结核分枝杆菌利福平依赖性的初步研究

目的 从耐利福平（RFP）结核分枝杆菌临床分离株中筛选RFP依赖的菌株，并在小鼠模型中验证RFP依赖现象，同时分析RFP依赖菌株的特征。 方法 采用7H11培养基绝对浓度法对46例临床分离菌株进行RFP依赖菌株筛选。以筛选得到的体外具有典型的RFP依赖现象的05116菌株建立气溶胶感染的小鼠模型，小鼠按照给药的种类和给药单位剂量分成对照组0.5%羧甲基纤维素钠（carboxymethyl cellulose sodium，CMC）、10 mg/kg Rfb（Rfb₁₀）组、10 mg/kg RFP（RFP₁₀）组、20 mg/kgRFP（RFP₂₀）组，每组各10只，观察给药4、8周的体质量、肺荷菌量及组织病理学改变在各组之间差异，以验证小鼠体内是否存在RFP依赖现象。比较RFP耐药株和依赖株的rpoB基因突变位点，以鉴定RFP依赖株的特征。数据以(x±s)表示，采用SPSS 11.0软件，组间比较采用单因素方差分析，以 P<0.01为差异有统计学意义。 结果 从46例RFP耐药的结核分枝杆菌临床分离株中筛选到18例RFP耐药伴依赖的菌株和28例RFP耐药非依赖的菌株。感染小鼠模型给药8周，各组小鼠的活菌计数从高到低依次为：RFP₂₀组（lgCFU为6.60±0.82）、RFP₁₀组（lgCFU为6.44±0.36）、Rfb₁₀组（lgCFU为6.32±0.55）、对照组(lgCFU为6.20±0.28)，接近于最初感染的菌量（lgCFU为5.93±0.14），但与对照组相比差异无统计学意义（F值分别为0.68、2.21、2.45，各组P值均大于0.05）。RFP耐药株8例和依赖株11例，分别有3/8和4/11的菌株在rpoB基因526位发生突变，2/8和6/11的菌株在rpoB基因531位发生突变。 结论 RFP临床耐药株中RFP依赖菌的检出率高，其RFP依赖现象在小鼠体内得到初步验证。RFP耐药株和依赖株的rpoB基因突变位点基本相同，因此RFP依赖的原因是表型变异还是其他因素尚有待进一步的实验证实。

Study on the rifampicin-dependent characteristics of Mycobacterium tuberculosis clinical isolate

Objective To validate the rifampicin(RFP)-dependent characteristics of M.tuberculosis clinical isolate in mice,and identify the characteristics of RFP-dependent strains.Methods RFP-dependent strains were selected from RFP-resistant clinical isolates on 7H11 medium(the final concentrations of rifampicin were 1 μg/ml and 10 μg/ml).The dependence was further studied in mouse TB model,in which the mice were treated with 0.5% carboxymethyl cellulose sodium(control group),10 mg/kg rifabutin(Rfb10 group),10 mg/kg RFP(RFP10 group) and 20 mg/kg RFP(RFP20 group),respectively.The differences of rpoB gene mutations between RFP-dependent and non-dependent strains were analyzed.Results 18 of 46 RFP-resistant isolates were RFP-dependent.After treatment with RFP or Rfb for 8 weeks in mouse model challenged by aerosol with RFP-dependent clinical isolate,the lung CFU counts showed a concentration-dependent increase(RFP20 group lgCFU were 6.60±0.82、RFP10 group 6.44±0.36、Rfb10 group 6.32±0.55、control group 6.20±0.28),but there were not significant difference in comparasion with control group(F value were 0.68、2.21、2.45,P>0.05).Five of 8 RFP-dependent strains and 10 of 11 non-dependent strains had mutations of rpoB codon 526 and 531,respectively.Conclusion RFP-dependent strains did exist and showed a high prevalence,and the dependence was further validated in mouse TB model.The RFP-dependent and non-dependent strains had common rpoB mutations,whether the dependence is a result of phenotype variation needs more evidence.