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

Aimee L Camplin,Sarah E Maddocks

doi:10.1186/1476-0711-13-19

Aimee L Camplin, Sarah E Maddocks

Open Access

https://doi.org/10.1186/1476-0711-13-19

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Abstract

BackgroundMedical 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.MethodsMinimum 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.ResultsWhen 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.ConclusionsP. 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.

Highlights

Medical grade manuka honeys are well known to be efficacious against Pseudomonas aeruginosa being bactericidal and inhibiting the development of biofilms; manuka honey effectively kills P. aeruginosa embedded within an established biofilm
Biofilms were cultured in 5 ml nutrient broth (NB) supplemented with manuka honey at the appropriate Minimum biofilm eliminating concentrations (MBEC) (50% w/v for ATCC 9207 and 45% w/v for 867) in a sterile plastic Petri dish for 48 h at 37°C, biomass was recovered by scraping the plate into 1 ml PBS, re-suspending by vortex and lawn plating onto nutrient agar (NA), these were incubated at 37°C for 48 h
Isolates recovered from biofilm biomass showed sustained, increased resistance to manuka honey treatment when cultured planktonically and as a biofilm The minimum inhibitory concentrations (MIC) for the reference strain ATCC 9027 was lower than the recovered isolate, being 25.6% (w/v) and 30.6%

Summary

Introduction

Medical grade manuka honeys are well known to be efficacious against Pseudomonas aeruginosa being bactericidal and inhibiting the development of biofilms; manuka honey effectively kills P. aeruginosa embedded within an established biofilm. Studies have demonstrated that with shortand long-term training experiments, no resistance to manuka honey treatment emerges [4,5]. This is of particular significance in an age where antimicrobial resistance outstrips the rate at which new antimicrobial treatments are discovered and where there is real concern that species resistant to all known antibiotics could become rife. The problem of resistance can be exacerbated, for example when microorganisms grow as a biofilm Such organisms are afforded intrinsic protection in the form of an extra-polysaccharide layer which restricts the diffusion of antimicrobials meaning that therapeutic doses do not reach all bacteria within the biofilm, causing infection to recur [6,7]. The biofilm mode of growth, by hindering diffusion, could provide an ideal environment whereby microorganisms are exposed to sub-lethal doses of antimicrobial providing a selective pressure for the emergence of resistance

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