药物外排泵基因表达与结核分枝杆菌耐药关系的探讨

Association of drug effiux pump gene expression with Mycobacterium tuberculosis drug resistance

目的 探讨MTB药物外排泵基因的表达与耐药表型、耐药相关基因突变型的关系.方法 从2007年天津市结核病控制中心参比室库存菌株中选择对异烟肼、利福平、链霉素和乙胺丁醇联合或单一耐药的全部耐药MTB菌株45株及对以上4种药物全敏感的MTB菌株26株（全敏组）.采用直接测序法检测异烟肼耐药相关基因katG、inhA、oxyR-ahpC、ndh,利福平耐药相关基因rpoB,链霉素耐药相关基因rpsL、rrs和乙胺丁醇耐药相关基因embB、embC、embA的突变情况.提取菌株RNA后逆转录并采用real-time PCR方法检测药物外排泵基因Rv1410c、Rv2136c、Rv0783c和Rv1819c的表达量,并用方差分析和t检验的方法分析药物外排泵基因表达量在不同菌株耐药表型、耐药相关基因突变型菌株中的差异.结果 Rv1410c表达量在耐链霉素组[（8.48±6.33）×10-5],耐异烟肼组[（8.43±6.38）×10-5],耐利福平组[（9.59±7.27）×10-5],耐多药组[（10.37±7.86）×10-5],耐异烟肼＋链霉素组[（9.39±6.81）×10-5],均高于全敏组表达量[（5.67±3.29）×10-5],差异有统计学意义（t′值分别为2.18、2.20、2.29、2.34、2.43,P均〈0.05）.Rv2136c表达量在耐异烟肼组[（3.51±2.43）×10-5],耐多药组[（4.21±2.94）×10-5],耐异烟肼＋链霉素组[（3.81±2.46）×10-5]均高于全敏组表达量[（2.50±1.44）×10-5],差异有统计学意义（t′值分别为2.03、2.22、2.28,P均〈0.05）.rpoB 531突变型菌株Rv0783c的表达量[（5.41±3.03）×10-6]高于野生型耐利福平菌株[（2.29±1.62）×10-6],差异有统计学意义（t′=2.81,P〈0.05）.结论 Rv1410c和Rv2136c表达量高低与MTB对多种药物耐药有关,Rv0783c的高表达与利福平耐药株rpoB 531位密码子突变可能相关.

Objectives To explore the associations between drug efflux pump gene expression and phenotypic drug resistance as well as gene mutation patterns related to drug resistance of Mycobacterium tuberculosis.Methods Forty-five Mycobacterium tuberculosis isolates resistant to one or more of drugs including isoniazid, rifampicin, streptomycin and ethambutol, and 26 isolates all sensitive to the above four drugs from Tianjin Tuberculosis Control Institute in 2007 were involved in this study. Direct sequencing was applied to detect the mutations in the corresponding resistance genes（isoniazid：katG, inhA, oxyR-ahpC, ndh, rifampicin：rpoB, streptomycin：rpsL, rrs, and ethambutol：embB, embC and embA）. After RNA extration and reverse transcription, real-time PCR was conducted to assess the expressions of putative drug efflux pump genes Rv1410c, Rv2136c, Rv0783c and Rv2136c, and Students＇ t test and ANOVA analysis were used to analyze the expression differences in Mycobacterium tuberculosis with different phenotypic drug resistance and drug resistance related gene mutation patterns.Results Compared to pan-sensitive isolates[（5.67±3.29）×10-5], Rv1410c showed higher expression in streptomycin[（8.48±6.33）×10-5, t＇=2.18, P〈0.05], isoniazid[（8.43±6.38）×10-5, t＇=2.20, P〈0.05], rifampicin[（9.59±7.27）×10-5, t＇=2.29, P〈0.05], multi-drug[（10.37±7.86）×10-5, t＇=2.34, P〈0.05] resistant isolates, and in isoniazid ＋ streptomycin resistant isolates[（9.39±6.81）×10-5, t＇=2.43, P〈0.05];Rv2136c showed higher expression in isoniazid resistant[（3.51±2.43）×10-5, t＇=2.03, P〈0.05], multidrug-resistant isolates[（4.21±2.94）×10-5, t＇=2.22, P〈0.05] and resistant to isoniazid＋streptomycin[（3.81±2.46）×10-5, t＇=2.28, P〈0.05] isolates . The expression of Rv0783c in rifampicin resistant isolates with rpoB 531 mutations [（5.41±3.03）×10-6] was higher than those with wild type of rpoB 531[（2.29±1.62）×10-6, t=2.81, P〈0.05].Conclusions The expression of Rv1410c and Rv2136c are associated with mutiple-drug resistance of Mycobacterium tuberculosis.The expression of Rv0783c in rifampicin resistant isolates is associated with mutation in rpoB 531.