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ABSTRACTExposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coliand Klebsiella pneumoniaemutants. E. coliand K. pneumoniaemutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabIgene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRIr) and triclosan-hypersusceptible (TRIhs) mutants of E. coliand K. pneumoniaewere obtained after selection with biocides and/or antibiotics. E. coliTRIrmutants, including those with mutations in the fabIgene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRIrmutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRIrmutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRIrmutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRIrmutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive traits.

ABSTRACTThe 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 qacgenes, 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 qacGgenes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacAand qacBincreased the chlorhexidine mode MIC. Isolates with a wild-type norApromoter or mutations in the norApromoter had similar biocide MIC distributions; notably, not all clinical isolates with norAmutations 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 of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitromutants displayed fitness defects in a killing assay in Galleria mellonellalarvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureusmutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitrotests appear not to be suitable for predicting levels of resistance that are clinically relevant.

ABSTRACTThe pneumococcal chromosome encodes about 140 transporters, many of which are predicted to be involved in efflux. In order to critically evaluate pneumococcal efflux, a series of transporter mutants were constructed, and their phenotypes were assayed by disk diffusion, microdilution drug susceptibility testing (MIC testing), growth of cultures at sub-MIC concentrations, and phenotype microarray analysis. Mutants with mutations in seven ATP binding cassette (ABC) transporters, three multiantimicrobial extrusion (MATE) family efflux pumps, and one major facilitator superfamily (MFS) transporter were obtained in Streptococcus pneumoniaestrain DP1004. The susceptibility of these 11 mutants to over 250 different substances was compared to that of the parent strain. Of the tested transporters, only the ABC transporter PatAB (SP2073-5) presented a clear multidrug resistance (MDR) profile, as the mutant showed significantly increased susceptibility to ethidium bromide, acriflavine, and berberine. Among the other transporters analyzed, the mutants devoid of the MATE efflux pump SP2065 exhibited reduced susceptibility to novobiocin, and those with mutations of the MATE family DinF transport system (SP1939) exhibited increased susceptibility to moxifloxacin, ciprofloxacin, and levofloxacin. This change in quinolone MIC was found to be independent from the competence-mediated effect of quinolones on the cinA-recA-dinFoperon. Furthermore, the dinFmutant, in contrast to the parental strain, allowed selection for quinolone-resistant mutants when exposed to moxifloxacin. These data confirm the clear MDR profile of the PatAB ABC transporter and suggest for the MATE DinF a phenotype associated with quinolone susceptibility, particularly for moxifloxacin.