黏质沙雷菌携带KPC-2型碳青霉烯酶的相关质粒特征分析

Characteristics of plasmids in KPC-2-producing Serratia marcescens

目的分析黏质沙雷菌携带KPC-2型碳青霉烯酶的相关质粒特征。方法4株碳青霉烯类抗生素耐药的黏质沙雷菌来自2016年我院肝胆病区的4位患者,使用仪器BD Phenix-100鉴定菌株并测定最小抑菌浓度(MIC),脉冲场凝胶电泳分析菌株之间的同源性,改良Hodge试验检测碳青霉烯酶表型,PCR及产物测序检测基因型,接合试验检测质粒的转移性,质粒全基因组测序后比对,并分析其特征,使用软件DNAMAN比对复制起始蛋白氨基酸序列,软件MEGA7.0 Neighobor-Joining法构建系统发育树。结果4株黏质沙雷菌对碳青霉烯类抗生素均表现耐药,菌株具有同源性,Hodge试验为阳性,碳青霉烯酶基因型为blaKPC-2,接合试验阳性;质粒为42742 bp的环状DNA,具有incX6质粒的类似骨架和相似的复制起始蛋白氨基酸序列,并和incX6具有共同的节点;blaKPC-2处于由Tn3家族转座子、重组酶基因、△ISKpn6和ISkpn27等插入元件构成的耐药核心区。结论未知质粒类型为incX6-like,blaKPC-2位于质粒的△Tn6296转座子上;携带KPC-2的此类质粒的细菌应引起重视。

Objective To analyze the characteristics of plasmids in KPC-2-producing Serratia marcescens(S.marcescens)isolates.Methods Four carbapenem-resistant S.marcescens strains were isolated from four patients admitted to the hepatobiliary ward of Affiliated Cancer Hospital of Zhengzhou University in 2016.BD Phenix-100 was used to identify the strains and detect the minimum inhibitory concentrations(MICs).Homology analysis was performed using pulsed-field gel electrophoresis(PFGE).The modified Hodge test was used to detect the phenotypes of carbapenemase.PCR and gene sequencing were used to detect the types of carbapenem resistance genes.The transferability of plasmids was detected by conjugation test.The characteristics of plasmids were analyzed by genomic alignment method after whole genome sequencing.DNAMAN V9 software was used to compare the amino acid sequences of the replication initiation proteins.A phylogenetic tree was constructed with neighbor-joining method using MEGA7.0.Results All of the four S.marcescens strains were resistant to carbapenem antibiotics.They were highly homologous according to PFGE.Hodge test results were all positive and the carbapenemase genotype was blaKPC-2.Conjugation test results were positive.The plasmid was a circular DNA of 42742 bp in length.It had the similar skeleton of incX6 plasmid and the similar amino acid sequence of replication initiation protein.Moreover,it and incX6 plasmid were at the same node in the phylogenetic tree.The blaKPC-2 was located in the core of drug resistance,which was composed of insertion elements including Tn3 family transposons,recombinant enzyme genes,△ISKpn6 and ISKpn27.Conclusions The plasmid was incX6-like.The blaKPC-2 gene was located in the transposon of△Tn6296.More attention should be paid to the bacteria carrying KPC-2 in incX plasmids.