耐碳青霉烯肺炎克雷伯菌基因检测及其同源性分析

Gene detection and homology analysis of carbapenem-resistant Klebsiella pneumoniae

目的探讨临床分离的肺炎克雷伯菌对碳青霉烯耐药机制,并进行同源性分析研究。方法用软件WHONET 5.6对连云港第一人民医院临床分离标本筛选出29株耐碳青霉烯类肺炎克雷伯菌进行耐药率统计分析;采用表型筛选试验对试验菌进行产A、B类碳青霉烯酶筛选;PCR法扩增确认相应的碳青霉烯酶酶型基因,目的产物经基因测序和BLAST网上比对确定其基因型;采用脉冲场凝胶电泳(PFGE)分析29株试验菌的同源性。选取其中6株菌作多位点序列分型(MLST)。结果 29株耐碳青霉烯肺炎克雷伯菌对常用抗菌药物均表现出高耐药性;碳青霉烯酶表型筛选试验全部呈阳性,28株测出相应的A、B类碳青霉烯酶型,另一株未测出酶型;28株筛选试验测出A、B类酶型的菌株中26株产A类碳青霉烯酶,经PCR确定酶基因型均为KPC-2型,2株产B类金属酶,经PCR确定一株基因型为NDM-1,一株基因型为VIM-2,未测出酶型的一株菌未扩增出A、B类相关基因;29株试验菌PFGE分型结果分为3群,26株产KPC-2型碳青霉烯酶的菌株为同一群,其中17株属于同一型。选取的6株做MLST分型的菌株,MLST分型结果均属于ST11型。结论连云港第一人民医院分离的耐碳青霉烯肺炎克雷伯菌主要机制为产碳青霉烯酶,以产KPC-2型碳青霉烯酶为主,同时有VIM-2和NDM-1型碳青霉烯酶的检出。PFGE和MLST分析结果表明:该院存在着耐碳青霉烯肺炎克雷伯菌的克隆传播,临床上应严格做好耐药菌的防控工作,避免耐碳青霉烯肺炎克雷伯菌医院感染暴发流行。

Objective To explore the carbapenem-resistant mechanisms of Klebsiella pneumoniae derived from clinical specimens and to perform homologous analysis. Methods A total of 29 strains of carbapenem-resistant Klebsiella pneumoniae （CRKP） were screened in the First People＇s Hospital of Lianyungang using WHONET5,6 software, and their resistance rates were statistically analyzed. The screening of A- and B- class carbapenemase phenotypes was performed by phenotypic screening test. The corresponding carbapenemase genes were amplified by PCR, the desired target products were sequenced, and were then compared with BLAST in Gen Bank to determine their genotypes. The homology of the 29 strains was analyzed using pulsed field gel electrophoresis （PFGE） and six strains of them were selected by multiple loci sequence classification （MLST）. Results All of the 29 strains of CRKP showed high resistance to commonly used antimicrobial agents. All of them were positive for the carbapenemase phenotypic screening test. Twenty-eight strains were detected as the corresponding A- and B- class carbapenemase types, and the left one was not measured. The related positive desired products were all amplified by PCR. Twenty- six strains producing carbapenemase of class A were determined to contain KPC-2 gene, and two strains producing metal enzymes of class B were determined to contain NDM-1 gene and VIM-2 gene, respectively. Another strain through class A and B carbapenemase phenotypic screening tests exhibited negative without related genes detected. PFGE classification showed three groups, 26 strains producing carbapenemase of class A belonged to the same group, and 17 of them belonged to the identical belt type. MLST classification for the selected 6 strains showed they were all ST11 type. Conclusion Producing carbapenemase was the major mechanisms of carbapenem-resistant Klebsiella pneumoniae in the First People＇s Hospital of Lianyungang, which mainly produced KPC-2 gene, and meanwhile, VIM-2 and NDM 1 type carbapenemase were detected. Results from PFGE and MLST showed that there was clone propagation of carbapenem-resistant Klebsiella pneumoniae, and the prevention and control of drug-resistant bacteria should be implemented strictly by the clinical practice to prevent infection outbreaks in the First People＇s Hospital of Lianyungang.