肺炎克雷伯菌blaCARB-2基因的分布及结构分析

Analyses on distribution and structure of bla_(CARB-2) in Klebsiella pneumoniae

为探讨β-内酰胺酶基因在临床分离肺炎克雷伯菌的分布及其相关可移动遗传元件的结构,本文利用基因组测序、PCR、分子克隆、接合转移和基因组学分析等方法,在对240株临床分离的肺炎克雷伯菌进行混合基因组测序的基础上,着重研究了bla_(CARB-2)等耐药性基因相关可移动遗传元件的结构及其在肺炎克雷伯菌基因组的定位,克隆获得耐药性质粒携带的若干耐药性基因并测定了它们的功能。结果显示,在240株肺炎克雷伯菌中检出11种β-内酰胺酶基因,其中1株肺炎克雷伯菌(KP1276)被检出bla_(CARB-2)基因,阳性率为0.42%(1/240);bla_(CARB-2)位于一个大小为182,450 bp的可接合转移质粒(pKP1276-182),pKP1276-182共编码222个基因,包含7个耐药性基因,分别是bla_(CARB-2)、bla_(KLUC)、aadA1、aadA2、cmlA1、dfrA1和sul2。bla_(CARB-2)是首次在肺炎克雷伯菌中被检出,且bla_(CARB-2)与其他3个耐药性基因一起构成一个新型结构的1型整合子(int-bla_(CARB-2)-aadA2-cmlA1-aadA1);对其中的3个耐药性基因bla_(CARB-2)、aadA2和CmlA1进行了分子克隆和耐药性测定,结果显示它们对相应药物都具有一定的耐药性,其中bla_(CARB-2)对青霉素类β-内酰胺药物具有较高的耐药性。本研究结果表明,bla_(CARB-2)基因已经在肺炎克雷伯菌中出现,其由1型整合子携带编码位于一个可接合转移的质粒上,bla_(CARB-2)基因有可能在相同及不同种属肠杆菌之间进行水平转移,引起耐药性播散。

In order to characterize the structure of the beta-lactamase gene and its corresponding mobile genetic elements in Klebsiella pneumoniae,the beta-lactamase genes from 240 clinical Klebsiella pneumoniae isolates were studied.blaCARB-2,a newly characterized gene,was extensively investigated utilizing next-generation sequencing,PCR,molecular cloning,conjugation,and comparative genomics analysis.We identified 11 beta-lactamase genes among the 240 clinical Klebsiella pneumoniae isolates;the blaCARB-2 gene exists only in one specific isolate(Klebsiella pneumoniae KP1276)(1/240,0.42%).The blaCARB-2 gene lies on a conjugative plasmid pKP1276-82,a 182,450-bp plasmid,which encodes 222 open reading frames.The plasmid has seven resistance genes,termed blaCARB-2,blaKLUC,aadA1,aadA2,cmlA1,dfrA1,and sul2.Among these genes,blaCARB-2 was identified for the first time in Klebsiella pneumoniae.Four of these resistance genes and an int gene form a class 1 integron(int-blaCARB-2-aadA2-cmlA1-aadA1).Further studies show that the blaCARB-2,aadA2,and cmlA1 genes are resistant to their corresponding antibiotics and the blaCARB-2 exhibits higher resistance activities to penicillin beta-lactams.These results reveal the possibility of horizontal transfer of the resistance genes and dissemination of resistance among bacteria of different genera or species of Enterobacteriaceae.