鸡大肠杆菌TEM和CTX-M型超广谱β-内酰胺酶基因分型研究

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

【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。

[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, F J405192, 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.