INTRODUCTION: The number of methicillin resistant S. aureus (MRSA) cases is increasing globally, with nearly 19000 deaths in the US alone in 2006. Coupled with the spread of vancomycin resistant strains, an alternative therapeutic approach is urgently needed. MRSA is frequently associated with severe biofilm infections, hence targeting the biofilm structure to potentially restore antibacterial susceptibility in MRSA is a promising area of study. We investigated the potential of an innovative combination therapy (CT) in treating MRSA and MSSA. This multi-targeted approach first degrades the extracellular polymeric matrix using an antioxidant and enzyme followed by appropriate antibiotic treatment.

METHODS and MATERIALS: MICs of potential CT formulae were first identified for MRSA and MSSA strains by testing different compositions and dosages on planktonic cultures. The effect of CT on MRSA and MSSA isolates was then determined by measuring biofilm viability using a resazurin assay. This was complemented with confocal microscopy to provide a detailed view of the proportion of live and dead bacteria in treated biofilms, as well as biofilm volume and thickness. Colony counts were conducted to enumerate remaining live bacteria post-treatment. Experiments were then repeated with antioxidants buffered to physiological pH. Fifteen different CT combinations were tested.

RESULTS: Data showed successful disruption of MRSA and MSSA biofilms in vitro. Formulations consisting of glutathione and an antibiotic of choice showed >99% inhibition of bacterial growth in planktonic culture and >95% disruption of preformed biofilm. Furthermore, a 1200-6300 fold decrease in cfu/ml was recorded after treatment. When the antioxidants were buffered to pH 7.4, a 10-50% decrease in biofilm viability occurred, depending on the antioxidant used.

CONCLUSIONS: The CT combinations tested show good promise in disrupting MRSA and MSSA biofilms. Further qualitative and quantitative investigations into the effects of CT on MRSA and MSSA biofilms are underway, including real-time observation of CT activity on biofilms as well as cytotoxicity studies on mammalian cells. Additionally different antioxidants have different targets, hence research is continuing to determine the most effective formulae and dosage.