重庆地区耐多药结核分枝杆菌对氟喹诺酮类药物耐药的相关基因特征分析

Prevalence and molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates resistant to fluo-roquinolones in Chongqing

目的分析重庆地区耐多药结核分枝杆菌对氟喹诺酮类（FQs）药物耐药的相关基因特征，以及与结核分枝杆菌基因型的相关性。方法收集2015年1月至2017年6月重庆市39个区（县）967例耐多药结核病（MDR-TB）可疑患者的所有耐多药（MDR）结核分枝杆菌临床分离株229株，通过微孔板Alamarblue显色法检测4种FQs药物[氧氟沙星（Ofx）、左氧氟沙星（Lfx）、莫西沙星（Mfx）、加替沙星（Gfx）]的耐药性，用PCR测序方法对FQs药物耐药相关基因删以、gyrB进行分析，并采用实时荧光定量熔解曲线方法进行北京基因型鉴定。结果在229株MDR菌株中，94株（41．0％，94／229）对任一FQs药物耐药。其中，Ofx耐药率最高（41．0％，94／229）；Lfx、Mfx耐药率居中，分别为31．4％（72／229）、30．6％（70／229）；Gfx耐药率最低（20．1％，46／229）。94株对FQs耐药菌株中，81株（86．2％，81／94）发生刖以基因突变，第94位密码子突变最常见（60．6％，57／94）；10株（10．6％，10／94）发生gyrB基因突变。7株gyrA基因双位点突变均显示为高水平耐药；9株gyrA与gyrB联合突变中，2株为高水平耐药。重庆地区对FQs耐药菌株中北京基因型80株（85．1％，80／94），其中，现代北京基因型占60．0％（48／80）。结论重庆地区MDR结核分枝杆菌对FQs耐药的菌株以现代北京基因型为主，其耐药相关基因突变主要发生于gytA基因。

Objective To analyze the molecular characteristics of fluoroquinolones （FQs） resistance-associated gene mutations and the correlation with the different genotypes among the MDR-TB strains in Chongqing. Methods Two hundred and twenty-nine MDR-TB clinical strains were tested for susceptibility to ofloxacin, levofloxacin, moxifloxacin, and gatifloxacin using microplate alamar blue assay （MABA）. PCR and sequencing were conducted for gyrA and gyrB genes conferring FQs resistance. The Beijing genotypes were identified with real time polymerase chain reaction （PCR） inching curve assay. Results Among the 229 MDR TB strains, 94 strains （41.0%, 94/229） were resistant to any FQ. The prevalence of FQs resistance in Chongqing were highest for ofloxacin （41.0%, 94/229）, intermediate for levofloxacin （31.4% ,72/229） and moxifloxacin （30.6%, 70/229）, and lowest for gatifloxacin （20.1% 46/ZZ9）. The 94 strains resistanv to FQs contained 81 gyrA mutation strains （86.2%, 81/94）, and the highest mutation frequency was at codon 94 （60.6% ,57/94）. Mutations with g）;rB were observed in 10 FQ-resistant strains （10.6%, 10/94）. Seven strains with double mutations in gyrA, and 2 of the 9 strains with Asp94Gly and Ala504Thr/Ala504Val double mutations in gyrA and gyrB respectively, showed high level FQ resistance. The proportion of Beijing genotype strains was 85.1% （80/94） among FQresistant strains in Chongqing. Of these, 60.0% （48/80） were modern-Beijing sublineage strains. Conclusion Modern-Beijing sublineage strains accounts for the majority of FQ-resistarit strains and the resistant related mutations mainly occurin gyrA gene.