Evaluation of Reduced Susceptibility to Quaternary Ammonium Compounds and Bisbiguanides in Clinical Isolates and Laboratory-Generated Mutants of Staphylococcus aureus

Abstract

ABSTRACTThe MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates ofStaphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened forqacgenes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence ofqacA,qacB,qacC, andqacGgenes increased the mode MIC, but not MBC, to benzalkonium chloride, while onlyqacAandqacBincreased the chlorhexidine mode MIC. Isolates with a wild-typenorApromoter or mutations in thenorApromoter had similar biocide MIC distributions; notably, not all clinical isolates withnorAmutations were resistant to fluoroquinolones.In vitroefflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region ofnorA, but these were distinct from this region of the clinical isolates. Still, none of thein vitromutants displayed fitness defects in a killing assay inGalleria mellonellalarvae. In conclusion, our data provide an in-depth comparative overview on efflux inS. aureusmutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, currentin vitrotests appear not to be suitable for predicting levels of resistance that are clinically relevant.