Role of resident and acquired multi-drug efflux pumps in reduced susceptibility to cationic biocides in Staphylococcus aureus

Objectives: In view of the requirements posed by licensing process of disinfectants according to the EC biocide directive, we evaluated the possibility to devise an in vitro test for evaluation of resistance to cationic antibacterial compounds including quaternary ammonium compounds and bisbiguanides. Methods: In three strains of S. aureus mutants were selected in vitro with benzalkonium chloride (BZC; quaternary ammonium compound), chlorhexidine (CHX; bisbiguanide), acriflavine (AF) and ethidium bromide (EB) and their phenotypes and norA promoter sequences were determined. Data on laboratory mutants was compared to molecular data from of 246 clinical S. aureus strains showing decreased susceptibility to BZC or CHX. Results: A survey for susceptibility to EB, AF, BZC and CHX on 75 clinical S. aureus isolates showed a bimodal distribution of susceptibility profiles for EB and AF and a normal distribution of susceptibility profiles to CHX and BZC. Mutation frequency in vitro to these compounds was found to be around 1E-10 for EB and AF, while no mutants could be selected in a single step protocol for BZC and CHX. Multiple passages on selective plates allowed to select also mutants with BZC and CHX. Irrespective the selective agent all mutants showed important increases in MIC and MBC to norfloxacin, ciprofloxacin, EB and AF. For BZC and CHX the MIC and MBC did either not change or increased by a single dilution. All mutans showed mutations in the promoter region of the NorA MDR efflux pump. Upon the 246 clinical isolates with reduced susceptibility to BZC or CHX, 77 were positive for qacA, 13 for qacC and 1 for qacG and all of these had increased MIC for EB. For 35 clinical strains with reduced EB or BZC susceptibility the norA promoter was sequenced. Out of these 9 had a short duplication, 5 a mutated and 21 a wt norA promoter region. In only one case a clinical strain matched to a mutation also generated in vitro. Conclusion: Our data show (1) that EB and AF are suitable agents for monitoring efflux phenotypes and related genotypes, while BZC and CHX not, (2) that standard mutation selection assays cannot be performed for BZC and CHX (3) and that the mutations selected in vitro by BZC and CHX do not match those detected in clinical isolates. Summarised these data indicate that for S. aureus an in vitro test for prediction of resistance development to BZC and CHX is not feasible and, in any case, would yield results of no clinical relevance.