The Establishment And Application Of Caenorhabditis Elegans Model Infected With Escherichia Coli Isolated From Bovine In Parts Of Guangxi

In order to establish an efficient and convenient animal model for detecting the virulence and drug resistance of bovine Escherichia coli,and to explore the impact of bovine Escherichia coli on its immune signaling pathway through the model and to quickly screen effective antibacterial drugs according to the model in vivo drug tests to provide a basis for the prevention and treatment of bovine disease and clinical medication.In this study,conventional methods were used to isolate and identify Escherichia coli from bovine disease materials,and modern molecular biology methods were used to detect virulence factors and drug resistance genes,respectively.Using the obtained Escherichia coli to construct Escherichia coli-Caenorhabditis elegans infection model and Escherichia coli-mouse pathogenic model.And use the constructed model to detect the effect of Escherichia coli on the Caenorhabditis elegans immune signaling pathway and the effect of antibacterial drugs on Caenorhabditis elegans,the results are as follows:1.13 strains of Escherichia coli were isolated from 13 disease materials collected in Nanning,Guilin,Liuzhou and other regions of Guangxi,of which O126 serotype accounted for 30.77 %(4/13),which was the dominant serotype,and O44 and O20 serotypes accounted for 15.38 %(2/13).2.19 virulence factors were detected from 13 strains of Escherichia coli,of which the detection rate of Escherichia coli housekeeping gene pho A and outer membrane protein omp A were all 100 %,followed by the detection rate of Stx1 76.92 %,The detection of uid A and h1 y A rate was 69.23 %,K88,ipa H and vat had the lowest detection rate,at 7.69 %.3.The detection of 15 drug resistance genes in 13 strains of Escherichia coli showed that the detection rate of quinolone resistance genes(Gyr A / Gyr B / Par C)were the highest,both were 100 %,and the detection of sulfonamide resistance genes(SUL1 / SUL2)were 76.92 % to 100 %,and the detection rate of macrolide resistance gene erm B was the lowest,at 7.69 %.Results of in vitro antibacterial tests of 13 strains of Escherichia coli: highly sensitive to ceftiofur and levofloxacin(84.62 % ? 96.31 %);moderately sensitive to gentamicin and tylosin(53.85 %);resistant to ceftriaxone Sodium,sulfamethoxine,and enrofloxacin(0 %).4.Construction of the Escherichia coli-Caenorhabditis elegans infection model results: According to the lethal rate of nematodes,three standards of Escherichia coli pathogenicity to Caenorhabditis elegans were established: strong,medium,and weak.It was reflected in the 2nd day to 4th day.Compared with the results of the Escherichia coli-mouse pathogenic model,Escherichia coli,which was not lethal to mice,had the lowest lethal rate to nematodes.Similarly,Escherichia coli,which was highly pathogenic to mice,had the highest lethal rate to nematodes.,while Escherichia coli with moderate pathogenicity to mice had a moderate lethal rate to nematodes.The results of the two infection models were consistent,and the Escherichia coli-Caenorhabditis elegans infection model was successfully constructed.5.RT-PCR was used to detect the effect of Escherichia coli on the C.elegans immune signaling pathway.The results showed that the pathogenic Escherichia coli had a greater effect on the nematode's TGF-? immune signaling pathway and insulin-like signaling pathway than the pathogenic Escherichia coli.The up-and down-regulation of antibacterial peptides Spp-1,Abf-2,Clec-85 and Lys-7 in nematodes had no obvious correlation with the pathogenicity.6.The use of antibacterial drugs on nematodes to study the toxicological effects of drugs on nematodes and the rescue effect of nematodes.The results showed that from the 27 antibacterial drugs,five antibacterial drugs with the least impact on the nematode survival test were selected,namely penicillin,Streptomycin,oxytetracycline,florfenicol and levofloxacin.Among them,levofloxacin and florfenicol could rescue nematodes infected with Escherichia coli,and levofloxacin had the most significant effect.Conclusions:1.Thirteen bovine Escherichia coli strains were isolated and identified from different areas of Guangxi.The dominant serotype of Escherichia coli was O126(30.77 %,4/13).A total of 19 virulence factors were detected in 13 strains of bovine Escherichia coli The detection rate of Escherichia coli housekeeping gene pho A,outer membrane protein omp A and Shiga toxin Stx1 virulence factor was the highest,being 100 %(13/13)and 100 %(13/13),76.92 %(10/13).2.The Escherichia coli-Caenorhabditis elegans model was successfully established.This model could replace the Escherichia coli-mouse infection model to achieve the detection of virulence and drug resistance of Escherichia coli.3.The resistance genes and resistance phenotypes of 13 strains of Escherichia coli were different.4.The pathogenic Escherichia coli had more influence on the nematode's TGF-? immune signaling pathway and insulin-like signaling pathway than the lower pathogenic Escherichia coli.The up-regulation and down-regulation of the antibacterial peptide genes Spp-1,Abf-2,Clec-85 and Lys-7 had no obvious correlation with the pathogenicity of Escherichia coli.