Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus

MIC and MBC for the biocides benzalkonium chloride and chlorhexidine was determined against 1602 clinical isolates of Staphylococcus aureus. Both compounds showed a unimodal MIC and MBC distribution (mode MIC of 2 and MBC of 4 or 8 mg/L, respectively), with no apparent subpopulation with reduced susceptibility. To further investigate, all the isolates were screened for qac genes and 39 of these also had the promoter region of the NorA MDR efflux pump sequenced. The presence of qacA, qacB, qacC and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride and only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions and, notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these showed comparable phenotypes to those selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct to that of the clinical isolates. Still none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting resistance of clinical relevance.