IncFII-FIA-FIB型多重耐药质粒pBTR-CTXM的结构基因组学分析

Structural genomics of IncFII-FIA-FIB multidrug resistant plasmid pBTR-CTXM

摘要:

【背景】IncFII-FIA-FIB型质粒广泛存在于肠杆菌科细菌中，介导了许多耐药基因的水平转移，并导致细菌多重耐药问题日益严重。【目的】分析IncFII-FIA-FIB型多重耐药质粒pBTR-CTXM的基因组结构，并研究其介导大肠杆菌BTR株的耐药基因水平转移机制。【方法】利用PCR进行耐药基因筛查；接合转移和电转化实验验证质粒pBTR-CTXM是否具备自主接合转移的特性；VITEK 2 Compact全自动细菌鉴定及药敏分析仪测定相关菌株对抗生素的药物敏感性；构建Mate Pair文库并进行细菌全基因组高通量测序和质粒结构基因组学分析。【结果】菌株BTR是携带blaNDM-1、blaCTX-M-15、blaTEM、qnrD、qnrS1、mph(A)、erm(B)和tetA(B)等耐药基因的多重耐药大肠杆菌，其中blaCTX-M-15、mph(A)、erm(B)和tetA(B)等耐药基因均位于大小为144 939 bp的质粒pBTR-CTXM (GenBank登录号MF156697)上，该质粒可与菌株BTR内质粒pNDM-BTR接合共转移到受体菌大肠杆菌EC600中。pBTR-CTXM具备IncFII-FIA-FIB型质粒典型的骨架区结构，其多重耐药(Multidrug-resistant，MDR)区由新的复合型转座子Tn6492、Tn2残余、Tn10残余、ISEcp1-blaCTX-M-15-Δorf477转座单元和一些插入序列组成。【结论】pBTR-CTXM中新复合型转座子Tn6492与Tn10残余和ISEcp1-blaCTX-M-15-Δorf477转座单元共同介导大肠杆菌BTR株的多重耐药与耐药基因的水平传播。

Abstract:

[Background] The IncFII-FIA-FIB incompatibility group plasmids are widely encountered in Enterobacteriaceae species. They mediate the horizontal transfer of many resistance genes and lead to the upsurge of multidrug-resistant strains. [Objective] To investigate the genomic characterization of the multidrug-resistant plasmid pBTR-CTXM assigned into IncFII-FIA-FIB incompatibility group and the plasmid-mediated horizontal transfer mechanism of resistance genes of Escherichia coli BTR. [Methods] The screening of antibiotic resistance genes was determined using PCR. The transferability of plasmid pBTR-CTXM was confirmed by conjugation experiments and electroporation experiments. The minimal inhibitory concentration (MIC) values were tested by VITEK 2 Compact system. The complete nucleotide sequence of pBTR-CTXM was determined using next-generation sequencing technology from a mate pair library. Structural genomics of pBTR-CTXM was analyzed subsequently. [Results] The multidrug-resistant E. coli BTR isolate harbored the blaNDM-1, blaCTX-M-15, blaTEM, qnrD, qnrS1, mph(A), erm(B), and tetA(B) genes. The blaCTX-M-15, mph(A), erm(B), and tetA(B) genes were located on pBTR-CTXM (GenBank accession number MF156697) with 144 939 bp in length. The pBTR-CTXM could be conjugatively mobilized to the recipient strain E. coli EC600 by pNDM-BTR, a conjugative plasmid existed in the E. coli BTR. pBTR-CTXM possessed typical backbones of IncFII-FIA-FIB plasmids and a multidrug-resistant (MDR) region, which was comprised of a novel composite transposon Tn6492, the Tn2 remnant, the Tn10 remnant, the ISEcp1-blaCTX-M-15-Δorf477 unit and some insertion sequences (IS) elements. [Conclusion] The novel composite transposon Tn6492, the Tn10 remnant, and the ISEcp1-blaCTX-M-15-Δorf477 unit mediated the horizontal transfer of resistance genes and the antibiotic resistance of E. coli BTR.