Study On Genomics Analysis And Biofilm Formation Mechanism Of Food-borne Campylobacter Jejuni

Campylobacter jejuni(C.jejuni)is an important food-borne pathogen and widely spreads in common foods such as chicken,milk and beef.In some developed countries,intestinal diseases caused by C.jejuni are even higher than Salmonella and Shigella.C.jejuni is a microaerobic caustic nutrient bacteria whose survival and transmission mechanisms in atmospheric environment are relatively poorly understood.The formation of biofilms is considered extremely important in this process.To further investigate the pathogenic risk and its survival of food-borne C.jejuni,this study used the technology of whole genome sequencing to sequence the genomic of 74 strains which were isolated from the main cities in China.The genome data were used to assess its pathogenic risk information.At the same time,the biofilm formation ability of all strains were evaluated,and the genes related to biofilm formation were screened by genome-wide association analysis(GWAS).Then,these genes Cj0423-Cj0425 which associated with biofilm formation were knocked out to verify their function.The main results are as follows:(1)Complete whole genome sequencing of 74 food-borne C.jejuni,and the GC content of all strains were about 30.5%.Most of these genome sizes is range from 1.6 M to 1.8 M.The L50 of most genomes was below 10.The genomes were approximately intact and could be used for following data analysis.(2)The genomic data were used for pathogenic risk analysis.We found that 74 C.jejuni carry 90-120 different kinds of virulence-related genes,and most of the strains carry about 100 virulence genes.Some strains carry wlaN that may cause Guillain-Barré syndrome in humans.Twenty-two different kinds of drug-resistance related genes were found in the genomic data.One strain even carried 19 different drug-resistance genes,and most of the other strains carried more than 5 drug-resistant genes.According to the results of the pan-genome analysis,these strains have genes which related to human diseases,all of which are distributed on core genes and accessory genes.From the results of MLST,the strains can be divided into 48 ST types,of which 26 are new STs.The dominant clonal complex is ST464,which is consistent with clinical strains in China.The results of core genomic SNP typing showed that the genomes of these strains are diverse.All strains can be roughly divided into 5 clusters,which have a long distance with the reference strain ATCC 700819.(3)Evaluation of biofilm formation ability of C.jejuni strains was performed by the method of crystal violet staining.The results showed that there were 38 strains with weak biofilm formation ability,11 strains with moderate biofilm formation ability,and 25 strains with strong biofilm formation ability.Confocal Laser Scanning Microscope(CLSM)and Scanning Electron Microscope(SEM)were used to observe some strong/weak biofilm formation ability strains,and the results were consistent with the results of crystal violet staining.(4)Using the biofilm phenotype and genome for genome-wide association analysis,642 biofilm-associated genes have been screened,of which 338 gene clusters were positively correlated with biofilm formation and 304 were negatively correlated.Enrichment analysis by DAIVD revealed that the enriched genes and GO were mostly related to membrane components and membrane proteins,and the enriched KEGG pathway was involved in capacity metabolism.(5)The Cj0423-Cj0425 gene cluster,which was screened by GWAS,was selected to construct the gene knockout mutant.The suicide vector was constructed by insertion/deletion method,which was transformed into C.jejuni cell by electroporation.The gene knockout system was successfully constructed and the gene cluster was successfully knocked out.Compared to wild-type strains,the biofilm formation capacity of mutants was significantly enhanced,which is consistent with the expected results of genome-wide association analysis.This thesis evaluated the pathogenic risk and biofilm formation ability of Campylobacter jejuni which were isolated from foods in China.At the same time,the GWAS method was used to screen biofilm formation-related genes,and the biofilm formation mechanism was initially studied.The Campylobacter jejuni knockout system was successfully constructed,which can be used for gene function verification in the future.