title: Contribution of phenotypic heterogeneity to adaptive antibiotic resistance.
abstract: Antibiotic-resistant isolates of Salmonella enterica were selected on plates containing lethal concentrations of rifampicin, kanamycin, and nalidixic acid. The stability of the resistance phenotype was scored after nonselective growth. Rifampicin-resistant (Rif(r)) isolates were stable, suggesting that they had arisen by mutation. Mutations in the rpoB gene were detected indeed in Rif(r) mutants. In contrast, a fraction of kanamycin-resistant (Km(r)) and nalidixic acid-resistant (Nal(r)) isolates showed reduced resistance after nonselective growth, suggesting that mechanisms other than mutation had contributed to bacterial survival upon lethal selection. Single-cell analysis revealed heterogeneity in expression of the porin gene ompC, and subpopulation separation provided evidence that reduced ompC expression confers adaptive resistance to kanamycin. In the case of Nal(r) isolates, mutations in the gyrA gene were present in most nalidixic acid-resistant isolates. However, the efflux pump inhibitor Phe-Arg-β-naphtylamide (PAβN) reduced the level of resistance in Nal(r) mutants, indicating that active efflux contributes to the overall level of nalidixic acid resistance. Heterogeneous efflux pump activity was detected in single cells and colonies, and a correlation between high efflux and increased resistance to nalidixic acid was found. These observations suggest that fluctuations in the expression and the activity of critical functions of the bacterial cell, alone or combined with mutations, can contribute to adaptive resistance to antibiotics.
authors.affiliation: Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Seville 41080, Spain.
authors.forename: María Antonia
authors.initials: MA
authors.lastname: Sánchez-Romero
authors.forename: Josep
authors.initials: J
authors.lastname: Casadesús
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D002469:Cell Separation; D027081:DNA Gyrase / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007612:Kanamycin / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D009154:Mutation; D009268:Nalidixic Acid / Q000494:pharmacology*; D010641:Phenotype; D018272:Porins / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology*; D019779:Salmonella enterica / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C113724:OmpC protein; D018272:Porins; C540081:rpoB protein, E coli; D049452:Green Fluorescent Proteins; D009268:Nalidixic Acid; D007612:Kanamycin; D012321:DNA-Directed RNA Polymerases; D027081:DNA Gyrase; D012293:Rifampin