结核分枝杆菌稳定L型利福平耐药性基因的研究

目的探讨细胞壁缺陷结核分枝杆菌形成利福平耐药性的分子机制。方法将结核分枝杆菌分别接种于含利福平与不含利福平的非高渗透压液体培养基内培养,逐日在倒置显微镜下观察L型的形成情况。L型形成和生长后,滤过培养物分离L型,在不含诱导剂的非高渗液体培养基内传代培养获得L型纯培养物。用结核分枝杆菌利福平耐药性相关基因(rpoB基因)引物分别对自发形成的稳定L型及诱导形成的稳定L型的染色体DNA进行PCR扩增,扩增产物分别以琼脂糖凝胶电泳及PCR-DS方法进行检测和分析。结果结核分枝杆菌既可在非高渗透压培养基内自发形成细胞壁缺陷细菌并成为稳定L型,也可在最低抑菌浓度的利福平作用下发生细胞壁缺陷并成为稳定L型。PCR扩增产物琼脂糖凝胶电泳可见自发形成和诱导形成的稳定L型均形成与其亲代细菌型一致的DNA条带,序列分析显示具有与其亲代细菌型相同的碱基序列。结论结核分枝杆菌在非高渗透压培养基内既可以自发地形成L型,也可以被利福平诱导形成L型。结核分枝杆菌稳定L型具有明显的利福平耐药性,但其相关rpoB基因并没有发生突变。提示结核分枝杆菌的利福平耐药性变异不一定同rpoB基因突变有关,细胞壁缺陷也是导致结核分枝杆菌形成利福平耐药性的一个重要机制。

Study on the rifampin resistance gene of the stable L-forms derived from Mycobacterium tuberculosis

Objective To probe the molecular mechanisms of rifampin resistance of the cell wall deficient Mycobacterium tuberculosis. Methods The strains of M. tuberculosis were inoculated separately into the non-high osmotic liquid medium with rifampin and without rifampin. When the L-forms were found, the cultures were filtrated and subculture in the non-high osmotic medium without any antibiotic to get the pure culture of stable L-form. The rpoB genes from the stable L-forms of M. tuberculosis were determined by the direct sequencing with 3730 automated sequencing system. Results The L-forms derived from M. tuberculosis would be not only induced by rifampin, but also formed spontaneously in the cultures of non-high osmotic medium. The rpoB gene of the L-forms was determined by the nPCR for the M. tuberculosis and the results showed the same band as that of their parental bacteria in agar gel electrophoresis. By the analysis of sequencing, the rpoB genes of the L-forms were showed the same sequences as that of their parental bacteria. Conclusion The sequence of rpoB gene of the L-forms derived form M. tuberculosis either formed spontaneously or induced by rifampin were same as that of their parental bacteria. It suggested that the RFP resistance of M. tuberculosis would not be connected with mutation of rpoB gene, the cell wall deficient variation was also the one important mechanism in the RFP resistance in M. tuberculosis.