Isolation And Identification Of Swine Extraintestinal Pathogenic Escherichia Coli Strains And Construction Of KpsM-iucB Deleted Mutant

According to its clinical symptoms and pathogenicity the academic circles divided Escherichia coli into three categories: Commensal Escherichia coli,Intestinal Pathogenic Escherichia coli and Extraintestinal Pathogenic Escherichia coli.Commensal Escherichia coli can be a lifetime and mutualism with the host,it spreads to the surrounding environment through feces, generally it does not cause the disease;Intestinal Pathogenic Escherichia coli exists widely in the host of intestinal and environment, generally will not cause extraintestinal infection immune function, but the immunity also can appear when parenteral infection;Extraintestinal Pathogenic Escherichia coli (ExPEC)can make a lot of different diseases such as urinary tract infection (UPEC),meningitis (NMEC),Septicemia, Pneumonia,Pleurisy and other parenteral soft tissue infections to hosts.So it was generally attention in the medical profession.For farming ExPEC can also cause mixed infection with other diseases and pose a great losses to Animal Husbandry.Based on the above dangers of Extraintestinal Pathogenic Escherichia coli, we conducted this study.We make a lot of experiments to get the clinical separation and master the epidemiological regularity of Extraintestinal Pathogenic E.coli, and lays the foundation for the diagnosis and prevention of the disease.We also carry out the pig source Extraintestinal Pathogenic E.coli ΔkpsM-AiucB double gene deletion bacteria vaccine strain to get effective control ExPEC method and hope through the genetic engineering vaccine development to effectively control the ExPEC. The main research was described as follows:1.We choose lung, liver, spleen, brain, trachea, joint which is separate from the disease pigs to take the culture,PCR identified,the results show that we get50Extraintestinal Pathogenic Escherichia coli strains. We also make other experiments such as virulence gene amplification,phylogenetic group identification and drug sensitive test. The results show that every of ExPEC strains has more than two virulence genes. And the phylogenetic group of ExPEC strains is A and B1.Sensitive drugs are cephalosporins for instance cefotaxime.But the clinical drug to work is limited, drug resistance present situation is serious.We use the E.coil donor strain X7213transformed with plasmid,pREAiucB,was conjugated with the recipient, ΔkpsM which is the PCN033gene deletion strain.Then we got the gene deleted strain ΔkpsM-ΔiucB and studied its biological characteristics and virulence.The Kunming mice were used to compare the pathogencity of PCN033and the PCN033AKpsM-ΔiucB double mutant strain.we describe the construction and characterization of the mutant PCN033ΔKpsM-ΔiucB,and study its biological characteristics, test of its pathogencity in Kunming mice.The result showed PCN033ΔKpsM-ΔiucB has the lower pathogencity, the LD50is about148times lower than PCN033.This laid the foundation for the clinical development of new Extraintestinal Pathogenic E. coli vaccine.