28株牛源分枝杆菌的耐药基因突变分析

Mutations of drug resistance genes in 28 bovine Mycobacterium strains

为明确分离自宁夏、甘肃和新疆地区的28株牛源分枝杆菌的耐药基因突变情况,通过采用16S rRNA、MPT64和多位点PCR方法进行基因分型鉴定,L-J比例法对其进行药敏试验,随后用DNA测序技术对10株耐药菌和4株敏感菌进行耐药基因检测及突变分析。分型鉴定结果表明,28株分离株均属于结核分枝杆菌复合群(MTBC),其中26株为牛分枝杆菌,2株为人型结核分枝杆菌。药敏试验结果表明,28株MTBC中18株对4种一线抗结核药物敏感,10株耐药。耐药菌株中,1株对利福平耐药;7株对异烟肼、利福平和链霉素三重耐药;2株对异烟肼、利福平、链霉素和乙胺丁醇四重耐药。耐药基因突变位点分析结果表明,rpoB 378位点的突变率为10.0%;katG 463位点的突变率为100.0%;rrs 311位点的突变率为11.1%;embB 378位点的突变率为100.0%;oxyR-ahpC和rpsL位点无突变。提示宁夏、甘肃和新疆地区奶牛场中MTBC主要以牛分枝杆菌为主,且存在耐多药情况,耐药突变位点以katG 463和embB 378为主。

To identify the drug-resistance gene mutations of 28 Mycobacterium bovis isolates isolated from Ningxia, Gansu and Xinjiang, 28 clinical strains were genotyped by 16 S rRNA,MPT64 and multi-site PCR.L-J drug sensitivity test was used to detect drug-resistant phenotypes of 28 clinical strains.Drug resistance gene mutation in 10 drug-resistant strains and 4 susceptible strains were analyzed by sequencing technique.Gene genotype results showed that 28 clinical strains were belonged to the Mycobacterium tuberculosis complex group(MTBC),or which 26/28 strains were Mycobacterium bovis and 2/28 strains were Mycobacterium tuberculosis.Drug sensitivity test results showed that 18/28 MTBC strains were sensitive to four first-line anti-tuberculosis drugs and 10/28 strains were drug-resistant.Among the drug-resistant strains, 1 strain was resistant to rifampin, 7 strains were triple resistant to isoniazid, rifampin and streptomycin, and 2 strains were quadruple resistant to isoniazid, rifampin, streptomycin and ethambutol.The mutation site analysis results showed that the mutation rate of rpoB gene on 378 site was 10.0%.The mutation rate of katG gene on 463 site was 100.0%.The mutation rate of rrs gene on 311 site was 11.1%.The mutation rate of embB gene on 378 site was 100.0%.There were no mutations in the oxyR-ahpC and rpsLgene in 28 strains.The results suggest that Mycobacterium bovis is the main strain of MTBC and there is widespread drug resistance of Mycobacterium bovis in dairy farms in Ningxia,Gansu and Xinjiang.The main drug-resistant mutation sites are KatG463 and embB378.