四川地区结核分枝杆菌喹诺酮耐药基因突变研究

目的 研究结核分枝杆菌耐喹诺酮类药物的分子机制，探索四川地区耐喹诺酮结核分枝杆菌临床分离株的耐药基因突变特征。方法 采用绝对浓度法检测结核分枝杆菌临床分离株的最小抑菌浓度(MIC)，再通过聚合酶链反应-单链构象多态性(PCR-SSCP)和直接测序检测结核分枝杆菌耐喹诺酮类药物临床分离株gyrA耐药决定区(QRDR)突变，并和标准株H37Rv比较。结果 68株临床分离株中，14株喹诺酮药物敏感株和8株低耐药株未发现gyrA耐药决定区基因突变，25株高耐药株、11株低耐药株和10株药物敏感株检测到gyrA基因突变，高耐药株突变率为100％，低耐药株突变率为58％，敏感株突变率为42％，总的突变率为68％。根据SSCP条带，gyrA突变可分成4种类型，测序发现分别为Ser95Thr、Asp94Gly、Ala90Val、Ala83Val突变。结论 结核分枝杆菌耐喹诺酮与gyrA基因突变密切相关，gyrA基因突变是耐喹诺酮结核分枝杆菌耐药性的主要分子机制。

Study on the Gene Mutation of Quinolone-resistant Mycobacterium tuberculosis Isolated from Sichuan Province

OBJECTIVE: To investigate the molecular mechanism of quinolone-resistance of M. tuberculosis and characterize the gene mutation in Sichuan Province. METHODS: Susceptibility of the clinical isolates to quinolones (ofloxacin, ciprofloxacin and sparfloxacin) was tested by the absolute concentration method. GyrA gene quinolone reasistance-determining region (QRDR) mutations M. tuberculosis were detected with PCR-SSCP and DNA sequencing. RESULTS: Of 68 clinical isolates of M. tuberculosis, 25 high-resistant, 11 low-resistant and 10 sensitive isolates were noted to have abnormal gyrA SSCP profile and different gyrA sequences from the standard strain H37Rv, and 14 sensitive and 8 low-resistant isolates were found with no mutation of gyrA gene. DNA sequencing unveiled Ser-->Thr mutation at codon 95, Asp-->Gly at codon 94, Ala-->Val at codon 90, and Ala-->Val at codon 83. CONCLUSION: This study confirmed the strong correlation between the quinolone-resistance and the mutation of gyrA gene, which might be a major molecular mechanism of quinolone-resistance in M. tuberculosis. The types of mutations exhibit no difference between Sichuan Province and other areas in China.