全基因组测序预测深圳市耐多药结核分枝杆菌的耐药性分析  

Whole-genome sequencing for drug resistance profile prediction in multidrug-resistant Mycobacterium tuberculosis in Shenzhen

作　　者：季乐财 张乐平[1] 吕建文[1] 李晓定 邹小飞[1] 朴玮 JI Le-cai;ZHANG Le-ping;LYU Jian-wen;LI Xiao-ding;ZOU Xiao-fei;PIAO Wei(Department of Tuberculosis,Shenzhen Center for Chronic Disease Control,Shenzhen 518020,China)

机构地区：[1]深圳市慢性病防治中心结核病科,518020 [2]中国疾病预防控制中心营养与健康所国家卫生与健康委员会微量元素营养重点实验室

出　　处：《中国防痨杂志》2021年第2期166-170,共5页Chinese Journal of Antituberculosis

基　　金：深圳市卫计委科研项目(SZXJ2017035);深圳市科技创新计划基础研究项目(JCYJ20160428145728055);深圳市医疗卫生三名工程(SZSM201611017、SZSM201611030)。

摘　　要：目的通过全基因组测序技术分析深圳市耐多药结核分枝杆菌(MDR-MTB)的耐药性检测情况,为MDR-TB患者的治疗及防控提供科学依据。方法选取2013-2017年存储于深圳市慢性病防治中心结核病实验室并成功复苏的420株MDR-MTB临床分离株,对其提取DNA后进行全基因组测序。使用内部Perl脚本分析原始数据,参考MTB耐药基因数据库、耐药决定突变数据库及无害突变数据库确定相关基因突变以判断菌株对异烟肼、利福平、链霉素、乙胺丁醇、氟喹诺酮类、吡嗪酰胺、乙硫异烟胺、对氨基水杨酸、卡那霉素、阿米卡星、卷曲霉素、利奈唑胺、贝达喹啉和氯法齐明的耐药情况,同时绘制菌株耐药情况弦图,以及准广泛耐药MTB(pre-XDR-MTB)及XDR-MTB菌株耐药情况热图。结果全基因组测序从420株菌株中检出23株XDR-MTB菌株(5.48%)和97株pre-XDR-TB菌株(23.10%);其中,所有菌株除对异烟肼、利福平全部耐药外,对链霉素的耐药率最高,达67.86%(285/420),其次为乙胺丁醇(66.19%,278/420)、氟喹诺酮(28.57%,120/420)、吡嗪酰胺(28.33%,119/420)、乙硫异烟胺(13.33%,56/420),对氨基水杨酸(7.38%,31/420)、卡那霉素(6.67%,28/420)、阿米卡星(5.48%,23/420)、卷曲霉素(5.48%,23/420)、利奈唑胺(0.24%,1/420),但未见对贝达喹啉和氯法齐明耐药。全基因组测序发现菌株有212种基因突变类型,以katG-315-S/T(81.43%,342/420)、rpoB-450-S/L(58.57%,246/420)、rpsL-43-K/R(65.96%,188/285)、embB-306-M/V(32.37%,90/278)突变居多。结论全基因组测序预测深圳市MDR-MTB临床分离株中的pre-XDR-MTB比例较高,对一线抗结核药品及氟喹诺酮类药品的耐药率较高,临床实践中应结合药敏试验结果谨慎使用;而对乙硫异烟胺、氯法齐明、对氨基水杨酸等二线抗结核药品的耐药率较低,考虑这些药品可作为MDR-TB患者治疗方案的组成部分。Objective To analyze the drug resistance spectrum of the clinical multidrug-resistant Mycobacterium tuberculosis(MDR-MTB)isolates in Shenzhen using whole-genome sequencing data,to provide scientific basis for the treatment and prevention of MDR-TB patients.Methods A total of 420 MDR-MTB strains were successfully recovered from all strains stored in the tuberculosis laboratory of Shenzhen Center for Chronic Disease Control from 2013 to 2017.Whole-genome sequencing were conducted for the genomic DNA extracted from these strains.The sequencing data were analyzed using an in-house pipeline.Drug-resistance to isoniazid,rifampicin,streptomycin,ethambutol,fluoroquinolones,pyrazinamide,ethionamide,p-aminosalicylic acid,kanamycin,amikacin,capreomycin,linezolid,bedaquiline,and clofazimine were determined based on MTB drug resistance gene database and drug resistance determining and harmless mutations database.Simultaneously,a string diagram of bacterial resistance and a heat map were used to visualize drug resistance spectrum of all strains and preXDR-MTB/XDR-MTB strains,respectively.Results Among the 420 clinical MDR-MTB strains,23(5.48%)were XDR-MTB strains and 97(23.10%)were pre-XDR-MTB strains;except for isoniazid and rifampicin resistance,the frequency of streptomycin resistance was the highest with 67.86%(285/420),followed by ethambutol(66.19%,278/420),fluoroquinolones(28.57%,120/420),pyrazinamide(28.33%,119/420),ethionamide(13.33%,56/420),p-aminosalicylic acid(7.38%,31/420),kanamycin(6.67%,28/420),amikacin(5.48%,23/420),capreomycin(5.48%,23/420),and linezolid(0.24%,1/420).No strain was resistant to bedaquiline or clofazimine.A total of 212 drug-resistance mutation types were revealed using the whole genome sequencing data,and the most common mutations were katG-315-S/T(81.43%,342/420),rpoB-450-S/L(58.57%,246/420),rpsL-43-K/R(65.96%,188/285),and embB-306-M/V(32.37%,90/278).Conclusion It was found that,among clinical MDR-MTB isolates in Shenzhen,the proportion of pre-XDR-MTB and the resistance rates to the first-line

关 键 词：结核 抗多种药物性 全基因组关联研究 高通量核苷酸测序 基因组 细菌 深圳 

分 类 号：R446.5[医药卫生—诊断学]