5株万古霉素敏感性减低耐甲氧西林金黄色葡萄球菌的graS基因及细胞超微结构分析

摘要

目的 分析在防耐药变异浓度(mutant prevention concentration, MPC)测定过程中筛选出的对万古霉素敏感性减低的耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)在graS基因及细胞超微结构上的变化. 方法 利用Sau-PCR、脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)对筛选菌株进行分析.扩增graS基因,并利用变性梯度凝胶电泳 (denaturing gradient gel electrophoresis, DGGE)技术检测在该基因上是否存在突变.通过电子显微镜技术观察细胞壁结构改变.结果 万古霉素敏感性减低菌株经Sau-PCR和PFGE分型检测,证实部分筛选菌株电泳条带与原始菌株存在有差异,且电镜结果也显示筛选菌株细胞壁有所增厚.DGGE检测未发现在graS基因上存在突变点.结论 试验中的菌株万古霉素敏感性的减低可能不是由graS基因突变造成的,可能存在其他基因位点的突变,或可能存在另外的耐药机制.%Objective The methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin which were selected during mutant prevention concentration(MPC) determination were characterized to compare the changes in the graS gene and cell ultrastructure.Methods The strains selected were characterized by pulsed-field gel electrophoresis (PFGE) and Sau-PCR assay.The graS gene was detected by polymerase chain reaction method;Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations in the graS gene.The electron microscopy was used to compare the differences on cell wall.Results Compared with the parental strains, some strains with reduced susceptibility to vancomycin had different DNA profiles by Sau-PCR assay and PFGE.Electron microscopy showed that the mutant strains had slightly thicker cell walls than those of the wild-type strains.DDGE was used to probe for mutations in the graS gene, but no different banding patterns were observed on the electrophoretogram spectrum.Conclusion The strains with reduced susceptibility to vancomycin were not caused by genetic mutations of graS, there may be mutations in genes other than the graS gene or existence of other resistant mechanisms.