Pathological Biology Characteristics Of Pic Deletion Strains Of Aquatic Enteroaggregative Escherichia Coli

In order to understand the pathogenic status of Aquatic pathogenic Escherichia coli(APEC)in Anhui Province,the isolation,identification and pathogenic analysis of E.coli were carried out from 2017 to 2018.In order to understand the ability of the pathogenic types of Aquatic pathogenic E.coli to break through the barrier of intestinal mucosa in mammals,a mixed oral challenge test was conducted.In order to understand the significance of pic as a typing marker gene of Enteroaggregative E.coli(EAEC)for pathogenicity of Escherichia coli,a pic-deleted strain was constructed by using CRISPR/Cas9 gene editing system,and its basic pathological and biological characteristics were studied.Specific research contents and results are as follows:1.Isolation,Identification and Pathogenic Analysis of APECIn 2017-2018,samples of infected fish,fish intestines,fish pond water and sediments,duck(or pig)-aquaculture system sediments,sick birds(chickens,ducks,geese),rabbits,sheep,rats,human tissues and feces were collected from Anhui province.The suspected E.coli strains were isolated by double selection of McConkay and Eosin-methylene blue selective medium,identified by 16S rDNA sequencing,and typed based on the marker virulence gene profile,methods of GB4789.6-201.The results showed that 76 strains of E.coli were isolated.10 strains were isolated from the anal swabs of pigs,sheep,rabbits and humans;53 strains were isolated from the anal swabs or liver of waterfowl,ducks,geese,chickens,etc;10 strains were isolated from the anal swabs,liver,spleen,brain or body surface of aquatic animals such as Quasipaa spinosa,Ctenopharyngodon idellus,Parabramis pekinensis,Acrossocheilus fasciatus,Aristichthys nobilis,Hypophthalmichthys molitrix and other aquatic animals;and 3 strains were isolated from pond sediment and water.There were 46 non-pathogenic strains,30 human diarrheagenic E.coli strains,including 4 Enteropathogenic E.coli EPEC strains,11 Enterotoxigenic E.coli ETEC strains,8 EAEC strains and 7 Enteroinvasive E.coli EIEC strains.2.The Effect of APEC on Intestinal Microecology and Intestinal Pathogenicity in Mice7 strains of APEC(2 EAEC strains,3 EPEC strains,1 ETEC strain and 1 non-pathogenic strain)were administered to Kunming mice by gavage.The clinical symptoms,anatomical changes,effects on intestinal microecology and the ability to break through the barrier of gastrointestinal mucosa were observed.The results showed that EAEC could be isolated from the spleen of mice in the experimental group 24 hours and 48 hours after infection.EAEC could also be detected in the genomic extracts of intestinal contents 12 hours,24 hours and 48 hours after infection.The virulence genes of EAEC were the same as those of 17J105.It can be confirmed that 17J105 strains isolated in our laboratory can cause enteritis and systemic infection in mice by oral route,and have advantages in pathogenicity of 7 strains.High-throughput 16S rDNA sequencing showed that the effect of APEC mixed challenge on the diversity of colonic and caecal flora in mice was mainly concentrated in 12 hours.After stopping gastric administration,the intestinal microecosystem would gradually return to normal level.3.Effects of pic Gene Deletion on Pathobiological Characteristics of EAEC 17J105 Strain from FishThe pic gene deletion mutant was constructed by CRISPR/Cas9 system.The effects of pic gene deletion on the biological characteristics and virulence of 17J105 were explored by bacterial dynamics and generation determination,drug sensitivity test,tissue loading and half lethal dose.The results showed that deletion of pic gene could prolong generation time,reduce tissue bacterial load,increase half lethality and decrease lethality to zebrafish.The resistance of pic deleted strains to neomycin,enrofloxacin,oxytetracycline and ciprofloxacin increased,while the resistance to azithromycin decreased.High-throughput 16S rDNA sequencing technique was used to detect and analyze the community diversity of intestinal contents in colon and caecum of mice challenged orally by 17J105 strain and pic deletion strain,respectively.The results showed that the community diversity of intestinal contents in the colon and caecum of mice challenged with 17J105 strain was significantly lower than that of the control group.The community diversity of pic-deleted strain was slightly higher than that of 17J105 strain,but still less than that of the control group.Differentially expressed genes in colonic and caecal tissues of mice challenged with 17J105 and pic deleted strains were screened by high-throughput RNA-Seq technique.Bioinformatics analysis was carried out and some differentially expressed genes were verified by fluorescence quantitative PCR.A total of 1161 differentially expressed genes were screened out in wild strain challenge group compared with pic deleted strain challenged group.Functional annotation results showed that differentially expressed genes were mainly involved in biological processes such as transcription and regulation;cytoplasm,cell membrane and its overall composition;protein binding,metal ion binding and other molecular functions.The main difference of KEGG pathway analysis is related to immune and disease pathways.The results of fluorescence quantitative PCR and transcriptome sequencing were basically consistent,which indicated the reliability of the sequencing data.The results showed that the pathogenic type of APEC was complex,and EAEC 17J105 strain from fish had the strongest ability to break through intestinal mucosal barrier.The deletion of pic gene in EAEC 17J105 strain weakens its pathogenicity and its ability to influence intestinal microecological homeostasis.The pic gene is involved in the regulation of intestinal immunity and disease-related response genes.