单耐氧氟沙星的结核分枝杆菌耐药机制研究  

Study on the mechanism of drug resistance in mono ofloxacin-resistant Mycobacterium tuberculosis

作　　者：王前[1] 宋媛媛[2] 池俊英[3] 王玉峰[2] 赵雁林[2] 

机构地区：[1]中国疾病预防控制中心结核病预防控制中心办公室,北京102206 [2]中国疾病预防控制中心参比实验室,北京102206 [3]中华人民共和国国家卫生和计划生育委员会与比尔及梅琳达·盖茨基金会结核病防治项目办公室

出　　处：《中国防痨杂志》2014年第5期350-355,共6页Chinese Journal of Antituberculosis

基　　金：基金项目：中国卫生部-比尔及梅琳达·盖茨基金会结核病防治项目（2009-04-01）

摘　　要：目的探讨单耐氧氟沙星的结核分枝杆菌中耐药相关基因突变及外排泵基因风坦两种不同耐药机制的作用。方法从国家结核病参比实验室2007年全国耐药基线调查菌株库中挑选单耐氧氟沙星的菌株17株。采用直接测序法检测利福平耐药相关基因删似和gyrB突变情况。提取gyrA和gyrB无突变菌株的RNA后反转录并采用Real—timePCR方法检测20个药物外排泵基因的表达量，对比菌株最低抑菌浓度（MIC）试验结果，筛选可能与氧氟沙星药物外排泵的相关基因，并选用大肠埃希菌为模式生物构建表达载体，检测过表达目的外排泵蛋白的大肠埃希菌对氧氟沙星的耐药程度加以验证。结果在17株单耐氧氟沙星的菌株中，有4株（4／17）检测到gyrA突变，其中包括90位点突变1株及94位点突变3株，上述突变均表现为高浓度耐药，其MIC均不低于4μg/ml。在gyrA和gyrB未突变菌株中，通过real-timePCR检测发现在高浓度耐药的2株菌株中，Rv0933和Rv2938的转录水平显著高于其他低浓度耐药菌株，较对照株H37Rv转录水平高16倍和5倍。在对照组和转入pEASY-E1-Rv2938的大肠埃希菌中MIC均小于0．125μg／ml，而转入pEASY-E1-Rv0933的大肠埃希菌的MIC为2μg／ml。结论本研究结果显示，gyrA耐药决定区基因突变与氧氟沙星高浓度耐药相关，PstB可能是氧氟沙星特异性的药物外排泵基因，其高水平表达与氧氟沙星高浓度耐药相关。Objective To investigate the function of the genes related with drug resistance and efflux pump in mono ofloxacin （Ofx）-resistant M. tuberculosis （Mtb） isolates. Methods Seventeen mono Ofx-resistant Mtb clini- cal isolates were obtained from National Tuberculosis Reference Laboratory. Direct sequencing was performed to de- tect the mutations in the gyrA/B genes. The total RNA was extracted from the Ofx-resistant strains without gyrA/B mutations. After RNA reverse transcription, the expression levels of 20 drug efflux genes were detected by Real- time PCR. In comparison with the corresponding MIC results, the Ofx-related efflux pumps were screened from 20 drug efflux genes, and the function of candidates was identified among transgenic E. coll. Results Of 17 mono Ofx-resistant strains, 4 （4/17） strains were found the mutations of gyrA, in which 1 isolates had mutation at codon 90 and 3 isolate had mutation at codon 94. In addition, the isolates harboring gyrA mutation displayed high-level of drug resistance, their MICs were more than 4μg/ml. Among the strains without gyrA/B mutations, the expression levels of Rv0933 and Rv2938 were higher in 2 high-level resistant strains, which was 16 and 5 folds higher than that of H37Rv strain, respectively. Furthermore, E. coli BL21 transferred with pEASY-E1 Rv0933 showed higher MIC level, 2μg/ml, compared with the control E. coli. Conclusion The mutations in gyrA are associated with high- level of Ofx resistance. Rv0933 may be the Ofx-specific efflux pumps in Mycobacterium tuberculosis. The high level of Rv0933 expression may correlated with high-level of Ofx resistance.

关 键 词：分枝杆菌 结核 荧光喹诺酮类 抗药性 细菌 DNA促旋酶 

分 类 号：R378.911[医药卫生—病原生物学]