肠球菌低水平利奈唑胺耐药的机制研究

Previous studies have demonstrated that the mechanisms of high level linezolid-resistant Enterococci (MIC≥16 mg/L) is correlated with the mutation of 23S rRNA, production of methyltransferase and deletion or mutation of gene coding for ribosomal protein L3/L4, but the mechanisms of low level linezolid-resistant Enterococci (MIC is 8 mg/L) is still unclear. In the current study, the whole genomic differences between clinical isolates of linezolid-sensitive and low level linezolid-resistant Enterococcus faecium will be detected by using high-throughput sequencing technology, and analyzed by using bioinformatics to screen the new resistance genes and gene mutation sites related to the development of low level linezolid-resistant Enterococcus faecium. Furthermore, recombinant DNA technology would be used to construct resistance genes / mutant genes knockout or high expression strains, and the sensitivity differences of recombinant strains to linezolid would be examined to explore the roles of different resistance genes / mutant genes in the development of low level linezolid-resistant Enterococcus faecium. The launching of this project will provide an important theoretical basis for the mechanisms of low level linezolid-resistant Enterococci, but also lay the foundation for the prevention and treatment of resistant Enterococci and drug development.

已往研究表明，高水平利奈唑胺耐药肠球菌（MIC≥16 mg/L）的发生主要与23S rRNA突变、甲基转移酶的产生和核糖体蛋白L3/L4的基因缺失或突变等有关，但关于MIC为4～8 mg/L的低水平利奈唑胺耐药肠球菌的发生机制目前尚不清楚。因此本课题拟在成功分离屎肠球菌低水平利奈唑胺耐药临床菌株的基础上，采用高通量测序技术对屎肠球菌利奈唑胺敏感株及低水平耐药株进行全基因组测序，结合生物信息技术在全基因组范围内筛选、定位与低水平利奈唑胺耐药形成有关的新的耐药基因和基因突变位点。在此基础上，利用基因重组技术构建耐药基因/突变基因敲除或高表达屎肠球菌菌株, 并检测不同重组菌株对利奈唑胺的敏感性差异，探讨不同耐药基因/突变基因在屎肠球菌低水平利奈唑胺耐药发生中的作用。本课题的开展将为揭示低水平利奈唑胺耐药肠球菌的发生机制提供重要的理论依据，同时也为耐药肠球菌的临床防治以及新药开发奠定基础。

利奈唑胺作为第一个应用于临床的噁唑烷酮类抗阳性菌药物,被用于各种耐药革兰阳性球菌感染治疗,但随着临床的广泛应用,细菌对利奈唑胺的快速耐药引起临床广泛关注。本研究通过对我院近几年的耐药性监测，筛选出4株低水平利奈唑胺耐药肠球菌。多位点序列分型硏究表明耐药菌株主要是ST78型。利用PCR扩增及测序方法筛选已知的最常见利奈唑胺耐药机制23sRNA基因突变，其中一株筛查出23sRNA G2576T突变。通过Illumina Hiseq2000平台测序，完成了4株利奈唑胺耐药肠球菌基因组重测序,基因组大小为2,935,722 bp-3,036,969bp，GC含量37.21-37.66%。在NCBI数据库选择与研究肠球菌菌株基因组序列同源性在95%以上参考序列进行比较基因组分析，物种进化分析，构建系统进化树及Ka/Ks分析。并利用相关软件及数据库完成了以下分析：基因成分分析及基因预测，基因功能注释（KEGG、COG、PAIDB），病原与宿主互作数据库注释（PHI），细菌致病菌毒力因子注释（VFDB），耐药基因注释（ARDB）。本研究对分离自临床的利奈唑胺耐药肠球菌的遗传特性、菌株的亲缘关系、毒性特征、耐药机制及其分子基础进行了研究,以期对耐药控制和临床用药提供依据。