携带blaKPC-3基因肺炎克雷伯菌新生儿分离株的研究

Study on characteristics of a blaKPC-3-harboring Klebsiella pneumonia clinical isolate recovered from a neonate

目的探讨肺炎克雷伯菌新生儿分离株QD167对碳青霉烯酶类抗菌药物耐药的分子机制。方法菌株QD167的鉴定和药敏采用梅里埃VITEK 2Compact自动系统。通过PCR扩增和产物测序获得菌株的多位点序列和携带的碳青霉烯酶耐药基因。质粒液相接合和S1-PFGE检测菌株携带的质粒特性。基于二代Illumina MiSeq和三代MinION平台获得接合子携带的质粒序列,使用BLASTN等软件分析质粒的耐药基因和移动元件。结果肺炎克雷伯菌QD167株携带blaKPC-3基因,多位点序列属于ST1型。另外,该菌能把携带blaKPC-3基因的质粒转移到大肠埃希菌J53Azi R而获得接合子QD167C,将其携带的质粒命名为pKPC-167。通过高通量测序获得了该质粒的全序列(GenBank:MH236906)。生物信息分析显示pKPC-167质粒大小为43333bp,属于IncX6型,携带有blaKPC-3和blaTEM-1基因。结论研究表明,肺炎克雷伯菌QD167株对碳青霉烯酶类抗菌药物耐药与其携带blaKPC-3基因有关。经检索,在ST1型肺炎克雷伯菌中检测到携带blaKPC-3和blaTEM-1基因的IncX6型质粒属于国内外首次报道。

Objective To explore the molecular mechanisms of carbapenemase-resistant Klebsiella pneumonia QD167strain isolated from a neonate in a university hospital in China.Methods Identification and antimicrobial susceptibility testing were determined using the VITEK-2Compact automated system.The genes encoding cabapenemases and multilocus sequence typing(MLST)were performed by polymerase chain reaction(PCR)and sequencing.Conjugation experiment of plasmid and S1-PFGE were performed to detect the transferability of plasmid.The sequence of plasmid in transconjugant QD167Cwas conducted using the Illumina MiSeq and Nanopore MinION technology.The resistance genes and mobile elements of the plasmid were carried out by using BLASTN and other softwares.Results PCR and sequencing demonstrated the presence of blaKPC-3genes in a ST1 Klebsiella pneumonia QD167strain.Conjugation experiments indicated that plasmid harboring blaKPC-3gene was transferable to E.coli J53Azi R.The complete genome sequence of plasmid in transconjugant QD167Cstrain was obtained by using the MiSeq and MinION platform and this plasmid was named as pKPC-167(GenBank Accession No.MH236906).Bioinformatics analysis showed that the pKPC-167,of 43333bp in size,is a IncX6plasmid harboring ablaKPC-3and a blaTEM-1gene.Conclusion Our results revealed that the Klebsiella pneumonia QD167strain was resistant to carbapenemase because it harbored the blaKPC-3 gene.To the best of our knowledge,this is the first report detecting a IncX6plasmid harboring ablaKPC-3and a blaTEM-1gene in a K.pneumoniae ST1strain in the world.