肺炎克雷伯菌质粒p1512-KPC的测序及比较基因组学分析

Sequencing and comparative genomics analysis of Klebsiella pneumoniae plasmid p1512-KPC

【目的】对多重耐药的肺炎克雷伯菌1512中的质粒p1512-KPC进行测序及比较基因组学的分析。【方法】利用16S rRNA基因测序进行菌种鉴定,根据7对管家基因(gapA、infB、mdh、pgi、phoE、rpoB和tonB)对菌株进行多位点序列分型(Multilocus sequence typing,MLST)。利用PCR进行耐药基因的筛查,通过接合转移实验及电转化实验将质粒转入受体菌大肠杆菌EC600。采用改良Carba NP法检测细菌碳青霉烯酶的活性以及类型,使用VITEK2Compact全自动细菌鉴定及药敏分析仪检测菌株最小抑菌浓度(Minimum inhibitory concentration,MIC)。最后通过高通量测序技术结合生物信息分析手段明确菌株1512及质粒p1512-KPC的耐药基因谱,并通过比较基因组方法对质粒p1512-KPC的基本结构、耐药基因遗传环境及移动元件等结构基因组学特征进行分析。【结果】菌株1512为产A类碳青霉烯酶的多重耐药肺炎克雷伯菌,MLST分型结果显示该菌为ST11型。经PCR筛查,菌株1512包含bla_(KPC-2)、dfrA1和sul1耐药基因,其中bla_(KPC-2)基因位于不可结合转移但电转成功的质粒p1512-KPC上。测序结果显示,质粒p1512-KPC长度为117.69 kb,同时包含IncFII型复制子和属于Rep_3家族但类型未知的复制子repB,并携带耐药基因bla_(KPC-2)、bla_(CTX-M-65)、bla_(TEM-1)及rmtB。其中bla_(KPC-2)、bla_(CTX-M-65)及bla_(TEM-1)分别存在于截短的Tn6296、Tn6367及截短的Tn2的基因环境中。【结论】携带bla_(KPC-2)、bla_(CTX-M-65)、bla_(TEM-1)及rmtB基因的质粒p1512-KPC介导了肺炎克雷伯菌1512对青霉素类、头孢菌素类、碳青霉烯类、单环β-内酰胺类及氨基糖苷类抗生素耐药,并能引起相应耐药基因的水平传播。此外,本研究还对IncFII型复制子和Rep_3家族复制子repB共存的质粒进行了比较基因组分析,为该类型质粒的多样性和进化提供了更深入的理解。

[Objective] Comparative genomics analysis of the fully sequenced plasmid p1512-KPC from the multi-drug resistant clinical Klebsiella pneumoniae 1512 isolate. [Methods] We used 16S rRNA gene sequencing to identify the bacterium. Seven housekeeping genes（gapA, infB, mdh, pgi, phoE, rpoB, and tonB） were used for the multilocus sequence typing（MLST） scheme. Resistance genes were screened by PCR amplification. The plasmid was transferred into recipient Escherichia coli EC600 using conjugal transfers and electroporation experiments. The activity and type of carbapenemase were detected using Carba NP, and the minimum inhibitory concentration（MIC） was determined using VITEK 2 compact system. Sequencing and bioinformatics analysis were used to characterize the plasmid p1512-KPC. [Results] The 1512 isolate was an ST11 multi-drug resistant K. pneumoniae strain showing Ambler class A carbapenemase. PCR demonstrated strain 1512 harbored blaKPC-2, dfrA1 and sul1 genes. The blaKPC-2 gene was located in non-self-transmissible plasmid p1512-KPC. Sequencing and bioinformatics analysis revealed that p1512-KPC, 117.69 kb in length, harbored IncFII replicon and replicon repB belonging to Rep3 family unknown incompatibility groups. In addition, p1512-KPC contained blaKPC-2, blaCTX-M-65, blaTEM-1and rmtB genes, among which blaKPC-2, blaCTX-M-65 and blaTEM-1 were carried by ΔTn6296, Tn6367 and ΔTn2 respectively. [Conclusion] The resistance of K. pneumoniae 1512 to penicillin, cephalosporin, carbapenem, monobactam and aminoglycoside were mediated by non-self-transmissible plasmid p1512-KPC. Moreover, the comparative genomics analysis provided a deeper insight into the diversification and evolution of those plasmids that harbored IncFII replicon and replicon repB belonging to Rep3 family unknown incompatibility groups.