resist honey treatment and become persistor organisms that are tolerant to other antimicrobial therapies.