肠杆菌科细菌中质粒介导的KPC-2型碳青霉烯酶的检测

Identification of plasmid-mediated carbapenem-hydrolyzing β-lactamase KPC-2 in Enterobacteriaceae

目的研究重症监护病房（ICU）出现的碳青霉烯耐药的黏质沙雷菌、肺炎克雷伯菌和大肠埃希菌等肠杆菌科细菌的分子流行病学及耐药机制。方法2006年4月-2007年2月，从我院2个ICU病房分离到对碳青霉烯耐药或敏感性降低的21株黏质沙雷菌、10株肺炎克雷伯菌和1株大肠埃希菌。用脉冲场凝胶电泳（PFGE）和肠杆菌基因间重复性一致序列-PCR（ERIC-PCR）分析这些菌株的分子流行病学。用接合试验、质粒消除试验、质粒图谱分析、等电聚焦电泳（IEF）、特异性PCR扩增和序列分析以及外膜蛋白分析等技术研究细菌对碳青霉烯耐药的分子机制。结果21株黏质沙雷菌为同一克隆株；10株肺炎克雷伯菌亲缘关系相近。亚胺培南和美罗培南对黏质沙雷菌、肺炎克雷伯菌和大肠埃希菌的MIC为2-8μg／ml，肺炎克雷伯菌K10为128和256μg／ml。所有菌株接合试验均获得成功，亚胺培南和美罗培南对接合子的MIC由原来的≤0．125μg／ml上升到1-2μg／ml。IEF、PCR扩增和序列分析证实黏质沙雷菌产KPC-2型碳青霉烯酶[等电点（pI）为6．7]和pI为6．5的β内酰胺酶；肺炎克雷伯菌产TEM-1（p15．4）、KPC-2、CTX-M-14（p17．9）和p1为7．3的β内酰胺酶；大肠埃希菌产KPC-2、CTX-M-15（pI9．0）和pI为7．3的β内酰胺酶；所有接合子均只产KPC-2一种β内酰胺酶。所有接合子质粒DNA的EcoRⅠ、HindⅢ和BcuⅠ限制性酶切图谱完全相同。外膜蛋白的十二烷基磺酸钠-聚丙烯酰胺电泳和基因序列分析发现肺炎克雷伯菌K10由于OmpK36基因中存在插入序列ISEcp1而导致OmpK36膜孔蛋白的缺失。结论我院ICU病房出现大量碳青霉烯耐药的黏质沙雷菌、肺炎克雷伯菌和大肠埃希菌；KPC-2是引起这些细菌对碳青霉烯耐药或敏感性降低的主要原因；KPC-2合并膜孔蛋白缺失可导致肺炎克雷伯菌对碳青霉烯高水平耐药；同一种编码blaKPC-2的质粒在3种不同属的细菌之间传播。

Objective To investigate the molecular epidemiology and mechanism of carbapenem resistance of Serratia marcescens, Klebsiella pneumoniae and Escherichia coli isolates from intensive care units （ICUs）. Methods Twenty-one S. marcescens, ten K. pneumoniae and one E. coli isolates with carbapenem resistance or reduced carbapenem susceptibility were recovered from two ICUs in our hospital from April 2006 to February 2007. Pulsed-field gel electrophoresis （PFGE） and enterobacterial repetitive intergenic consensus-PCR （ERIC-PCR） were performed to analyze the molecular epidemiology of isolates. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments were carried out in mixed broth cultures. Plasmid DNA was obtained by using an alkalinelysis technique and was digested by various endonucleases. Ehmination of plasmid from S. marcescens isolates were performed by repeated SDS treatment. The crude β-lactamase extracts of original isolates and E. coli transconjugants were subjected to isoelectrie focusing （IEF） ; Specific PCRs and DNA sequencing were preformed to confirm the genotype of β-lactamases. Results ERIC-PCR indicated that all S. marcescens isolates belonged to a clonal strain. PFGE indicated that ten IC pneumoniae isolates were indistinguishable or closely related to each other. The MICs of imipenem and meropenem for all isolates were 2 to 8 μg/ml except K. pneumoniae K10 （ 128 and 256 μg/ml）. Conjugation studies with E. coli （EC600） resulted in the transfer of reduced carbapenem susceptibility from original isolates （MICs： from ≤0. 125 μg/ml to 1-2 μg/ml）. IEF, PCR and DNA sequence analysis confirmed that S. marcescens isolates produced KPC-2 （ pI of 6. 7 ） and a β-lactamase （ pI 6. 5）. K. pneumoniae isolates produced TEM-1 （ pI 5.4）, KPC-2, CTX-M-14 （ pI 7.9）, and a β- lactamase （pI 7.3）. E. coli E1 produced KPC-2, CTX-M-15 （ pI 9.0）, and a β-lactamase （pI 7.3）. Only a KPC-2 was detected in E. coli transconjugants. Plasmid restriction analysis using EcoR Ⅰ , HindⅢ, and Bcu Ⅰ showed identical restriction patterns among all E. coli transconjugants. SDS-PAGE and ompK 35/36 gene sequence analysis of OMPs revealed that K. pneumoniae K10 failed to express OmpK36 because of insertional inactivation by an insertion of ISEcpl. Conclusions Carbapenem-non-susceptible S. marcescens, K. pneumoniae and E. coli were epidemic in two ICUs in our hospital. Resistance or reduced susceptibility to carbapenems in these strains is mainly due to production of KPC-2. Presence of KPC-2 combined with porin deficiency result in high-level carbapenem resistance in K. pneumoniae. The same blaKPC-2-encoding plasmid spreads among the three different genera.