高毒力肺炎克雷伯菌中KPC-2型碳青霉烯酶对细菌毒力的影响  

Effect of KPC-2 type carbapenemase on the virulence of hypervirulent Klebsiella pneumoniae

作　　者：祝俊英 魏清 沈震 袁挺[1] 李敏[1] ZHU Junying;WEI Qing;SHEN Zhen;YUAN Ting;LI Min(Department of Laboratory Medicine,Renji Hospital,School of Medicine,Shanghai JiaoTong University,Shanghai 200127,China)

机构地区：[1]上海交通大学医学院附属仁济医院检验科,上海200127

出　　处：《中国感染与化疗杂志》2021年第6期697-702,共6页Chinese Journal of Infection and Chemotherapy

基　　金：国家自然科学基金青年项目(81702062);上海市卫生健康委员会科研课题(20204Y0288)。

摘　　要：目的了解高毒力肺炎克雷伯菌获得KPC-2型碳青霉烯酶后对细菌毒力的影响。方法收集3株碳青霉烯类敏感的高毒力肺炎克雷伯菌野生株(hvKP)作为实验菌株并调查其临床信息,采用PCR方法检测其毒力基因、荚膜血清分型及ST分型等。采用过表达实验将blaKPC-2克隆至pHSG396质粒并转化至hvKP野生株构建blaKPC-2转化株,采用血清抵抗试验和生物膜实验比较hvKP野生株和blaKPC-2转化株对血清中杀菌物质的抵抗能力及生物被膜形成能力,低速度离心沉降实验及糖醛酸测定比较二者黏液性及荚膜多糖含量,荧光定量PCR检测荚膜多糖结构基因及调控基因转录水平表达情况。结果3株hvKP野生株中1株为ST23 K1型,所检测的10种毒力基因均为阳性。另外2株均为ST65 K2型,毒力基因rmpA、rmpA2、iroB、iutA、ybtS、entB、mrkD均为阳性,magA、allS及kfu均为阴性。与hvKP野生株相比,blaKPC-2转化株对血清中杀菌物质的抵抗能力明显下降,生物被膜形成能力明显下降,细菌黏液性下降,荚膜多糖含量下降,荚膜多糖结构基因及调控基因转录水平下降。结论hvKP野生株获得KPC-2型碳青霉烯酶后可导致细菌荚膜多糖产生减少,可能导致细菌毒力降低。Objective We aimed to characterize the effect of blaKPC-2 type carbapenemase on the virulence of hypervirulent Klebsiella pneumoniae(hvKP).Methods A total of 3 strains of carbapenem-sensitive hvKP were selected for experiment.The clinical data of these strains was investigated.All the 3 isolates were assessed for virulence genes,capsular serotype,and ST types by PCR.blaKPC-2 transformants were developed by overexpression of blaKPC-2 and transformed into hvKP.Serum resistance and biofilm assays were performed to compare hvKP and blaKPC-2 transformants.Low-speed centrifugation and standard curve of glucuronic acid were used to measure mucoviscosity and capsular polysaccharide(CPS)quantification.Transcriptional level of CPS structural genes and regulatory genes were assessed by real-time PCR.Results One hvKP isolate belonged to ST23,K1 type and positive for all the 10 virulence genes.The other 2 isolates were both ST65 and K2 type,and positive for virulence genes rmpA,rmpA2,iroB,iutA,ybtS,entB,mrkD,but negative for magA,allS,and kfu genes.Compared to hvKP parental strains,blaKPC-2 transformants exhibited markedly reduced resistance to the bactericidal substance in normal human serum and smaller amounts of biofilm formation.Furthermore,blaKPC-2 transformants showed reduced mucoviscosity and CPS production.RT-PCR results showed reduced expression of CPS structural genes and regulatory genes.Conclusions Acquisition of blaKPC-2 leads to reduced CPS production and impaired virulence.

关 键 词：高毒力肺炎克雷伯菌 毒力 KPC-2型碳青霉烯酶 荚膜多糖 

分 类 号：R378.996[医药卫生—病原生物学]