重庆地区耐多药结核分枝杆菌三种二线注射类药物耐药相关基因特征分析

Molecular analysis of resistance to three second-line injectable drugs in multidrug-resistant Mycobacterium tuberculosis strains in Chongqing, China

目的分析重庆市耐多药结核病(MDR-TB)临床菌株二线注射类药物耐药相关基因突变特征,为本地区合理应用分子生物学技术进行注射类药物耐药性检测提供科学依据。方法收集2015年1月至2017年6月重庆市39个区(县)967例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Alamar blue显色法检测3种二线注射类药物[卡那霉素(Km)、阿米卡星(Am)、卷曲霉素(Cm)]的耐药性,用PCR测序方法对其耐药相关基因rrs、eis启动子区、tlyA进行分析,并采用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,35株(15.3%,35/229)对三种二线注射类药物任一耐药,Km、Am和Cm耐药率分别为14.8%(34/229)、11.8%(27/229)、8.3%(19/229)。在34株Km耐药株中,21株发生rrs基因A1401G突变,1株rrs基因G1484T突变,4株eis启动子区G(-10)A突变及1株C(-14)T突变。在27株Am耐药株中,20株发生rrs基因A1401G突变,1株rrs基因G1484T突变,1株eis启动子区G(-10)A突变。在19株Cm耐药株中,13株发生rrs基因A1401G突变,1株rrs基因G1484T突变,1株tlyA基因137位碱基插入改变。北京与非北京基因型间注射类药物耐药及相关基因突变情况差异均无统计学意义。结论重庆地区MDR结核分枝杆菌Km、Am和Cm各耐药相关基因呈现不同的突变频率特征,其中rrs基因突变频率最高,可作为三种注射类药物的耐药诊断标记物。北京基因型与二线注射类药物耐药无相关性。

We analysed the molecular characteristics of second-line injectable drugs resistance-associated gene mutations among MDR-TB isolates in Chongqing,to provide the scientific evidences for rational application of molecular diagnosis technologies for detection second-line injectable drugs resistance in this area.In this study,two hundred and twenty-nine MDR-TB clinical strains were tested for susceptibility to kanamycin,amikacin and capreomycin using micropte alamar blue assay(MABA).PCR and sequencing were conducted for rrs,tlyA and the promoter region of the eis genes conferring second-line injectable drugs resistance.The M.tuberculosis.Beijing genotypes were identified with real-time polymerase chain reaction(PCR)melting curve assay.The results showed that 35 strains(15.3%,35/229)were resistant to any second-line injectable drug among the 229 MDR-TB strains.The percentage of resistance to kanamycin was the highest,accounting for 14.8%(34/229),and the percentages of resistance to amikacin and capreomycin were 11.8%(27/229),8.3%(19/229)respectively.Among the 34 strains resistant to Km,21 strains harbored A1401G mutations and 1 strain with G1484T mutation in rrs,4 strains carried an G(-10)A mutations and 1 strain carried an C(-14)T mutation of the eis promoter.Among the 27 strains resistant to Am,mutations in the rrs included the A1401G(n=20)and G1484T(n=1),and the mutation in the eis promoter was G(-10)A(n=1).Among the 19 capreomycin resistant strains,mutations in the rrs included the A1401G(n=13)and G1484T(n=1),and 1 strain harbored an insertion of G at position 137 There were no statistically significant differences in the 3 second-line injectable drugs resistance and resistance-associated gene mutations between Beijing and non-Beijing genotypes.Therefore,the distributions of gene mutations associated with Km,Am and Cm resistance were different among MDR-TB isolates in Chongqing.The most frequent mutation is A1401G in the rrs gene served as good marker for the 3 second-line injectable drugs resistance.Beijing genotype strains are not associated with the 3 second-line injectable drugs resistance.