Comparative Genomics Studies Of Pathogenic Escherichia Coli

E.coli is a conditionally pathogenic bacterium present in the intestinal tract of humans and animals,which can become pathogenic under certain circumstances.Pathogenic E.coli is one of the main pathogens causing diseases in piglets,often causing diarrhea in piglets,which has seriously affected the development of the pig industry in China and caused huge economic losses.In this study,five strains of E.coli P211,P111,P555,P444 and P32 isolated from piglets with diarrhea and two strains S10670 and E24190 purchased from China Microbial Strain Conservation Center were used to identify the pathogenicity of the strains by mouse infection test,and the whole genome of the seven strains was sequenced and analyzed by comparative genomics research method.The genetic variants associated with virulence differences were analyzed by comparative genomics,and a theoretical basis was laid for the molecular pathogenesis of E.coli.The main results are as follows.1.7 strains were purified by selective medium,and then examined by Gram stain microscopy,physiological and biochemical identification and molecular identification,and the identification results were that all 7 strains were more than 99.5% similar to Escherichia coli,which were Escherichia coli.2.The results of the infection test on mice with the same concentration of bacterial suspension showed that the seven strains of the bacterium caused different lethality of the mice,and the dissection of the infected mice showed that the mice had different degrees of tissue lesions,including enlargement and darkening of the heart,liver,spleen,lung and kidney;the intestine was thin,with a large amount of contents and a foul smell.Pathological sections were made from the lesioned tissues,which showed that the myocardial fibers in the heart of the experimental mice were disorganized,irregularly arranged,and partially fractured and disintegrated with hemorrhage;the liver tissues were congested,with swollen cells and localized red masses and blurred tissue boundaries;the spleen tissues were congested;the lung tissues were partially indistinct and hemorrhagic,with red cells in the alveolar cavity and thickened alveolar walls;the kidney tissues were congested,and the glomerular structure was not obvious.The glomerular structure was not obvious;the intestinal epithelium was damaged and enlarged,the intestinal mucosa was necrotic,and the intestinal villi were detached.The pathogenic E.coli were classified into strong virulent strains: S10670 and E24190;moderate virulent strains: P211 and P555;and weak virulent strains: P32,P444 and P111 according to the lethality of experimental mice,mouse autopsy and histopathological observation.3.The genomic sequences were assembled after whole-genome sequencing using second-generation high-throughput sequencing technology,and the results showed that the genome lengths of the seven strains ranged from 4.6 Mbp to 5.3 Mbp,N50 from 86.348 kb to 148.7kb,GC content was around 50%,and the number of genes ranged from 4,376 to 5,124.The number of genes annotated to be functional ranged from 3,183 to 3,482,and the COG classification yielded 22 categories,with the largest number of genes annotated to be related to metabolism,which are mainly related to energy,carbon source and biosynthesis in E.coli,allowing the bacteria to survive and reproduce better in the host intestine,making them more pathogenic.Strains S10670 and E24190 annotated to the highest number of genes and both had more replication,recombination,and repair genes(K)than the other strains,which cause the bacteria to increase the frequency of exogenous genes acquired from outside,making the genome larger.4.The number of virulence genes annotated to strains S10670,E24190,P211,P555,P32,P444,and P111 was 126,61,52,55,47,44,and 38,respectively,mainly involved in bacterial invasive adhesion,secretion system,and iron ion uptake system.The potent strain S10670 contained strong toxin genes for shiga toxin(Stx),hemolysins(hly A,B,C,D,E/A),tight adhesins(eae,tir,tox B),iron uptake related virulence genes(iro B,chu A,X,T,S,W,U,Y)and a large number of secretion system related genes(Nle,Esc,Sep L,Sep Q,Esp).The strong strain E24190 contained the enterotoxin gene(LT),the hemolysin gene(hly A),the CFA/I hair gene(cfa A,B,C,D,E),and the ABC transporter protein gene(aat A,B,C,D,P).Intermediate strain P211 contained CFA/I bacterial hair genes(cfa A,B,C,D,E),P bacterial hair genes(pap A,C,D,H)and iron uptake system genes(sit A,B,C,D);P555 contains 12 VI secretion system genes(aec15?aec19,aec22?aec32).Weakly virulent strains P32,P444 and P111 contained virulence genes(e.g.,adhesionin Fim,invasin Ibe,etc.)were present in other strains.5.Using E.coli.str.K-12 as the reference genome,the number of SNPs and In Dels of the seven strains in this study were revealed at the genomic level,and the number of SNPs of S10670,E24190,P111,P211,P32,P444,and P555 were 58,230,18,346,22,245,33,042,13,807,18,994,12,320,and the number of In Del was 169,85,70,114,36,67,46,and the genes with mutations were enriched for KEGG pathways,resulting in 77 pathways,with 7 pathways with P-Value < 0.05,namely fatty acid degradation signaling pathway,monomycin biosynthesis pathway,RNA degradation pathway,vitamin B6 metabolic pathway,glycine,serine and threonine metabolic pathway,valine,leucine and isoleucine degradation pathway,microbial metabolic pathway in different environments,most of these pathways are metabolic synthetic pathways.In summary,the differences in pathogenicity of the seven strains of E.coli were studied by mouse infection test,and then whole-genome resequencing and comparative genomics were performed to resolve the causes of the differences in pathogenicity of the seven strains of E.coli at the genomic level,and the results of this study can provide a theoretical basis for in-depth study of the pathogenesis of E.coli,which is of great significance.