基于全基因组测序的72株耐多药结核分枝杆菌耐药基因突变特征分析  

Molecular characterization of mutations associated with resistance among 72 multidrugresistant strains of Mycobacterium tuberculosis by whole genome sequencing

作　　者：穆成[1] 孙蕊[1] 王志锐[1] 赵慧[1] 王春花[1] MU Cheng;SUN Rui;WANG Zhirui;ZHAO Hui;WANG Chunhua(Tianjin Center for Tuberculosis Control,Tianjin Key Laboratory of Pathogenic Microbiology of Infectious Disease,Tianjin 300011,China)

机构地区：[1]天津市结核病控制中心,天津市传染病病原微生物重点实验室,天津300011

出　　处：《中国热带医学》2023年第7期725-729,741,共6页China Tropical Medicine

基　　金：天津市卫生健康科技项目(No.ZC20199)。

摘　　要：目的 利用全基因组测序(whole genome sequencing, WGS)了解天津地区72株耐多药结核分枝杆菌(multidrug-resistant tuberculosis, MDR-TB)的耐药基因突变特征,评估WGS预测MDR-TB耐药性的效果。方法 收集2020年1-9月天津市结核病控制中心门诊部就诊患者的临床菌株,使用硝基苯甲酸(PNB)培养基进行菌种鉴定和固体比例法药敏试验。排除重复菌株后,筛选出72株MDR-TB进行WGS。使用在线数据库进行数据分析,对表型药敏结果与WGS预测的耐药谱进行比较。结果 72株MDR-TB基因型均为L2分支,准广泛耐药(pre-extensive drug-resistant tuberculosis, pre-XDR-TB)在现代株和古代株中的差异无统计学意义(χ^(2)=0.287,P=0.592)。共发现与12种药物耐药相关基因的突变类型81种,对不同药物耐药的菌株中常见突变类型分别为链霉素:rpsL Lys43Arg;异烟肼:katG Ser315Thr;利福平:rpoB Ser450Leu;乙胺丁醇:embB Met306Val;氧氟沙星、左氧氟沙星、莫西沙星:gyrA Asp94Gly;卡那霉素、卷曲霉素、阿米卡星:rrs 1401a>g;对氨基水杨酸:folC Ile43Thr。丙硫异烟胺耐药株9种突变类型,每种1株。WGS预测不同药物耐药的灵敏度和特异度分别为链霉素:98.15%,88.89%;异烟肼:90.28%,-;利福平:98.62%,-;乙胺丁醇:79.49%,75.76%;氧氟沙星:97.30%,85.71%;卡那霉素:85.71%,98.46%;对氨基水杨酸:27.27%,95.08%;丙硫异烟胺:81.82%,60.66%;卷曲霉素:60.00%,98.51%;左氧氟沙星:97.22%,83.33%;莫西沙星:97.30%,85.71%;阿米卡星:100.00%,100.00%。结论 WGS方法快速,与表型药敏一致性较高,具有良好的应用前景。Objective To understand the characteristics of mutations associated with resistance among 72 multidrugresistant tuberculosis(MDR-TB)strains using whole genome sequencing(WGS)and to evaluate the performance of WGS for predicting MDR-TB drug resistance.Methods The clinical strains isolated from patients who visited the outpatient department of Tianjin Center for Tuberculosis Control from January to September in 2020 were collected.Identification tests using p-nitrobenzoic acid(PNB)medium were performed.Drug susceptibility tests(proportion method)on L-J medium were performed.After excluding duplicate strains,72 MDR-TB strains were selected for WGS.Data were analyzed by using online databases and the phenotypic drug susceptibility test results were compared with resistance profiles predicted by WGS.Results All of 72 MDR-TB strains belonged to linage 2,and there was no significant difference in rate of pre-extensive drug-resistant tuberculosis(pre-XDR-TB)between modern type and ancestral type(χ^(2)=0.287,P=0.592).A total of 81 mutation types were found from resistance-related genes for 12 anti-tuberculosis drugs,and the common mutation types in different drug-resistant strains were:streptomycin(SM):rpsL Lys43Arg;isoniazid(INH):katG Ser315Thr;rifampicin(RIF):rpoB Ser450Leu;ethambutol(EMB):embB Met306Val;ofloxacin(OFX),levofloxacin(LFX),moxifloxacin(MFX):gyrA Asp94Gly;kanamycin(KAM),capreomycin(CAP),amikacin(AMK):rrs 1401a>g;para-aminosalicylic acid(PAS):folC Ile43Thr.Nine mutation types were found in 9 prothionamide(PTO)-resistant strains,one type for each strain.The sensitivity and specificity of WGS for predicting resistance to different drugs were SM:98.15%and 88.89%,INH:90.28%and-,RIF:98.62%and-,EMB:79.49%and75.76%,OFX:97.30%and 85.71%,KAM:85.71%and 98.46%,PAS:27.27%and 95.08%,PTO:81.82%and 60.66%,CAP:60.00%and 98.51%,LFX:97.22%and 83.33%,MFX:97.30%and 85.71%,AMK:100.00%and 100.00%,respectively.Conclusion WGS is a rapid and promising method which has high consistency with the phenotypic drug sensitivity test.There

关 键 词：结核 全基因组关联研究 高通量核苷酸测序 突变 

分 类 号：R378.91[医药卫生—病原生物学]