1株鹑鸡肠球菌产ESBLs和AmpC酶的基因型鉴定

为检测1株临床分离的七彩鸟鹑鸡肠球菌的ESBLs和AmpC酶的基因型,探讨其耐药基因的进化机制;采用两倍稀释法测定此菌株对18种常用药物的敏感性,并用9对特异性引物进行PCR扩增、基因克隆及测序分析,确定鹑鸡肠球菌ESBLs和AmpC酶的基因型及基因亚型.结果表明,鹑鸡肠球菌为产ESBLs和AmpC酶菌,除对3代、4代头孢,碳青霉烯类和磷霉素体外敏感外,对其他常用药物均呈现耐药,具有多重耐药特性;该菌所产 ES-BLs的TEM序列与AJ847364(TEM-116)序列相比发生了2处碱基突变,即512T→A和695A→C,引起了相应氨基酸的突变171Ile→Lys、232Lys→Thr,是一种新的TEM亚型,GenBank注册号DQ849329;AmpC酶与序列EF078894(ACT-like型)有97%的同源性,GenBank登录号是DQ849330.这表明该株鹑鸡肠球菌ESBLs为一种新TEM亚型,来源于同时分离的鹑鸡克雷伯菌TEM型的ESBLs;ACT型AmpC酶的存在可能与七彩鸟鹑鸡接触过β-内酰胺类药物有关.

Genotype identifications of ESBLs and AmpC β-Iactamase of a Enterococcus gallinarum isolated from chloebia gouidiae

The genotypes of extended spectrum β-laetamases(ESBLs) and AmpC β-lactamases produced by Enterococcus gallinarum isolated from chloebia gouldiae were determined to elucidate the evolution mechanism of the resistant genes.The minimum inhibitory concentrations (MICs) of 18 antibacterial drugs against the Enterococcus gallinarum were detected with two dilution method,and the ESBLs and AmpC β-lactamases from the bacterium were amplified by PCR using the primers of TEM,SHV,CTX-M,ACC,CIT,DHA,EBC,FOX and MOX,respectively.The PCR products were cloned and then the cloned fragments were sequenced to identify their genotypes and subtypes.The bacterium was proved to be a ESBL-producing and AmpC β-lactamase-producing bacterium,showing severe resistant to the other drugs,except the third and forth cephalosporins,carbopenems and fosfomycin.Compared with that of AJ847364 (TEM-116),the sequence of the TEM-type was characterized by two nucleotide mutations (512T→A and 695A→C),which led to two mutations of amino acids(17111e→Lys and 232Lys→Thr),showing that the detected TEM-type was a new genotype,the sequence of the AmpC β-lactamase was similar to that of EF078894 (ACT-like type)with a 97% homology.The genotype of ESBLs of Enterococcus gallinarum was a new TEM-type derived from the TEM-type ESBLs of klebstella pneumoniae isolated from the same avian.The genotype of AmpC lactamase was ACT-type,which probably concerned with β-1actam antibiotics used.