鸡源大肠杆菌毒力基因检测及分子流行特征

Virulence genes and molecular characteristics of chicken-derived Escherichia coli

摘要:

[目的] 本试验旨在阐明鸡源大肠杆菌致病性及分子流行特性，为探索大肠杆菌流行途径制定合理的防控策略提供新思路。[方法] 2018–2019年在河北省采集病死鸡肝脏样品，通过选择培养基筛选、生化鉴定、血清凝集试验对分离菌株进行系统鉴定，应用PCR方法检测分离株中毒力基因流行情况。参考系统发育群分类方法对大肠杆菌进行分群分析，并参照McMLST网站数据库提供的7对管家基因序列进行多位点序列分型（multilocus sequence typing，MLST）分析。[结果] 结果显示，56株分离株符合大肠杆菌生化特征，分为8个生化表型，B4（30.36%）、B5（25%）和B2（23.21%）为主要生化表型。56株分离株大肠杆菌血清凝集试验均呈阳性，分为11种血清型，O₇₈（26.79%）、O₂（23.21%）、O₁₅₇（17.86%）和O₁（14.29%）为主要流行血清型。56株大肠杆菌共检测出15种肠外大肠杆菌毒力基因，未检出papC、ibeA和ibeB基因。黏附相关基因fimC和抗血清存活因子相关基因ompA携带率为100%。aatA、yijP、irp2、mat、iss，检出率分别为98.21%、98.21%、98.21%、96.43%、92.86%。同时，大肠杆菌与铁转运相关基因iroN、fyuA、iucD、irp2检出率均在80%以上。56株大肠杆菌中有20株属于肠出血型大肠杆菌（enterohemorrhagic E.coli，EHEC），其次是肠聚集型大肠杆菌（enteroaggregative E.coli，EAEC）（n=4）、肠产毒素型大肠杆菌（Enterotoxigenic E.coli，ETEC）（n=2）。这些菌株D群分离株较多，其次是B2群。通过MLST分型分析，共分为22个ST型，其中ST88（n=7）、ST85（n=6）、ST243（n=6）型为主要流行型。[结论] 结果显示大肠杆菌血清型多样，毒力因子种类繁多，致病性大肠杆菌同时携带多种毒力基因，表明动物源大肠杆菌具有较强的毒力基础。

关键词:大肠杆菌;毒力基因;MLST;系统发育群

Abstract:

[Objective] The purpose of this test is to elucidate the pathogenicity and molecular epidemic characteristics of chicken-derived Escherichia coli, and to provide new ideas for exploring reasonable ways to prevent and control E. coli. [Methods] Livers of dead chicken samples were collected in Hebei Province from 2018 to 2019, and the isolated strains were systematically identified through selection of media selection, biochemical identification, and serum agglutination test. Detection of virulence genes in isolates was conducted by PCR. Cluster analysis of E. coli was performed with reference to phylogenetic classification. Multi-sequence typing analysis of housekeeping gene sequences with reference to 7 databases provided on the McMLST website database. [Results] The results show that 56 isolates conformed to the biochemical characteristics of E. coli were divided into 8 biochemical phenotypes. Among them, B4 (30.36%), B5 (25%), and B2 (23.21%) were the main biochemical phenotypes. 56 isolates of E. coli were positive for serum agglutination test, divided into 11 serotypes. O₇₈ (26.79%), O₂ (23.21%), O₁₅₇ (17.86%), and O₁ (14.29%) were the main epidemic serotypes. A total of 15 E. coli virulence genes were detected in 56 strains of E. coli, but no papC, ibeA, and ibeB genes were detected. The gene carrying rate of Adhesion-related gene fimC and antiserum survival factor-related gene ompA in all isolates is 100%. The detection rates of the genes of aatA, yijP, irp2, mat, and iss were 98.21%, 98.21%, 98.21%, 96.43% and 92.86%. The detection rates of iroN, fyuA, iucD and irp2 of E. coli and iron transport-related genes were all above 80%. Of the 56 strains of E. coli, 20 are Enterohemorrhagic E. coli (EHEC), followed by Enteroaggregative E. coli (EAEC) (n=4) and enterotoxigenic E. coli (ETEC) (n=2). These strains have more D group isolates, followed by B2 group. According to MLST typing analysis, there are 22 ST types in total, of which ST88, ST85 and ST243 are the main epidemic types. [Conclusion] The serotypes of E. coli were diverse and the virulence factors were various. The pathogenic E. coli also carried multiple virulence genes, indicating that animal-derived E. coli has a strong virulence basis.

Key words:Escherichia coli;virulence gene;MLST;phylogenetic group