| 区炳明 1,2，陈晓洁 1
，李清青 1,2，陈锦洪 1
，萧碧扬 1
，钟炜楠 1
，林 雪 1
，刘蕾蕾 1
，张敏瑜 3.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[J].广东农业科学,2024,51(5):115-124 |
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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 |
| Isolation, Identification, Analysis of Growth Characteristics and Genomic Evolution of Non-pathogenic and Non- antibiotic Resistant Escherichia coli Isolated from Pig Intestines |
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| DOI：10.16768/j.issn.1004-874X.2024.05.010 |
| 中文关键词: 猪 原生大肠杆菌 毒力基因 抗生素耐药性 生长特性 遗传进化 |
| 英文关键词: pig native Escherichia coli virulence gene antibiotic resistance growth characteristics genetic evolution |
| 基金项目:国家自然科学基金（32102703）；广东省基础与应用基础研究联合基金（2019A1515111186）；肇庆学院
农村科技特派员工作站项目（2022N001）；肇庆大学大学生创新创业培训计划项目（X202310580123） |
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| 中文摘要: |
| 【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。 |
| 英文摘要: |
| 【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. coli
strains (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. |
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