Role of the CmeABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter under selection pressure.
Role of the CmeABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter under selection pressure.
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DOI：
10.1093/jac/dkl412
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发表时间：
2006-12
期刊：
影响因子：
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通讯作者：
Meiguan Yan;O. Sahin;Jun Lin;Qijing Zhang
中科院分区：
文献类型：
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作者：
Meiguan Yan;O. Sahin;Jun Lin;Qijing Zhang
本研究的目的是确定在不同水平的选择压力下，CmeABC外排泵对空肠弯曲菌中氟喹诺酮（FQ）耐药突变体的出现的贡献。方法在野生型C.空肠NCTC 11168及其同基因cmeB突变体和cmeR突变体（过表达cmeABC）。选择代表性的环丙沙星抗性突变体用于gyrA序列分析和MIC测定。使用荧光分光光度法测量环丙沙星在弯曲杆菌细胞中的蓄积。结果cmeB突变体在10倍和32倍MIC的环丙沙星下显著降低了耐药突变体的出现频率，而cmeR突变体在32倍MIC下的突变体出现频率比野生型菌株增加了约17倍。在5x和10 x MIC下选择的抗突变体中，gyrA中发生了各种点突变，而在32 x MIC下选择的突变体中，Thr-86->Ile突变占主导地位。Thr-86 → Ile的改变赋予了对FQs的高水平抗性，但其他突变仅赋予了中等水平的FQs抗性。相反，所有类型的gyrA突变在CmeABC过表达的背景下赋予高水平的耐药性环丙沙星。过表达cmeABC显着减少了细菌细胞内积累的环丙沙星的量。结论CmeABC不仅是维持高水平抗性的重要因素，而且也是产生耐药突变体的重要因素。抑制这种外排泵可以防止临床相关的耐药弯曲杆菌突变体的出现。
OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.