耐第三代头孢菌素革兰阴性杆菌超广谱β-内酰胺酶及耐药基因分析

Enzyme Activities and Encoding Genes of Extended-spectrum β-Lactamases from Clinical Isolates of Gram-negative Bacilli

目的了解革兰阴性杆菌对第三代头孢菌素耐药的分子机制。方法采用常规琼脂2倍稀释法测定11株(CR01-CR11)临床分离革兰阴性杆菌,对13种β-内酰胺类抗菌药物的敏感性,纸片扩散法检测耐药菌的ESBLs表型,通过PCR扩增检测其耐药基因。结果MIC结果表明,11株病原菌对几种常见的第三代头孢菌素具有明显的耐药性;表型确证实验表明,除CR07外,其余10株均为产ESBLs菌株;酶的水解底物轮廓实验表明,10株产ESBLs株菌所产生的β-内酰胺酶均能高效水解头孢哌酮和头孢孟多,而对头孢他啶水解活性较低。结论10株产ESBLs革兰阴性杆菌的酶活性、等电点及耐药基因在一定程度上揭示了病原菌产ESBLs与其耐药性之间的关系。

OBJECTIVE To study the molecular mechanism of the drug-resistance of Gram-negative bacilli to the third generation of cephalosporins.METHODS MICs of 13 β-lactams to the eleven Gram-negative bacilli clinical isolates were detected with the standard agar dilution technique.K-B disc confirmatory method was conducted to determine the ESBLs phenotype of the eleven isolates.The ESBLs encoding genes were analyzed by using PCR.RESULTS The eleven isolates were all resistant to the third generation of cephalosporins（MIC≥64 μg/ml）.Disk confirmatory test showed that 10 isolates produced ESBLs.The hydrolytic activity of the ESBLs from the 10 isolates to cefoperazone and cefamandole was very high.However,the hydrolytic activity of the ESBLs from the 10 isolates to ceftazidime was very low.CONCLUSIONS The enzyme activities and the genes of extended-spectrum β-lactamases from 10 Gram-negative bacilli clinical isolates are preliminarily analyzed.These results provide the basis for further study on the molecular mechanism of the drug-resistence of Gram-negative bacilli.