Isolation And Identification And Comparative Genomics Analysis Of Extraintestinal Pathogenic Escherichia Coli

Extraintestinal pathogenic Escherichia coli(ExPEC)is one of Escherichia coli cloned at intestinal but can cause infection at outside the intestinal tract,leading to different systems diseases of people and animals based on its various virulence factors.Animal derived ExPEC causing serious economic loss,also have public health significance.This study aims to confirm the biological characteristics of prevalent animal-derived ExPEC in some areas of our country by isolation and identification of pathogen.Based on the characteration,representative strains were selected and subjected to comparative genome analysis to predict possible and special virulence genes,to lay the foundation for the research of molecular pathogenic mechanism of ExPEC,and also provid theoretical basis to prevention and control of ExPEC infection.Firstly,E.coli isolated and identified from 132 extra-int estinal tissues of infected animals,such as swine,poultry and mink,of which 53 isolates carry at least two specific virulence genes of pap,sfa/foc,afa/dra,iut A and kpsMT II,and other virulence factors such as fimH,ibeA,hlyA,malX,iss and iroN,and so on,suggested that were ExPEC.53 strains ExPEC were showed different multiple antimicrobial resistances to Chloramphenicol,Kanamycin and Polymyxin B,and so on.E.coli O-antisera was mainly distributed in O1,O2,O8,O11,O38,O78 and O120serogroups.Phylogenetic analysis showed that ExPECs mainly belonged to group A and B1,among of 53 strains 28 isolates belonged to group A,19 isoaltes belonged to group B1.20representative ExPEC isolates were selected and carried out by intraperitoneally injected into mice at different dosage,the results showing that 11 strains ExPECs could cause all fatal of mice at the highest infection dose 3×10~7 CFU/mice.4 of 20 strains toxicity test was carried out,and the LD50 of ExPEC53,ExPEC52,ExPEC42 and ExPEC6 strains in mice was 2.38×10~7 CFU/mL,9.48×10~6 CFU/mL,3.49×10~6 CFU/mL and 9.49×10~5 CFU/mL,respectively.The strains of ExPEC53,ExPEC52 and ExPEC5 were subjected to complete genome sequencing and analysis by the technology of Illumina HiSeq.ExPEC53 and ExPEC52 complete genome include one p1asmid sized 98,080 base pairs and 207,975 base pairs,respectively,and both of them have tetracycline resistance gene,but ExPEC5 complete genome not carry plasmid.The predicted genes of isaolated strains of ExPEC53,ExPEC52 and ExPEC5 inclued the numbers of 747,667 and 703 virulence factors,respectively,that involved the invasion,adhere,secretion system and inorganic iron uptake systems,aslo included the numbers of unknown functions coding sequences proteins 232,242 and 232.Compared with bacterial strain PCN033(CP006632)reference genome,the specific gene numbers of genome of ExPEC53,ExPEC52and ExPEC5 were 718,393 and 239,and the fragments of insertions or deletions(length≤50 bp)were 365,367 and 339,respectively.In comparison of genome with reference PCN033,there were,and 6,541,6,498 and 6,493 non-synonymous mutation of single nucleotide polymorphism(SNP)at area of coding sequences.ExPEC53,ExPEC52 and ExPEC5 were subjected to establish orthologous of and constructed the evolution tree,and the results showing ExPEC52and ExPEC5 genetic relationship was closer,the ExPEC53 second.In conclusion,this study isolated and identified 53 ExPEC strains,confirmed the multiple drug resistance,E.coli O antisera,phylogenetic group of isaolates and pathogenicity of mice.3isaolates including mink-drived ExPEC and pig-drived strains with the O11 antisera serotype were selected to carry out whole-genome re-sequencing and comparative genomics analysis,and their characteratons of genomes,specific genes and virulence genes were predicted and analyzed.All of above results could lay the foundation for the research of molecular pathogenic mechanism of ExPEC,and also provide theoretical basis to prevention and control infection of ExPEC.