猪肠道非致病性且无耐药性大肠杆菌的分离鉴定、生长特性和基因组进化分析

【目的】从猪肠道中分离出无毒力基因且无抗生素耐药性的大肠杆菌，并分析其生长性能和遗传进化规律，以期为开发猪用微生态制剂或口服疫苗奠定基础。【方法】采集广东省 2 个地区养猪场的 11 头和 15 头健康成年猪的粪便样本。首先，采用培养方法从麦康凯培养基中分离出疑似大肠杆菌的菌株，通过 16S rRNA 序列分析鉴定其是否为大肠杆菌菌株；然后，采用 PCR 方法验证所选大肠杆菌是否含有 13 种常见的猪致病性大肠杆菌毒力基因，并以 7 类（14 种）抗生素进行药敏试验；最后，对筛选出的优质大肠杆菌菌株进行生长动力学分析，并提取其全基因组 DNA，构建全基因组系统进化树。【结果】在 260 株疑似为大肠杆菌菌株中，经 16S rRNA 序列分析确认后有 206 株属于大肠杆菌。其中，107 株大肠杆菌不含 13 种常见毒力基因，25 株大肠杆菌对 7 类（14 种）抗生素敏感。在这 25 株菌株中，有 23 株非致病性且无抗生素耐药性大肠杆菌（编号为 2-9、3-2、3-4、5-1、6-1、6-2、6-9、6-10、8-2、8-9、10-5、B-4、B-6、B-7、B-10、D-10、E-2、E-10、J-1、J-4、K-6、L-6 和 O-9）的生长性能与大肠杆菌 Nissle1917、MG1655 相似，其他 2 株大肠杆菌（编号为 6-4 和 5-2）的生长速度高于 Nissle 1917 和 MG1655。此外，有 10 株菌株（编号为 E-10、E-2、6-4、6-9、8-2、O-9、3-2、6-2、J-1、K-6）耐酸性能较好，可能适合长期定殖于猪胃肠道内。基于 SNP 核心基因组系统进化树分析，发现这 25 株菌株中有 9 株菌株（编号为 3-2、6-10、10-5、6-1、8-9、5-2、L-6、O-9、K-6）在系统进化树开端处属于同一大类群，表明它们极可能为猪原生大肠杆菌的祖先群。【结论】综合上述大肠杆菌的抗生素敏感性、生长性能和全基因组进化树和胃液环境耐受性能分析，菌株 O-9 在这 4 个方面均具备优势，该菌株与大肠杆菌 Nissle 1917、MG1655 的生长动力学相似甚至更优，意味着 O-9 菌株可能是优质的猪肠道原生大肠杆菌，具有极大研究潜力。

Isolation, Identification, Analysis of Growth Characteristics and Genomic Evolution of Non-pathogenic and Non- antibiotic Resistant Escherichia coli Isolated from Pig Intestines

【Objective】The study aimed to isolate Escherichia coli without virulence genes and antibiotic resistance from pig intestines and to analyze their growth performance and genetic evolution, with a view to laying a foundation for the development of microecologics or oral vaccines for pigs. 【Method】Fecal samples of 11 and 15 healthy adult pigs were collected from two pig farms in Guangdong Province, respectively. First, E. coli strains were isolated by McConkey plate and identified by 16S rRNA gene sequencing. Then, PCR was used to verify whether the selected E. coli contained 13 common virulence genes of pig pathogenic E. coli. Antimicrobial susceptibility tests were performed with 7 classes (14 kinds) of antimicrobial agents. Finally, the growth curves of selected E. coli strains were analyzed, and a phylogenetic tree was constructed based on the extracted genome sequences.【Result】Among 260 suspected E. coli strains, 206 strains were identified as E. coli by 16S rRNA gene sequencing analysis. 107 strains of E. coli did not contain 13 common virulence genes, of which 25 strains were sensitive to 7 classes (14 kinds) of antibiotics. Among the 25 E. coli strains, 23 non-pathogenic and non-antibiotic resistant E. coli strains (numbered as 2-9, 3-2, 3-4, 5-1, 6-1, 6-2, 6-9, 6-10, 8-2, 8-9, 10-5, B-4, B-6, B-7, B-10, D-10, E-2, E-10, J-1, J-4, K-6, L-6 and O-9) showed similar growth performance to E. coli Nissle 1917 and MG1655, and 2 E. colistrains (numbered as 6-4 and 5-2) showed higher growth rates than those of E. coli Nissle 1917 and MG1655. Among them, 10 strains (numbered as E-10, E-2, 6-4, 6-9, 8-2, O-9, 3-2, 6-2, J-1, K-6) had good acid tolerance and may be suitable for long-term colonization in the gastrointestinal tract of pigs. The phylogenetic tree analysis based on SNP core genome showed that 9 strains (numbered as 3-2, 6-10, 10-5, 6-1, 8-9, 5-2, L-6, O-9 and K-6) of the 25 strains were in the same group at the beginning of the phylogenetic tree, which were most likely to belong to the ancestral group of pig native E. coli. 【Conclusion】Based on the comprehensive analysis of antibiotic sensitivity, growth performance, genome-wide evolutionary tree and gastric environment tolerance, one strain (numbered as O-9) was dominant in these four aspects. The growth kinetics of this strain is similar to or even better than that of E. coli Nissle 1977 and MG1655, which means that O-9 strain of E. coli isolates was likely to be high-quality pig native E. coli strain, with great research potential.