肠球菌新型利奈唑胺耐药基因optrA传播机制研究

Enterococci are important nosocomial pathogens causing a variety of infections. The emergence and rapid spread of multidrug resistant strains especially the vancomycin-resistant enterococci represent a particular challenge. Linezolid is an important alternative for treatment of infections caused by such strains, and linezolid resistance therefore becomes a critical issue. Mutations in 23S rRNA gene and ribosomal proteins and presence of cfr gene are the main mechanisms of linezolid resistance in enterococci. Recently, we identified a novel linezolid resistance gene optrA in enterococci. optrA gene can located either on plasmid or in chromosomal DNA, however, the transmission mechanism of optrA gene remains unclear. Forty-six optrA-positive enterococci were collected from multiple hospitals in the current study. The location of optrA gene will be determined by S1-nuclease digested PFGE and Southern blot hybridization. The characteristics of optrA-carrying plasmid will be investigated by conjugation and transformation experiments, and plasmid typing. The sequence of optrA-carrying plasmid and sequence surrounding optrA gene will be obtained by high-throughput whole-plasmid sequencing and whole-genome sequencing. The structure characteristics of optrA-carrying plasmids, genetic structure surrounding optrA gene, and mobile genetic elements involving the transfer of optrA gene will be analyzed by bioinformatics. This study aims to reveal the transmission mechanism of optrA gene in enterococci and provide the scientific basis for preventing the spread of resistance genes and delaying the development of resistance.

肠球菌是重要的院内感染病原菌，多重耐药尤其是万古霉素耐药菌株的出现和传播给临床抗感染带来很大挑战，利奈唑胺是治疗这类细菌感染的重要选择，因此其耐药问题备受关注。肠球菌对利奈唑胺耐药的主要原因包括23S rRNA基因和核糖体蛋白氨基酸突变以及携带cfr基因。本课题组最近发现一个新型利奈唑胺耐药基因optrA，该基因可定位于质粒或染色体上，然而其传播机制尚不清楚。本项目收集到46株optrA基因阳性的肠球菌，拟通过Southern杂交对optrA进行基因定位；通过接合和转化试验、质粒分型等研究optrA质粒特性；通过全质粒测序和全基因组测序获得optrA质粒序列及optrA基因侧翼序列；采用生物信息学技术研究optrA质粒结构特征、optrA基因所在遗传结构特点以及介导optrA基因转移的可移动元件，阐明optrA基因在肠球菌中的传播机制，为控制耐药基因的播散和延缓耐药性的发展提供科学依据。

肠球菌是引起院内感染重要的病原菌，多重耐药尤其是万古霉素耐药菌株的出现和传播给临床抗感染带来很大挑战，利奈唑胺是治疗这类细菌感染的重要选择，利奈唑胺的耐药问题也因此受到极大关注。2015年课题组报道了一个新型的利奈唑胺耐药基因optrA，本项目通过optrA基因定位、optrA质粒转移能力测定和质粒分型、全基因组测序获取optrA基因侧翼序列、介导optrA基因转移的可移动元件分析等手段对该基因的传播机制进行了研究。研究结果显示，optrA基因可定位于染色体或质粒上，optrA质粒可通过接合试验在不同肠球菌之间传播。OptrA蛋白可分为20种变异体，以EDM（n=40）最为常见，其次为WT（n=23）和RDK（n=20），不同种类肠球菌所携带OptrA变异体的类型有所差异。携带WT、RDK、KLDP、D和KD等变异体的菌株，利奈唑胺MIC值大多为8μg/ml甚至更高。不同的optrA基因变异体其侧翼结构也有所差异，WT型optrA基因大多定位于染色体上，其上游存在fexA基因及Tn554转座子。D、KD、DP、EDP、KLDP和RDK等变异体，optrA基因双侧或单侧存在转录方向相同或相反的IS1216E转座酶基因，反向PCR证实两个同向的IS1216E插入序列可形成含有optrA基因的环状中间体。EDM变异体的侧翼结构变化较大，其上游大多含有araC基因及Tn558转座子，EYDNDM变异体则位于“Tn558-top-mob-araC-optrA”结构中。本研究明确了optrA基因的定位和基因侧翼结构，揭示了IS1216E插入序列可介导optrA基因发生转移，而Tn554和Tn558转座子在optrA基因的传播过程可能发挥了重要作用。