结核分枝杆菌及其稳定L型的katG基因研究

Study on KatG gene of Mycobacteritun tuberculosis and the stable L-forms

目的探讨细胞壁缺陷对结核分枝杆菌异烟肼敏感性的影响以及结核分枝杆菌细胞壁缺陷形成异烟肼耐药性的分子基础。方法采用非高渗培养基体外药敏试验,检测自发形成及诱导形成的结核分枝杆菌稳定L型的异烟肼敏感性；用异烟肼耐药性相关结构基因katG的特异性引物,对自发形成及诱导形成的结核分枝杆菌稳定L型及其亲代细菌型的染色体DNA进行PCR扩增与序列分析。结果不论对异烟肼敏感还是耐药的结核分枝杆菌自发或诱导形成稳定L型后,都能够在含0.2μg/ml异烟肼的非高渗液体培养基内生长和传代培养。结核分枝杆菌稳定L型katG基因PCR扩增产物经琼脂糖凝胶电泳,可见形成与其亲代细菌型一致的DNA条带。PCR-DS分析结果显示,结核分枝杆菌稳定L型katG基因扩增产物具有与其细菌型相同的核苷酸序列。结论无论是自发形成的还是异烟肼诱导形成的结核分枝杆菌稳定L型都可形成异烟肼耐药性,但其katG基因的核苷酸序列并没有发生改变。提示结核分枝杆菌稳定L型的异烟肼耐药性与katG基因突变无关,细胞壁缺失也是导致结核分枝杆菌形成异烟肼耐药性的一个重要机制。

Objective To probe the influence on isoniazid susceptibility of mycobacterium tuberculosis （MT） by cell wall deficiency and the molecular mechanisms of the isoniazid resistance of the stable L-forms. Methods The stable L-forms of MT were induced by isoniazid or formed spontaneously in the non-high osmotic liquid medium and then the variants were subcultured in the medium with isoniazid. The DNA of the MT and the stable L-forms were amplified and determined by PCR with the specific primers of the katG gene and produces of the PCR were analyzed by direct sequencing. Results All strains of the stable L-forms derived from either induced sensitive or resistant MT were unsusceptibility to isoniazid and they could grow and be subcultured in the non-high osmotic liquid medium which contained isoniazid （0. 2μg/ml）. The molecular weight and the base sequences of the katG gene from the stable L-forms were same as that of their parental bacteria by analysis PCR and PCR-DS. Conclusion The isoniazid resistance of the stable L-form was not connected with mutation of katG gene, and the cell wall deficiency would be the important mechanisms of the isoniazid resistance of MT.