鸡大肠杆菌TEM和CTX-M型超广谱β-内酰胺酶基因分型研究

 【目的】检测鸡大肠杆菌超广谱β-内酰胺酶(ESBLs)的基因型和基因亚型,了解河南省产酶鸡大肠杆菌ESBLs基因型分布。【方法】 对河南省7个地区不同鸡场临床分离28株产ESBLs鸡大肠杆菌分别用TEM、SHV和CTX-M等3种通用引物进行PCR扩增及测序分析,确定其基因型和基因亚型。【结果】 21株鸡大肠杆菌检出TEM型基因,2株检出CTX-M型基因,5株检出TEM型和CTX-M型两种基因,未检出SHV型。TEM基因亚型以TEM-1变异型为主,与AY293072(TEM-1)相比同源性为98%—99%,核苷酸序列除发生18T→C沉默突变外,尚有3株分别有另一处碱基发生变化：783G→A(C3菌,序列号为FJ405207)、21T→A(X2菌,序列号为FJ405208)和269G→A(F4菌,序列号为FJ405211),其中前两处为沉默突变,F4菌的碱基突变引起92甘氨酸变为天冬氨酸,为TEM-57型。CTX-M基因亚型包括两种：CTX-M-65亚型4株(C2、C3、C4和K1,序列号分别为FJ405191,FJ405192,FJ405212和FJ405214)和CTX-M-14亚型3株(F2、X3和B5,其中F2菌的相关序列已上传至GenBank,获序列号为FJ405213)。【结论】 目前河南省产ESBL鸡大肠杆菌基因亚型以TEM-1变异型为主,CTX-M-65和CTX-M-14型次之。

Genotypes of TEM-Type and CTX-M-Type Extended-Spectrum β-Lactamases Produced by Escherichia coli Isolated from the Fow

【Objective】 The genotypes and subtypes of ESBLs produced by Escherichia coli isolated from the fowl were detected to elucidate their distributions in Henan Province. 【Method】 Twenty-eight ESBL-producing Escherichia coli were collected from different chicken farms of seven districts in Henan Province, and were amplified by PCR using the primers of TEM, SHV and CTX-M, respectively. Their PCR products were cloned and then the cloned fragments were sequenced. The nucleotide sequences were analyzed by DNAStar software and GenBank database. 【Result】 Twenty-one strains were TEM-type, two isolates were CTX-M-type, and two genotypes were detected in five strains, including TEM-type and CTX-M-type. No SHV-type was found. TEM-1 variant was the most common subtype, and the sequences of 26 isolates shared 98%-99% homology with that of AY293072 (TEM-1 β-lactamase). Except a silent mutation of 18T→C of all, the sequences of C3, X2, and F4 were characterized by another nucleotide base variations (783G→A, 21T→A, and 269G→A, respectively), and only the last variation led to the shift of amino acid (92Gly→Asp). These three sequences were submitted to Genbank database and obtained the accession No (FJ405207, FJ405208, and FJ405211, respectively). The CTX-M-type of ESBLs included two subtypes: three isolates (F2, X3, and B5) CTX-M-gene sequences were identical with EF158299 (CTX-M-14), the others (C2, C3, C4, and K1) CTX-M-gene belonged to CTX-M-65 type (FJ405191, FJ405192, FJ405212, and FJ405214, respectively). 【Conclusion】 The predominant genotype of ESBLs is TEM-1 variant in chicken farms of Henan Province, and then are CTX-M-65 and CTX-M-14.