title: Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus.
abstract: The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus 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 for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while 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; 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 mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region 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 levels of resistance that are clinically relevant.
authors.affiliation: LAMMB, Dipartimento Biotecnologie, Università di Siena, Siena, Italy.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Furi
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: Ciusa
authors.forename: Daniel
authors.initials: D
authors.lastname: Knight
authors.forename: Valeria
authors.initials: V
authors.lastname: Di Lorenzo
authors.forename: Nadia
authors.initials: N
authors.lastname: Tocci
authors.forename: Daniela
authors.initials: D
authors.lastname: Cirasola
authors.forename: Lluis
authors.initials: L
authors.lastname: Aragones
authors.forename: Joana Rosado
authors.initials: JR
authors.lastname: Coelho
authors.forename: Ana Teresa
authors.initials: AT
authors.lastname: Freitas
authors.forename: Emmanuela
authors.initials: E
authors.lastname: Marchi
authors.forename: Laura
authors.initials: L
authors.lastname: Moce
authors.forename: Pilar
authors.initials: P
authors.lastname: Visa
authors.forename: John Blackman
authors.initials: JB
authors.lastname: Northwood
authors.forename: Carlo
authors.initials: C
authors.lastname: Viti
authors.forename: Elisa
authors.initials: E
authors.lastname: Borghi
authors.forename: Graziella
authors.initials: G
authors.lastname: Orefici
authors.forename: Ian
authors.initials: I
authors.lastname: Morrissey
authors.forename: Marco Rinaldo
authors.initials: MR
authors.lastname: Oggioni
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001548:Benzalkonium Compounds / Q000494:pharmacology; D002710:Chlorhexidine / Q000494:pharmacology*; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D024881:Drug Resistance, Bacterial; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009036:Moths / Q000382:microbiology; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D011401:Promoter Regions, Genetic; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D012692:Serial Passage; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001548:Benzalkonium Compounds; D002710:Chlorhexidine; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000644:Quaternary Ammonium Compounds; C075040:NorA protein, Staphylococcus; C068498:qacA protein, Staphylococcus aureus