人工诱导耐恩氟沙星金黄色葡萄球菌grlA和gyrA基因突变研究

Study on gyrA and grlA genes in stepwise-selected enrofloxcin-resistant mutants of Staphylococcus aureus

通过恩氟沙星人工多步诱导标准金黄色葡萄球菌 2 80 0 1产生MIC值为 4～≥ 1 2 8μg/ml的耐药变异株 ,选其 3株提取染色体DNA ,对gyrA和grlA基因进行PCR扩增 ,产物与pMD1 8 T载体连接 ,转化至JM1 0 9大肠埃希氏菌感受态细胞 ,筛选阳性克隆 ,测序。序列分析结果表明 ,低水平 (MIC≤ 1 6μg/ml)的耐药突变株只发生grlASer80 (TCC)→Phe (TGC)或grlA ,Ser80 (TCC)→Tyr (TAC)点突变 ,高水平 (MIC≥1 2 8μg/ml)的耐药突变株需要grlASer80 (TCC)→Phe (TGC)和gyrASer84(TCA)→Leu (TTA)等两个位点的突变。三株耐药株均发生grlASer80→Tyr (Phe)点突变 。

Fluoroquinolone resistant mutants (MIC: 4～≥128μg/ml) were obtained in vitro from Staphylococcus aureus 28001 by stepwise selection on increasing concentrations of enrofloxacin. The chromosomal DNAs of three(MIC: 4, 16 and ≥128μg/ml) of these strains were obtained, PCR was employed to amplify the gyrA and grlA genes;the PCR product were aligned to pMD18 T vector; transformation in E.coli JM109 competent cell were performed; positive clones were chosen and sequenced. The sequence analysis showed that grlA Ser80 (TCC)→Phe (TGC) or grlA , Ser80 (TCC)→Tyr (TAC) point mutation was involved in low resistant mutation strains (MIC≤16μg/ml); grlA Ser80 (TCC)→Phe (TGC) and gyrA Ser84 (TCA)→Leu (TTA) multiple point mutation were involved in high resistant strains(MIC≥128μg/ml). All three resistant mutation have grlA Ser80→Tyr (Phe) point mutation. Results suggested that the primary target enzyme to Staphylococcus aureus of enrofloxacin was topoisomerase IV.