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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 study the molecular mechanism of drug resistance to imipenem in Serratia marcescens. Methods One imipenem-resistant S. marcescens was isolated clinically [minimum inhibitory concentration (MIC): 64 μg / ml]. Serratia marcescens and Escherichia coli conjugation test, the use of agar dilution method to detect Serratia marcescens and E. coli before and after the drug MIC; extracted Serratia marcescens and E. coli crude enzyme conjugate after the merger Isoelectric focusing electrophoresis and three-dimensional experiments; specific PCR amplification and DNA sequence analysis confirmed the genotype of β-lactamase that caused serine resistance to imipenem. Results In addition to carbapenem antibiotics, serosuramin also resistant to penicillins, cephalosporins and monoamides and other antibiotics, quinolones and aminoglycoside antibiotics sensitive; joint test allows E. coli The results of isoelectric focusing electrophoresis showed that there were two kinds of β-lactamases in isolates of Serratia marcescens with isoelectric point (PI) of 6.5 and 6.7, respectively. The conjugate Escherichia coli There was a β-lactamase with a PI of 6.7; specific PCR amplification and DNA sequence analysis confirmed that the β-lactamase with a PI of 6.7 is carbapenemase KPC-2, whose nucleotide and amino acid sequences have been submitted to GenBank, PI 6.5 of the enzyme to be further studied; in one-dimensional test, clavulanic acid, ethylenediaminetetraacetic acid (EDTA) and cloxacillin can not inhibit the KPC-2 enzyme Jiepeipan hydrolysis activity. Conclusion For the first time, carbapenemase KPC-2 was found in Serratia marcescens, which is the main reason for the resistance of Serratia marcescens to imipenem.