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目的阐明体外诱导利奈唑胺耐药粪肠球菌的核糖体蛋白位点变异特征。方法收集1株血流感染的粪肠球菌利奈唑胺敏感株,通过体外浓度倍增法诱导利奈唑胺耐药;挑取单克隆,经E-test条测定MIC值,获得各菌株的耐药浓度梯度;提取耐药菌株基因组DNA,PCR扩增核糖体蛋白L3和L4(对应rpl C和rpl D基因),扩增产物经测序后与野生株比较,获得核糖体蛋白及对应氨基酸的突变位点。结果经体外多步法诱导利奈唑胺耐药的不同MIC值粪肠球菌共13株。PCR测序分析2株母株均无变异位点,rpl C基因对应的氨基酸位点不尽相同,rpl D基因普遍存在T301C位点变异,对应的氨基酸为Phe101Leu。结论体外多步法可诱导粪肠球菌利奈唑胺耐药,耐药机制与核糖体蛋白位点突变密切相关,但仍需进一步研究证实突变位点与耐药的关系。
Objective To elucidate the variation of ribosomal protein loci induced by linezolid resistant Enterococcus faecalis in vitro. Methods One HIV-susceptible strain of Enterococcus faecalis infected with bloodstream was collected and treated with linezolid in vitro to induce linezolid resistance. The monoclonal antibody (MIC) was selected by E-test to determine the drug resistance of each strain The genomic DNA was extracted from the resistant strains. The ribosomal proteins L3 and L4 (corresponding to the rpl C and rpl D genes) were amplified by PCR. The amplified products were sequenced and compared with the wild type to obtain the ribosomal protein and corresponding amino acid mutation sites . Results In vitro multi-step method of linezolid resistance induced by different MIC values of Enterococcus faecal 13 strains. PCR analysis of the two strains showed no mutation sites, rpl C gene corresponding amino acid sites are not the same, rpl D gene prevalence T301C site variation, the corresponding amino acid Phe101Leu. Conclusion In vitro multi-step method can induce genistein-resistant linezolid, the mechanism of resistance is closely related to the mutation of ribosomal protein. However, further studies are needed to confirm the relationship between the mutation and drug resistance.