耐碳青霉烯类铜绿假单胞菌第1类整合子可变区启动子检测及同源性分析

Detection of promoters of variable regions for classⅠintegron in carbapenem-resistant Pseudomonas aeruginosa and their homology analysis

目的了解医院耐碳青霉烯类铜绿假单胞菌(CR-PA)中第1类整合子可变区启动子的种类和排列方式,分析其与介导的耐药基因表达之间的关系;并明确整合子阳性菌株的同源性情况。方法运用聚合酶链反应(PCR)、琼脂糖凝胶电泳以及测序的方法,以2012-2014年医院临床分离42株第1类整合子阳性CR-PA为对象,检测其可变区启动子和同源性。结果 42株实验菌株中强启动子PcS为2株、弱启动子PcW为2株,剩余38例皆为杂合1型启动子PcH1;位于Pc下游的P2启动子42例皆为-35到-10区间隔有14个碱基序列的无活性型的启动子;42株CR-PA经肠杆菌基因间重复共有序列PCR(ERIC-PCR)基因分型,主要分为A、B、C、D、E 5型,其中A1型32株,A2型6株,B、C、D和E型各为1株,A型菌株主要是来源于ICU及神经外科。结论本地区临床分离CR-PA中第1类整合子可变区的启动子主要以杂合1型启动子PcH1为主,P2启动子全为无活性型;整合子阳性菌株的基因分型间呈现高度的同源性,可能存在着医院的克隆传播。

OBJECTIVE To understand the types and arrangement of the promoters of variable regions for class Ⅰ integron in carbapenem-resistant Pseudomonas aeruginosa （CR-PA） in hospital,analyze the relationship between the promoters and the expression levels of drug-resistant genes that they mediated,and clear the homology of the integron positive isolates.METHODS A total of 42 strains of class Ⅰ integron in CR-PA isolated from 2012 to 2014 were used as research objects,and polymerase chain reaction（PCR）,agarose gelelectrophoresis and sequencing were used to detect the promoters of variable regions and the homology.RESULTS The analysis of the promoters of variable regions for 42 strains of class Ⅰ integron positive strains showed that two isolates were PcS（strong promoter）,two isolates were PcW（weak promoter）,and the rest of 38 isolates were PcH1 type.The 42 strains of P2 promoters located at the downstream of Pc were no active promoters with 14 bases in the region of-35 to-10.The genotyping results of 42 isolates of CR-PA by ERIC PCR showed that there were mainly five genotypes,including A,B,C,D and E,respectively,which included 32 isolates of type A1,6 isolates of type A2,and one isolate of type B,C,D,and E,respectively.Type A strains were mainly from ICU and neurosurgery department.CONCLUSION In our region,the promoters of variable regions for class Ⅰ integron in carbapenem-resistant P.aeruginosa are mainly PcH1,and P2 promoters were no active types.It has a high geneotyp homology within the class Ⅰ integron positive isolates,and these strains are likely to be clonally spread in the hospital.