Objective: To detect the effects of four efflux pump inhibitors on the minimum inhibitory concentration of clarithromycin (CLA) against Mycobacterium abscessus (M. abscessus) in vitro, and to explore the role of efflux pump in CLA resistance of M. abscessus. Methods: Four frequently-used efflux pump inhibitors (Carbonyl Cyanide 3-chlorophenylhydrazone, CCCP, N, N'-dicyclohexylcarbodiimide, DCC, Verapamil, VP, Reserpine, RSP) were evaluated in this study. The minimum inhibitory concentration (MIC) values of clarithromycin against M. abscessus reference strain and 60 clinical strains with or without efflux pump inhibitors were detected by Alamar Blue method. Sequence analysis of erm(41) and rrl genes known to be associated with CLA resistance in M. abscessus was performed to analyze the correlation between the effect of efflux pump inhibitors on MIC and mutation of resistance-related genes. Results: CCCP, DCC, VP and RSP could reduce the MIC of M. abscessus to CLA, and the effect of RSP was weaker than the other three efflux pump inhibitors. Among the sixty M. abscessus clinical strains, ten strains were resistant to clarithromycin, seven of which had rrl gene mutation. The CLA resistance rate of smooth phenotype isolates was higher than that of rough phenotype isolates. At 3 day of clarithromycin incubation, the MICs of resistant strains were all reduced by efflux pump inhibitors. Compared with the strains with rrl gene mutation, efflux pump inhibitors had a greater effect on the strains without rrl gene mutation. At 14 day of clarithromycin incubation, 83% of M. abscessus subsp. abscessus, were induced to be resistant, and all of them were T28 sequence type of erm(41). With the occurrence of induced drug resistance, the effect of efflux pump inhibitor on CLA MIC decreased. Efflux pump inhibitors had no statistically significant diffence in the effect of effcux pump inhibitors on CLA MIC levels in different phenotypes of isolates. Conclusions: Efflux pump is involved in the resistance process of M. abscessus to CLA. Efflux pump inhibitors reduce the drug resistance to clarithromycin against M. abscessus in different degrees. The use of efflux pump inhibitors may provide a new way to alleviate the drug resistance of M. abscessus.
目的： 检测四种常见外排泵抑制剂对克拉霉素（CLA）抗脓肿分枝杆菌体外最低抑菌浓度的影响，探讨药物外排泵在脓肿分枝杆菌对CLA耐药中所起的作用。 方法： 本研究自2008年1月至2012年12月，纳入脓肿分枝杆菌标准菌株和60株脓肿分枝杆菌临床分离株。选择四种常见外排泵抑制剂：氰化羰基-3-氯苯腙（CCCP）、二环己基碳二亚胺（DCC）、维拉帕米（VP）和利血平（RSP），采用Alamar Blue法检测CLA对上述菌株的体外最低抑菌浓度（minimum inhibitory concentration，MIC），比较有无外排泵抑制剂作用时，CLA抗菌的MIC改变情况；对脓肿分枝杆菌的CLA耐药相关基因erm（41）和rrl基因进行序列分析，分析外排泵抑制剂对MIC的影响与耐药相关基因突变与否及突变类型之间的相关性。 结果： CCCP、DCC、VP和RSP均能降低CLA抗脓肿分枝杆菌的MIC，但RSP作用效果弱于其他三种外排泵抑制剂。60株脓肿分枝杆菌临床分离株中，包含10株CLA耐药菌株，其中7株发生rrl基因突变。光滑表型分离株的CLA耐药率（21.2%和66.7%）高于粗糙表型分离株的CLA耐药率（11.1%和51.9%）。当CLA药敏孵育时间为3 d时，外排泵抑制剂对CLA耐药菌株的MIC影响较大，均可使约50%菌株的MIC降低2倍以上，且相较于发生rrl基因突变的菌株，外排泵抑制剂对未发生rrl基因突变菌株的MIC影响更大；当CLA药敏孵育时间为14 d时，有24株携带erm（41）基因T28序列型的脓肿亚种分枝杆菌发生诱导耐药，随着诱导耐药的发生，外排泵抑制剂对CLAMIC影响减小。外排泵抑制剂对不同表型分离株的CLAMIC水平影响差异无统计学意义。 结论： 外排泵参与脓肿分枝杆菌对CLA耐药过程，外排泵抑制剂可不同程度降低脓肿分枝杆菌对CLA的耐药水平。外排泵抑制剂的使用为脓肿分枝杆菌病的临床治疗提供了新的思路和方法。.