利奈唑胺诱导金黄色葡萄球菌耐药株50S核糖体蛋白突变位点分析

摘要

Abstratc:Objective It is to investigate the characteristics of genetic mutations in 50S ribosomal protein gene of linezolid-induced Staphylococcus aureus in vitro . Methods Two S.aureus strain ATCC29213 and ATCC25923 named S2 and S3, two i-solated from the breast milk and blood ( named S5 and S7) were induced by linezolid with series dilution methods in vitro . Such four strains were sensitive to Linezolid and Methicillin .Four mother strains , and series of LZD -nonsusceptible ones were isolated and the MIC value was identified by E -Test.Then, the 50S ribosomal protein gene of these strains was ampli-fied and sequenced .Results 24 linezolid-nonsusceptible S .aureus induced in vitro was isolated and identified .Then, PCR and sequencing indicated the genetic mutations in 50S V ribosomal protein gene of these strains contained multiple point muta-tions in L3 ribosomal protein, while in L4, the common amino acid mutation was Ala56Asp except for S2 series strains.Con-clusion Linezolid-resistant S.aureus can be induced in vitro.Linezolid resistance in S.aureus was closely associated with the genetic mutations in 50S ribosomal protein gene , but the resistant mechanism needed to be further clarified .%目的：阐明体外诱导利奈唑胺耐药金黄色葡萄球菌的核糖体蛋白位点变异特征。方法收集2株金黄色葡萄球菌质控菌ATCC29213和ATCC25923、1株乳汁来源的金黄色葡萄球菌和1株血流感染的金黄色葡萄球菌（编号分别为S2、S3、S5和S7菌株，均为甲氧西林和利奈唑胺敏感株），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rplC和rplD基因），扩增产物经测序后与野生株比较，获得核糖体蛋白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值金黄色葡萄球菌共24株。 PCR测序分析4株母株均无变异位点，各菌株L3对应的氨基酸位点不尽相同，S2组菌株L4未见统一的氨基酸位点，余S3、S5、S7各组菌株普遍存在Ala56Asp 氨基酸位点变异。结论体外多步法可诱导金黄色葡萄球菌利奈唑胺耐药，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。