Evidence for Active Efflux as the Primary Mechanism of Resistance to Ciprofloxacin in Salmonella enterica Serovar Typhimurium

作者：Étienne Giraud, Axel Cloeckaert, Dominique Kerboeuf, Elisabeth Chaslus-Dancla · 发表于：Antimicrobial Agents and Chemotherapy · 年份：2000 · DOI：10.1128/aac.44.5.1223-1228.2000 · 被引用次数：229 · 研究领域：Antibiotic Resistance in Bacteria、Salmonella and Campylobacter epidemiology、Bacterial Genetics and Biotechnology

The occurrence of active efflux and cell wall modifications were studied in Salmonella enterica serovar Typhimurium mutants that were selected with enrofloxacin and whose phenotypes of resistance to fluoroquinolones could not be explained only by mutations in the genes coding for gyrase or topoisomerase IV. Mutant BN18/21 exhibited a decreased susceptibility to ciprofloxacin (MIC = 0.125 microg/ml) but did not have a mutation in the gyrA gene. Mutants BN18/41 and BN18/71 had the same substitution, Gly81Cys in GyrA, but exhibited different levels of resistance to ciprofloxacin (MICs = 2 and 8 microg/ml, respectively). None of the mutants had mutations in the parC gene. Evidence for active efflux was provided by a classical fluorimetric method, which revealed a three- to fourfold decrease in ciprofloxacin accumulation in the three mutants compared to that in the parent strain, which was annulled by addition of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone. In mutant BN18/71, a second fluorimetric method also showed a 50% reduction in the level of accumulation of ethidium bromide, a known efflux pump substrate. Immunoblotting and enzyme-linked immunosorbent assay experiments with an anti-AcrA antibody revealed that the resistance phenotype was strongly correlated with the expression level of the AcrAB efflux pump and suggested that decreased susceptibility to ciprofloxacin due to active efflux probably related to overproduction of this pump could occur befor...