浙东地区耐多药结核分枝杆菌耐药特性及分子机制研究

目的 对浙东地区耐多药结核分枝杆菌（MDR-TB）耐药性及分子机制进行研究，为治疗耐多药结核病提供理论依据。方法 收集2018 年1 月～2019 年12 月浙江东部9 家结核病定点医院临床分离的结核分枝杆菌。采用比例法检测异烟肼（INH）、利福平（RIF）、氧氟沙星（OFL）、链霉素（SM）、乙胺丁醇（EMB）、阿米卡星（AK）、对氨基水杨酸（PAS）、卷曲霉素（CM）和丙硫异烟胺（TH）对MDR-TB 的耐药性。通过基因芯片方法检测耐多药结核分枝杆菌rpoB、katG、inhA 突变位点，PCR 扩增OFL 耐药的MDR-TB 的gyr 耐药基因并测序。结果 耐多药结核分枝杆菌对OFL、SM、EMB、AK、PSA、CM、TH、INH 和RIF 耐药率分别为38.1%、54.8%、28.6%、11.9%、8.3%、9.5%、13.1%、100.0%和100.0%。耐多药结核分枝杆菌的突变位点为rpoB 511（9 例）、rpoB 513（3 例）、rpoB 516（3 例）、rpoB 526（25 例）、rpoB 531（38 例）、rpoB 533（2 例），KatG 315（71 例），inhA-15(4 例)，KatG 315 与inhA-15 同时突变（9 例）。检测到26 株gyrA 基因和2 株gyrB 基因发生突变，突变类型为Thr478Asn、Asn477Thr、Ala90Val、Ser91Pro、Asp94Ala、Asp94His、Asp94Asn 和Asp94Gly。结论 耐多药结核分枝杆菌利福平耐药以rpoB 基因531位点突变为主；异烟肼耐药以KatG 基因315 位点突变为主；MDR-TB 对喹诺酮类药物的耐药机制以gyrA 基因Ala90Val、Ser91Pro、Asp94Gly 突变类型为主。

Study on drug resistance characteristics and molecular mechanism of MDR-TB in eastern Zhejiang Province

Objective To study the drug resistance and molecular mechanism of multi-drug resistant mycobacterium tuberculosis (MDR-TB) in eastern Zhejiang province,and to provide theoretical basis for the treatment of MDR-TB.Methods Mycobacteria tuberculosis that were clinically isolated in 9 designated tuberculosis hospitals in eastern Zhejiang province from January 2018 to December 2019 were collected.The drug resistance of MDR-TB to isoniazid(INH),rifampicin(RIF),ofloxacin(OFL),streptomycin(SM),ethambutanol(EMB),amikacin(AK),para-aminosalicylic acid(PAS),capreomycin(CM) and propyl thionisocyanamine(TH) was detected by the ratio method.The rpoB,katG and inhA mutation sites of MDR-TB were detected by the gene chip method.The gyr resistance gene of OFL-resistant MDR-TB was amplified by PCR and sequenced.Results The drug resistance rates of MDR-TB to OFL,SM,EMB,AK,PSA,CM,TH,INH and RIF were 38.1%,54.8%,28.6%,11.9%,8.3%,9.5%,13.1%,100.0% and 100.0%,respectively.The mutational sites of MDR-MTB were rpoB 511(9 cases),rpoB 513(3 cases),rpoB 516(3 cases),rpoB 526(25 cases),rpoB 531(38 cases),rpoB 533(2 cases),KatG 315(71 cases),inhA-15(4 cases),KatG 315 and inhA-15 simultaneously(9 cases).Mutations were detected in 26 gyrA genes and 2 gyrB genes,and the mutation types were Thr478Asn,Asn477Thr,Ala90Val,Ser91Pro,Asp94Ala,Asp94His,Asp94Asn and Asp94Gly.Conclusion The major mutation site of genes in MDR-TB resistant to rifampicin is rpoB 531.The major mutation site of genes in MDR-TB resistant to isoniazid is KatG 315.The resistance mechanism of MDRTB to quinolones is mainly gyrA gene Ala90Val,Ser91Pro and Asp94Gly mutations.