耐异烟肼结核分枝杆菌临床分离株耐药相关基因突变研究

Characterization of the katG, inhA, ahpC, kasA,and oxyR gene mutations in isoniazid-resistant and susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing

目的阐明结核分枝杆菌耐异烟肼临床分离株katG、inhA、ahpC、kasA及oxyR基因突变特点。方法对144株结核分枝杆菌临床分离株(耐异烟肼菌株101株;异烟肼敏感株43株)的katG、inhA、kasA、ahpC及oxyR基因进行DNA片断扩增及DNA序列分析,与GeneBank中结核分枝杆菌标准序列进行比较。结果(1)耐异烟肼菌株中未发现katG完全缺失,81株耐药株(80.2%)katG存在点突变、缺失或插入,其中16个突变位点未见报道;39株(38.6%)耐药株第315位点突变,低耐药菌株(1μg/ml)第315位点突变率显著高于高耐药菌株(10μg/ml;χ2=9.31,P<0.05);58株(57.4%)耐药株第463位点突变。23株(53.3%)敏感株第463位点突变。(2)5株(4.9%)耐药株inhA发生突变。敏感株inhA无突变。(3)3株(2.9%)耐药株ahpC发生突变。敏感株ahpC无突变。(4)17株(16.8%)耐药株kasA发生突变。敏感株中3株菌株Gly312Ser突变。(5)在全部菌株中未发现oxyR基因突变。(6)综合本项研究中各基因的突变情况,共有91株耐异烟肼菌株发生与异烟肼耐药相关的突变。结论本项研究进一步证实了结核分枝杆菌耐异烟肼与katG、inhA、ahpC及kasA基因突变之间的关系,并且提示还有其他机制参与异烟肼耐药。

Objective To study the characteristics of katG, inhA, ahpC, kasA, and oxyR gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. Methods A total of 101 isoniazid-resistant and 43 susceptible strains of Mycobacterium tuberculosis were analyzed by PCR and sequence analysis of their katG, inhA, ahpC, kasA, and oxyR genes. Results (1) Sequencing of katG from 101 INH-resistant strains showed point mutations, small deletions or insertions in 81 isolates(80.2%), but no complete deletions were identified. The mutations at 16 position were found for the first time. Point mutations at position 315 were found in the genomes of 38.6%(39/101) of isoniazid-resistant strains. Low level isoniazid resistant strains (1 μg/ml) had higher mutation frequency at 315-Ser than high level isoniazid resistant strains (10 μg/ml; χ 2=9.31,P<0.05). Mutations at position 463 were detected in 58 (57.4%) isoniazid-resistant strains. Arg463leu was also present in 23 of 43 susceptible strains. (2) Mutations in inhA genes were identified in 5 isoniazid-resistant isolates (4.9%). None of the susceptible strains contained any mutation in inhA genes. (3) Only 3 isolates in the 101 (2.97%) isoniazid-resistant clinical isolates had mutations in ahpC genes. No mutations were identified in the ahpC genes in 43 isoniazid-susceptible isolates. (4) Mutations in kasA genes were present in 17 of 101 (16.8%) isoniazid-resistant isolates. However, G312S was also present in 3 of 43 susceptible strains. (5) None of the isoniazid-resistant strains and susceptible isolates contained oxyR gene mutation. (6) Taken together, 91 of 101(90%) isoniazid-resistant strains had mutations in katG, inhA, ahpC, and kasA genes which were associated with drug resistance. Conclusion These studies provide further evidence supporting the association between katG, inhA, ahpC, and kasA gene mutations and INH resistance in Mycobacterium tuberculosis, while other mechanisms of INH resistant may exist.