武汉地区结核分枝杆菌临床分离株乙胺丁醇耐药分子特征研究

Molecular characterization of ethambutol-resistant Mycobacteria luberculosis isolates recovered from Wuhan

探讨武汉地区结核分枝杆菌乙胺丁醇耐药基因embB的分子特征,为深入研究结核分枝杆菌乙胺丁醇(EMB)耐药分子机制、快速检测技术的建立和个体化临床用药奠定基础;同时探索武汉地区"北京基因型"菌株的传播类型,为结核防控提供参考。收集85株临床分离株,采用PCR直接测序法分析embB基因的突变特征;采用RD105基因缺失检测法鉴定85株分离株中"北京基因型"菌株。结果显示22例EMB耐药株中,15(68.2%)例在embB基因检测到突变,其中14例为306位点突变,突变类型为M306V ATG→GTG和M306I ATG→ATA,1例为319位点突变,突变类型为Y319C TAT→TGT;同时,在63例EMB敏感株中也检测到了5株(7.9%)发生了embB基因突变,其中4例在306位点,1例在319位点。85例临床分离株中78株为"北京基因型"菌株,占全部菌株的91.8%,非"北京基因型"菌株有7株,占8.2%。研究表明embB306位点突变不仅在乙胺丁醇耐药菌株中,而且在乙胺丁醇敏感菌株中也能检测到。由于乙胺丁醇耐药菌株同时还耐其他药物,故不能排除其他药物与embB306位点突变的关系,因此embB306位点突变与结核分枝杆菌耐药的相关性有待深入探讨。本研究发现武汉地区结核分枝杆菌乙胺丁醇耐药株中embB基因突变类型并不存在地域差异,而在敏感株中却存在明显的地域差异性。本研究同时表明,"北京基因型"菌株在武汉地区呈主要流行趋势,应该引起我们的重视。

The research aims to explore the molecular characteristics of Mycobacterium tuberculosis ethambutol resistance in Wuhan, to establish a rapid test method about the EMB resistance of Mycobacterium tuberculosis and to understand the transmission of Beijing genotype. PCR amplification was performed on the 85 isolates to characterize the embB gene. At the same time, the identification of Beijing genotype in the 85 clinical isolates was performed using RD105 deletion-targeted multiplex PCR. The results show that 15 （68.2%） of the 22 ethambutol resistant isolates were found to have a mutation in the embB gene and among the 15 isolates, fourteen had a mutation at codon 306, the mutation types were M306V ATG---~GTG and M306I ATG---＊ATA, the other one was at codon 319, Y319C TAT---~TGT; Meanwhile, five of the 43 EMB susceptible isolates had also found a mutation in embB gene, four was at codon 306 and one was at codon 319. Of the 85 clinical isolates, 78（91.8% ） were Beijing genotypes and 7 （8.2%） were non-Beijing genotypes. According ethambutol resistant isolates, but also in ethambutol are also resistant to other drugs at the same time, to the research, mutations in embB are not only found in susceptible isolates. Because the ethambutol resistant isolates we can＇t exclude the relationship between other drugs and mutations in embB at codon 306. Therefore, the role of embB306 mutations remains unclear. In addition, ＂Beijing genotype＂ strains of Mycobacterium tuberculosis were typically prevalent in Wuhan, which should arouse our more attention.