结核分支杆菌耐喹诺酮分子机制的研究

Studies of molecular mechanism of quinolone-resistant in Mycobacterium tuberculosis isolates

目的了解结核分支杆菌耐喹诺酮药物的分子机制,建立快速的药敏的方法。方法通过16SrRNA聚合酶链反应-单链构象多态性(PCR-SSCP)技术分析77株分支杆菌临床分离株;通过PCR-SSCP和直接测序技术(PCR-DS)分析结核分支杆菌的gyrA基因突变的情况。结果75株为结核分支杆菌复合群。以结核分支杆菌标准菌株H37Rv和卡介苗为对照,30株喹诺酮敏感株的gyrA基因的SSCP图谱均泳动正常,测序分析与对照株相同。45株耐喹诺酮的菌株中,34株(75.6%)gyrA基因SSCP图谱泳动异常;测序证实10株为90位密码子的突变,24株为94位密码子突变,所有gyrA突变株的氧氟沙星4μg/ml≤MICs≤32μg/ml,左氧氟沙星2μg/ml≤MICs≤16μg/ml。结论gyrA基因突变,尤其90位和94位密码子突变是结核分支杆菌耐喹诺酮的主要分子机制,PCR-SSCP可能成为测定部分结核分支杆菌喹诺酮耐药基因型的简便、快速的方法,并有望直接用于临床标本喹诺酮敏感性试验。

Objective To study the molecular mechanism in M.tuberculosis quinolones-resistant ~isolates , and develop a new method for detection of drug resistance. Methods Seventy-seven clinical isolates were identified for their mycobacteria species, and the gyrA genes mutation of M.tuberculosis were analyzed with PCR-SSCP and PCR-direct sequencing. Results Seventy-five isolates were identified as M.tuberculosis. The standard strain of M.tuberculosis H37Rv and BCG as control were used. All of 30 quinolones-sensitive ~isolates had the same gyrA SSCP profiles as the controls. Among 45 quinolones-resistant isolates, 34(75.6%) displayed abnormal gyrA SSCP profiles; DNA direct sequencing of gyrA gene was performed, the mutation at codon 90 of gyrA occurred in 10 quinolones-resistant isolates; the mutation at codon 94 of gyrA occurred in 24 quinolones-resistant isolates, the MICs of ofloxacin were higher than 4μg/ml and lower than 32 μg/ml, while the MICs of levofloxacin were higher than 2μg/ml and lower than 16μg/ml in all mutational isolates. Conclusion Quinolones resistances in some M.tuberculosis isolates were due to mutation on gyrA genes, especially the mutations at 90 and 95 codons. PCR-SSCP method might become a simple and rapid diagnostic test for genotypes of M.tuberculosis quinolones-resistance.