耐吡嗪酰胺结核分枝杆菌基因突变研究

目的 研究吡嗪酰胺酶编码基因pncA突变与结核分枝杆菌吡嗪酰胺(pyrazinamide,PZA)耐药的关系.方法 将临床分离的36株结核分枝杆菌进行常规PZA药敏试验和pncA基因序列分析.采用绝对浓度法进行PZA药敏试验;PCR扩增pncA基因及其上游、下游碱基序列,纯化回收PCR产物克隆到T载体并进行全自动测序.结果 36株临床分离株中25株PZA耐药,11株PZA敏感.5株高耐PZA(PZA浓度为250 μg/ml)临床分离株4株pncA基因突变;20株低耐PZA(PZA浓度为50 μg/ml)6株pncA基因突变;25株耐PZA结核分枝杆菌pncA基因突变率为40.0%.3株PZA敏感临床分离株pncA基因出现突变、其中2株为同义突变.11株耐PZA结核分枝杆菌pncA基因上游调控序列突变,其中5株同时有pncA基因突变;2株PZA敏感结核分枝杆菌pncA基因上游序列突变.结论 pncA基因突变可引起结核分枝杆菌对PZA耐药,但pncA基因突变只是结核分枝杆菌耐PZA的一种机制,可能还存在PZA的其他耐药机制.

PncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates

Objective To evaluate the frequency of pncA gene mutations in pyrazinamide-resistant (PZA-resistant)Mycobacterium tuberculosis(M.TB)isolates.Methods The isolates were tested for PZA susceptibility with absolute concentration method.The pncA gene was amplified and the products were cloned into T-vector,followed with sequencing.Results In all the 36 M.TB isolates,there were 25 PZA-resistant strains and 11 PZA-susceptible strains.Mutations of the pncA gene were found in 10 PZA-resistant isolates,four of which belonging to high PZA-resistant strains.The wild type pncA sequence was present in 3 PZA-susceptible strains, synonymous mutations of pncA gene were detected in two PZA-susceptible strains.Additionally,11 PZA-resistant strains and 2 PZA-susceptible ones showed putative regulatory mutations in the upsteam of pncA gene.Condusions The mutation of the pncA gene can cause the PZA resistance.However.there ale other drug resistance mechanism except this mutation.