Role of the CmeABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter under selection pressure

Abstract

The objective of this study was to determine the contribution of the CmeABC efflux pump to the emergence of fluoroquinolone (FQ)-resistant mutants in Campylobacter jejuni under various levels of selection pressure. The frequency of emergence of ciprofloxacin-resistant mutants was measured in wild-type C. jejuni NCTC 11168 and its isogenic cmeB mutant and cmeR mutant (overexpressing cmeABC) using plates containing various concentrations of ciprofloxacin. Representative ciprofloxacin-resistant mutants were selected for gyrA sequence analysis and MIC determination. Accumulation of ciprofloxacin in Campylobacter cells was measured using spectrofluorometry. Mutation of cmeB drastically reduced the frequency of emergence of FQ-resistant mutants at 10x and 32x the MIC of ciprofloxacin, while the cmeR mutant displayed an approximately 17-fold increase in the frequency of emergence of the mutants at 32x the MIC when compared with the wild-type strain. Various point mutations occurred in gyrA in the FQ-resistant mutants selected at 5x and 10x the MIC, while the Thr-86-->Ile mutation was predominant in the mutants selected at 32x the MIC. The Thr-86-->Ile change conferred a high-level resistance to FQs, but other mutations only conferred an intermediate-level FQ resistance. In contrast, all types of gyrA mutations in the CmeABC-overexpressed background conferred high-level resistance to ciprofloxacin. Overexpression of cmeABC significantly reduced the amount of ciprofloxacin accumulated within bacterial cells. CmeABC is not only important for maintaining high-level resistance to FQs but also contributes significantly to the emergence of FQ-resistant mutants. Inhibition of this efflux pump may prevent the emergence of clinically relevant FQ-resistant Campylobacter mutants.