摘要
目的探讨单耐利福平结核分枝杆菌中耐药相关基因突变以及药物外排泵两种不同耐药机制的功能。方法从国家结核病参比实验室2007—2008年全国耐药基线调查菌株库中挑选单耐利福平的菌株23株。采用直接测序法检测利福平耐药相关基因rpoB突变情况。提取rpoB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(minimum inhibitory concentration,MIC)实验结果,筛选可能与利福平药物外排相关的基因,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的大肠埃希菌对利福平的耐药程度以验证。结果在23株单耐利福平的菌株中,有16株(69.6%)检测到rpoB突变,其中包括531位点突变9株,526位点突变6株及533位点突变1株。Ser531Leu、His526Asp表现为高浓度耐药,其MIC分别为192μg/ml和256μg/ml;而突变类型His526Gly、His526Leu、His526Arg和Leu533Pro表现为低浓度耐药,其MIC分别为0.75μg/ml、4μg/ml、2μg/ml和0.5μg/ml。在rpoB未突变菌株中,通过real-time PCR发现Rv2936、Rv0783和Rv0933的转录水平与MIC呈正相关。在对照组和转入pEASY-E1-Rv0933的大肠埃希菌中MIC均为8μg/ml,而转入pEASY-E1-Rv2936和pEASY-E1-Rv0783的大肠埃希菌的MIC均有不同程度的提高,分别为32μg/ml和16μg/ml。结论不同耐药突变类型导致的耐药程度不同,Rv2936和Rv0783可能是与利福平耐药相关的的药物外排泵基因。
Objective To investigate the roles of drug-resistant related gene mutation and efflux pump genes in mono-rifampicin resistance of Mycobacterium tuberculosis isolates. Methods Twenty-three mono-rifampicin resistant clinical isolates identified in national survey of drug-resistant tuberculosis in 2007—2008 were obtained from National Tuberculosis Reference Laboratory.Direct sequencing was performed to detect the mutations in the rpoB gene.The total RNA of the rifampicin resistant strains without rpoB mutation was extracted and reverse-transcribed.The transcript level of 20 drug efflux genes was detected by real-time PCR and the RFP-related efflux pumps were screened by comparing with the corresponding results of minimum inhibitory concentration（MIC）.The function of candidates in RFP-resistance was confirmed by transgenic E.coli. Results Of 23 mono-rifampicin resistant strains,the mutations of rpoB were detected in 16（69.6%） strains,including 9 isolates with mutation located in 531 codon,6 isolates in 526 codon and 1 isolate in 533 codon.The isolates with Ser531Leu and His526Asp mutation displayed high-level rifampicin resistance,the MIC of which was 192 μg/ml and 256 μg/ml,respectively,while the mutations including His526Gly,His526Leu,His526Arg and Leu533Pro were related to low-level rifampicin resistance,the MIC of which was 0.75 μg/ml,4 μg/ml,2 μg/ml and 0.5 μg/ml,respectively.In the strains without rpoB mutation,the expression levels of Rv2936,Rv0783 and Rv0933 by PCR positively correlated to MIC.Moreover,the BL21 transferred with pEASY-E1-Rv2936 and pEASY-E1-Rv0783 showed higher MIC level,32 μg/ml and 16 μg/ml,respectively,than 8 μg/ml in the control E.coli and the E.coli transferred with pEASY-E1-Rv0933. Conclusion The different mutations in rpoB are associated with MIC level.And Rv2936 and Rv0783 may be the efflux pumps related to rifampicin resistance in Mycobacterium tuberculosis.
出处
《中国防痨杂志》
CAS
2012年第5期275-279,共5页
Chinese Journal of Antituberculosis
基金
"十一五"国家重大科技专项(2008ZX10003-009)
关键词
分枝杆菌
结核
利福平
抗药性
细菌
Mycobacterium tuberculosis
Rifampicin
Drug resistance
bacterial
