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.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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