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    大肠埃希菌质粒编码的blaKPC-2基因介导碳青霉烯类药物低水平耐药的分子机制

    Molecular mechanism of plasmid-encoded blaKPC-2 gene mediating low-level resistance to carbapenems in Escherichia coli

      摘要
    • 摘要:
      目的 明确碳青霉烯酶编码质粒p21QH43K-KPC从肺炎克雷菌Klebsiella pneumoniae转移到大肠埃希菌Escherichia coli后介导碳青霉烯类药物低水平耐药的分子机制。
      方法 采用构建EZ-Tn5转座子突变文库、基因敲除、转录组测序和全基因组测序(WGS)等手段，探究blaKPC-2基因在大肠埃希菌中对美罗培南产生低水平耐药的分子机制。
      结果 通过构建接合子EC600/p21QH43K-KPC的EZ-Tn5转座子突变文库，获得1株对美罗培南耐药水平增强的转座子突变株EC600/p21QH43K-KPC-130(MIC=8.000 mg/L)。WGS结果和Mauve分析显示，该转座子插入到染色体ompR基因，同时发现了2个点突变基因ltrA和iron。基因敲除发现，只有ompR缺失突变体对碳青霉烯的敏感性降低，并且可以通过基因回补恢复这一表型。
      结论 质粒编码的blaKPC-2基因在大肠埃希菌中介导碳青霉烯类药物低水平耐药与ompR对blaKPC-2基因的调控有关。

       

      Abstract:
      Objective To elucidate the molecular mechanism by which the carbapenemase-encoding plasmid p21QH43K-KPC is transferred from Klebsiella pneumoniae to Escherichia coli, and mediates low-level resistance to carbapenem drugs in E. coli.
      Method The construction of EZ-Tn5 transposon mutation library, CRISPR-Cas9-mediated gene knockout, transcriptome sequencing and whole genome sequencing (WGS) were used to determine the regulatory mechanism of low-level resistance to meropenem mediated by the blaKPC-2 gene in E. coli.
      Result A transposon mutant EC600/p21QH43K-KPC-130 with enhanced meropenem resistance (MIC=8.000 mg/L) was obtained by constructing the EZ-Tn5 transposon mutation library of the EC600/p21QH43K-KPC conjugon. WGS and Mauve analysis revealed that the transposon had inserted into one chromosomal gene ompR, and two point mutation genes of iron and ltrA were found. However, through gene knockout, only ompR deletion mutants exhibited reduced sensitivity to carbapenem that could be restored by gene complementation.
      Conclusion The molecular mechanism of the blaKPC-2 gene encoded on the plasmid mediating low-level resistance to carbapenems in E. coli is related to the regulation of blaKPC-2 gene by ompR.

       

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