儿童产IMP-4及KPC-2型碳氢霉烯酶肺炎克雷伯菌分子流行病学分析

Molecular epidemiological analysis of KPC-2 and IMP-4 carbapenemase possessing Klebsiella pneumonia isolated from children

目的调查碳氢霉烯类非敏感肺炎克雷伯菌中获得性碳氢霉烯酶的分布情况，探讨其在医院感染流行病学中的作用。方法收集2008年11月至2009年3月武汉市儿童医院住院患儿临床分离的非重复的碳青霉烯类抗生素非敏感肺炎克雷伯菌20株，使用生物梅里埃公司生产的GNS．142检测菌株的MIC值，PFGE技术分析耐药菌株间的同源性，改良的Hodge试验筛选产碳青霉烯酶阳性菌株，亚胺培南-EDTA，美罗培南-EDTA及头孢他啶-EDTA协同试验筛选产金属酶菌株，PCR扩增常见获得性碳氢霉烯酶及整合酶基因并进行基因测序，质粒接合转移试验研究细菌耐药的传播方式和Southem杂交定位耐药基因。结果PFGE共检出4种细菌基因型，包括A型5株（A1型3株、A2型2株）、B型2株、C型12株、D型1株。A型及C型为主要克隆株。8株细菌同时携带KPC-及IMP-4两种碳氢霉烯酶基因，10株只携带IMP-4基因，2株只携带KPC-2基因。未检出NDM-1、G1M、SPM、SIM、OXA-23、VIM型碳氢霉烯酶基因。所有20株菌株均携带Intl基因，Southern杂交提示Intl及IMP-4基因均定位于染色体。结论IMP-4及KPC-2基因是武汉地区肺炎克雷伯菌中最主要的获得性碳氢霉烯酶基因，Intl介导1MP-4耐药基因的水平传播及C型耐药克隆株在武汉市儿童医院临床多个科室间的传播是肺炎克雷伯菌碳青霉烯类耐药性传播的主要方式。

Objective To investigate the distribution of acquired carbapenemases in carbapenem- resistant strains of Klebsiella pneumon/ae, and explore its role in epidemiology of nosocomial infection. Methods From November 2008 to March 2009, twenty clinical isolates of carbapenem-resistant Klebsiella pneumoniae were collected from children hospitalized in Wuhan children＇s hospital. MICs of antibiotics were tested by using the bioM^rieux company＇s GNS-142 cards. PFGE was performed to analyze the homogeneity of genomic DNA of Klebsiella pneumoniae. Modified Hodge test was used to screen strains producing carbapenemases, combined imipenem （IPM）-EDTA , meropenem （MEM）- EDTA and ceftazidime （CAZ）- EDTA double-disk synergy test （DDST） were used to detect metallo-13-1actamase-producing. PCR amplification of the carbapenemase and integrase genes, and sequencing were performed. Plasmid conjugation transfer experiments and Southern hybridization were applied to study the mode of drug resistance transmission~ Results Four types of Klebsiella pneumoniae were detected by PFGE, type A consisted of 5 strains, including 3 strains of typeA1 and2 strains of type A2）, type B （2 strains）, type C （12strains） and type D （ 1 strain）. Type A and C were the main drug resistant clones. Eight strains of Klebsiella pneumoniae carried both KPC-2 and IMP-4 genes, 10 strains carried IMP-4 gene, 2 strains carried KPC-2 gene. None of NDM- 1, GIM, SPM, SIM, 0XA-23, and VIM carbapenemase genes was detected in 20 isolates. All of 20 isolates carried lntl which were found to be located on bacterial chromosome by Southern blot. Conclusions KPC-2 and IMP-4 genes are the major carbapenemase genes in Klebsiella pneumoniae isolated in Wuhan.Transmission of drug resistance is mainly through vertical transmission of type C resistant clone and horizontal transmission of lntl on bacteria chromosome.