广谱β-内酰胺酶TEM-141的克隆及特性研究

Cloning and Characterization of ESBLs TEM-141

目的:对临床分离阴沟肠杆菌株EC002所产TEM型β-内酰胺酶进行克隆及原核表达,并了解其相关特性。方法:以琼脂二倍稀释法检测阴沟肠杆菌EC002的耐药情况,以双纸片法筛选及确证试验检测超广谱β-内酰胺酶(ESBLs),以等电聚焦电泳(IEF)法测定酶的等电点(pI),以聚合酶链反应(PCR)扩增酶编码基因并将TEM型β-内酰胺酶基因进行原核表达及表型鉴定。结果:阴沟肠杆菌株EC002对所试多数β-内酰胺类抗菌药物耐药,ESBLs表型鉴定及质粒接合试验结果均为阳性。IEF显示该菌产2种pI分别为8.7及5.4的β-内酰胺酶,测序表明其为CTX-M-22及一种新的TEM亚型,其克隆菌株所表达酶的表型为非ESBLs。该酶的编码基因已被GenBank命名为TEM-141。结论:临床分离阴沟肠杆菌株EC002所产TEM-141为一种新型质粒介导的广谱β-内酰胺酶。

OBJECTIVE： To clone, prokaryotic express and characterize the TEM - type β-lactamase produced by Enterobacter cloacae clinical isolate EC002. METHODS： The drug susceptibility of Enterobacter cloacae clinical isolate EC002 was detected by agar double dilution, double disk screening and confirmatory test were employed to detect the ESBLs. The isoelectric point （pI） of enzyme was detected by isoelectric focusing electrophoresis （IEF）, the genes were coded by PCR amplification enzyme, and the prokaryotic expression and phenotype of the TEM-type β- lactamase were detected. RESULTS ： Enterobacter cloacae EC002 were resistant to most of the β- lactamases. Positive results were noted for the phenotype identification and plasmid conjugation test. IEF showed that Enterobacter cloacae EC002 produced two β-lactamases with pI value at 8.7 and 5.4 respectively, which were confirmed to be CTX - M - 22 and a new TEM - subtype β-lactamase by DNA sequencing, and the phenotype of the expressed enzyme of the cloned strains was non - ESBLs. The TEM- type β-lactamase was named as TEM-141 by GenBank. CONCLUSION： The TEM-141 produced by Enterobacter cloacae EC002 was a new type of plasmid- mediated broad-spectrum β-lactamase.