弗氏枸橼酸杆菌中发现介导bla_(KPC-2)传播的新型质粒及比较基因组学分析

Emerging and comparative genomic analysis of a novel plasmid carrying bla_(KPC-2) in Citrobacter freundii

目的探讨来自弗氏枸橼酸杆菌的产碳青霉烯酶的新型不相容群组质粒pNY2385-KPC的耐药机制及基因结构特征。方法从宁波市医疗中心李惠利医院急诊病房的发热患者尿液样本中获得1株多药耐药菌。采用16S rRNA基因扩增和平均核苷酸一致性(ANI)进行菌种鉴定;采用全自动细菌鉴定及药敏分析系统检测细菌MIC值;通过“三代”测序方法获得基因组序列,然后通过BLASTP/BLASTN、RefSeq、ConservedDomains、ResFinder、Isfinder等对基因功能进行详细注释及比较基因组学分析。结果弗氏枸橼酸杆菌NY2385携带的质粒pNY2385-KPC为一种新型不相容群组质粒,包含bla_(KPC-2),具有接合转移(Ⅳ型分泌系统)基因,可发生接合转移。NCBI中共检索到同型质粒15个,来源于弗氏枸橼酸杆菌、黏质沙雷菌、大肠埃希菌、阴沟肠杆菌、肺炎克雷伯菌和植生拉乌尔菌等细菌,为泛宿主质粒。通过对来自弗氏枸橼酸杆菌中6个该新型质粒进行序列比较分析,发现该新型质粒结构具有一定的保守性,具有携带bla_(KPC-2)的Tn6296变体结构,而且质粒pCF1807-3同时具有repApNY2385-KPC和repAIncX8。结论弗氏枸橼酸杆菌中的pNY2385-KPC型质粒均携带bla_(KPC-2)耐药基因,分为repApNY2385-KPC单复制子和repApNY2385-KPC/repAIncX8双复制子两种亚型,属于泛宿主质粒,作为可移动遗传元件促进了bla_(KPC-2)的传播。

Objective To explore the drug resistance mechanism and gene structure characteristics of a carbapenemase-producing novel incompatibility group plasmid pNY2385-KPC from Citrobacterfreundii.Methods A multi-drug resistant strain was obtained from urine samples of patients with fever in the emergency ward of Li Huili Hospital,Ningbo Medical Center.Bacterial species was preliminary identified and finally confirmed by 16S rRNA gene amplification and the average nucleotide identity alignment,respectively.The minimum inhibitory concentrations of the antimicrobial agents were determined by VITEK 2 Compact System.The complete genome sequence was obtained by"third-generation"sequencing methods,and then detailed annotation of gene function and comparative genomic analysis of plasmid structure were carried out by BLASTP/BLASTN,RefSeq,ConservedDomains,ResFinder,Isfinder,etc.Results The pNY2385-KPC carried by citrobacter freundii NY2385 belonged a novel incompatibility group,and contained bla_(KPC-2) and conjugative transfer(typeⅣsecretory system,T4SS)genes,which could induce conjugative transfer.A total of 15 plasmids of the same type as pNY2385-KPC were retrieved by NCBI,which were from Citrobacterfreundii,and the rest were from Serratia marcescens,Escherichia coli,Enterobacter cloacae,Klebsiella pneumoniae,Raoultella planticola and other bacteria,and were broad-host-range plasmids.The sequence comparative analysis of all 6 of the novel plasmid from Citrobacterfreundii showed that the structure of the novel plasmid had certain conserved property,with Tn6296 variant structure carrying bla_(KPC-2),and plasmid pCF1807-3 had both repApNY2385-KPC and repAIncX8.Conclusion The pNY2385-KPC type plasmids in Citrobacterfreundii carried bla_(KPC-2) resistance gene,which were divided into two subtypes:repApNY2385-KPC single replicator and repApNY2385-KPC/repAIncX8 complex replicator,belonging to broad-host-range plasmids.And as a mobile genetic element,the plasmids promote the spread of bla_(KPC-2).