对亚胺培南耐药粘质沙雷菌中质粒介导KPC-2型碳青霉烯酶的研究分析  

Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in an imipenem-resistant Serratia marcescens isolate

作　　者：张嵘[1] 蔡加昌[1] 周宏伟[1] 陈功祥[1] 

机构地区：[1]浙江大学医学院附属第二医院临床检验中心,杭州310009

出　　处：《中华微生物学和免疫学杂志》2007年第8期734-738,共5页Chinese Journal of Microbiology and Immunology

摘　　要：目的研究粘质沙雷菌对亚胺培南耐药的分子机制。方法临床分离到1株亚胺培南耐药的粘质沙雷菌[最小抑菌浓度(MIC)为64μg/ml]。将粘质沙雷菌和大肠杆菌进行接合试验,采用琼脂稀释法检测粘质沙雷菌和接合前后大肠杆菌对药物的MIC;提取粘质沙雷菌和接合后大肠杆菌的酶粗提液进行等电聚焦电泳和三维试验;特异性PCR扩增和DNA序列分析确认引起粘质沙雷菌对亚胺培南耐药的β-内酰胺酶的基因型。结果除碳青霉烯类抗生素外,粘质沙雷菌还对青霉素类、头孢菌素类和单酰胺类等抗生素耐药,对喹诺酮类和氨基糖甙类抗生素敏感;接合试验可使大肠杆菌获得与粘质沙雷菌相似的耐药谱;等电聚焦电泳显示粘质沙雷菌中存在等电点(PI)分别为6.5和6.7的两种β-内酰胺酶,接合后的大肠杆菌存在PI为6.7的β-内酰胺酶;特异性PCR扩增和DNA序列分析证实PI为6.7的β-内酰胺酶为碳青霉烯酶KPC-2,其核苷酸及氨基酸序列已递交到GenBank,PI为6.5的酶有待进一步研究;在一维试验中,克拉维酸、乙二胺四乙酸(EDTA)和氯唑西林均不能抑制KPC-2酶对业胺培南的水解活性。结论首次在粘质沙雷菌中发现碳青霉烯酶KPC-2,该酶是引起粘质沙雷菌对亚胺培南耐药的主要原因。Objective To investigate the molecular mechanism of carbapenem resistance in Serratia marcescens（S, marcescens）. Methods An imipenem-resistant S. marcescens （MIC： 64 μg/ml） was investigat- ed. Antibiotic sensitivity were performed by agar dilution method; conjugation experiments were carried out in mixed broth cultures; the crude β-1actamase extracts of S. marcescens and E. coli transconjugant prepared were subjected to analytical isoelectric focusing（IEF） and three-dimensional tests; specific PCR and DNA sequence analysis were preformed to confirm the β-lactamase type. Results The S. marcescens isolate was resistant to carbapenems, penicillins, cephalosporins and monobactams, but sensitive to quinolones and aminoglycosides ; isoelectric focusing demonstrated two β-lactamases, with PIs of 6.7（KPC-2） and 6.5（unidentified）, respectively; conjugation studies resulted in the transfer of reduced carbapenem sensitivity from S. marcescens. IEF and plasmid profile results of the E. coli transconjugant indicated that the β- lactamase with a PI of 6.7 （ KPC-2 ） was encoded by a conjugative plasmid; the presence of blaKpc.2 gene was confirmed by specific PCR and DNA sequence analysis; three-dimensional test and three-dimensional inhibition test showed that the KPC-2 enzyme activity against imipenem and meropenem was not inhibited in the presence of clavulanic acid, EDTA or cloxacillin. Conclusion It is the first detection of carbapenem-hydrolyzing β-lactamase KPC-2 in S. marcescens. Production of KPC-2 enzyme mainly contributed to reduced imipenem sensitivity of the S. marcescens.

关 键 词：粘质沙雷菌 耐药性 碳青霉烯酶 KPC-2 

分 类 号：R516[医药卫生—内科学]