Pathogenicity Of Vibrio Parahaemolyticus In Six Kinds Of Foods And Its Relationship With Gut Microbiota

Vibrio parahaemolyticus(Vp)is a food-borne pathogen that inhabits marine waters.It has been isolated from many seafood types.There is new evidence that Vp can also contaminate freshwater fish,poultry,pork,fish,eggs and their products and cause infections in humans.The difference in symptoms of food infections are mostly related to pathogenicity of the infecting organism.It is now established that the pathogenicity of Vp is related to bacterial dose and and it’s toxic metabolites.However,the pathogenicity of Vp in different foods and the pathogenic mechanism in vivo are still unclear.Therefore,my thesis aimed to conduct research to address the following.1.In vitro test to characterize the growth and virulence factors of Vp in six food matricesFollowing Vp inoculation of shrimp,oyster,freshwater fish,pork,chicken and egg fried rice,the growth,TDH activity,total hemolytic activity,expression of virulence genes and extracellular enzymes activities were detected by plate counting method,using blood agar plates,relative hemolytic activity method,q-RT-PCR and extracellular enzymes detection.Results showed that the growth rate of Vp was higher in shrimp,oyster,freshwater fish and egg fried rice than in pork and chicken,even though there was a similar number of Vp(10~9 CFU/g)in all of these samples at the stationary phase.During the 6 to12 h incubation period,the total hemolytic activity of Vp increased linearly with time in shrimp,oyster,freshwater fish and egg fried rice,and the rate constant was greater than0.052,while in pork and chicken the rate constant was 0.023 during the linear growth range from 6 to18 h.Later,the total hemolytic activity of Vp reached a maximum at 24 h and remained stable in all foods tested.At the end of incubation,Vp showed the highest expression of tdh and tlh genes in egg fried rice and hence the highest TDH activity and total hemolytic activity followed by shrimp>freshwater fish>chicken>oyster>pork.But the exoenzyme activities were not consistent with the hemolytic activity profile showing significantly higher gelatinase,caseinase,phospholipase,DNase,urease and amylase activities in shrimp>freshwater fish>chicken>oyster>pork>egg fired rice.2.Mice in vivo test to investigate the pathogenicity of Vp in six food matricesThe mice were challenged with Vp cultured by different foods.The clinical symptoms including mortality,and changes in blood profiles,liver and kidney function,and organ coefficients(OC)were observed.Initial symptoms observed in mice were slow movement,fluffy hair and ultimately death in shrimp,freshwater fish and chicken.The mortality rates of mice in shrimp,oyster,freshwater fish,pork,chicken and egg fried rice group were41.7%,16.7%,33.3%,0%,25%and 8.4%,respectively.The white blood cell(WBC)and red blood cell(RBC)counts and hemoglobin(Hb)were significantly decreased(p<0.05)(WBC-64.2%,RBC-45.4%,Hb-18.7%)but the platelets were significantly increased(+48.6%)in shrimp followed by freshwater fish and chicken.In oyster,pork and egg fried rice groups,only WBC and RBC decreased.The BUN,ALP,ALT and AST were significantly increased(p<0.05)in shrimp with values of 91.7%,208.5%,109.1%,200%respectively.In oyster,freshwater fish and chicken,BUN,ALT and AST were significantly increased(p<0.05)but not ALP.There was no significant change in liver and kidney function parameters in pork and egg fried rice groups.In the intestine,OC was significantly increased(p<0.05)in all groups but in the stomach and liver,OC was increased only in shrimp and freshwater fish groups.There were significant differences in virulence of Vp in different foods,with the virulence in shrimp>freshwater fish>chicken>egg fired rice>oyster>pork.3.Self-constructed pseudo germ-free mouse model to test whether the gut microbiota promote the pathogenesis of Vp in vivo.The mice were gavaged with a mixture of antibiotics to remove gut microbiota and construct pseudo germ-free mouse model.These mice and control mice were then challenged with a suspension of Vp.The intestinal damage,body inflammation,and liver and kidney functions were monitored.The control mice with intact gut microbiota showed marked intestine damage than the pseudo germ-free mice.The changes in inflammatory factors IL-1β,IL-6,TNF-αand IL-10 in control normal mice were~2.5-fold higher than in the pseudo germ-free mice,and the liver(ALT,AST,ALP)and kidney(BUN)function indexes were~1.6-fold higher.Following Vp infection,the normal mice showed marked more seriously intestine damage,inflammation reaction and liver-kidney damages than pseudo germ-free mice.To our knowledge,this is the first report that gut microbiota increased the virulence of Vp.4.Metagenomics:16S rDNA sequencing method to identify gut microbiota biomarkers associated with pathogenicity of Vp in six food matrices.The 16S rDNA sequencing of mice gut microbiota was used to investigate the pathogenicity of Vp fed to mice following culture in different food matrices.The diversity parameters,species relative abundance,principal coordinate analysis and predicted community function were used to identify the target gut microbiota associated with Vp pathogenicity.Theα-diversity index was different between Vp-infected and non-infected group,with lower Shannon,Chao1 and Ace indexes in Vp-infected mice.There were significant differences in abundance of gut microbiota.At the phylum level,the proportion of sequences assigned to Bacteroidetes was significantly reduced(p<0.05)in all Vp-infected groups compared with non-infected groups,whereas reads assigned to Firmicutes and Proteobacteria were increased.At the genus level,the relative abundance of Bacteroides,Lactobacillus,Bifidobacterium,Rikenellaceae and Lachnospiraceae were decreased in all Vp-infected groups,whereas the relative abundance of Enterococcaceae,Streptococcus,Clostridiales and Vibrio were higher than non-infected groups.Especially in the highly pathogenic groups of shrimp,freshwater fish and chicken,the proportion of sequences assigned to Akkermansia(a probiotic bacteria),Ruminococcaceae(producing butyric acid)and Faecalibacterium(producing short chain fatty acids)was decreased while the reads assigned to Prevotella(promoting inflammation)and Sutterella(causing liver damage)were increased compared to the non-infected groups.Furthermore,the PICRUSt analysis revealed that there were significant changes in microbial communities function,including metabolism,genetic information processing and bacterial infection pathways in all Vp-infected groups.However,changes in the bacterial invasion epithelial cells pathway,cAMP signaling pathway and bile acid synthesis pathway were observed only in shrimp,freshwater fish and chicken.The pathogenicity of Vp in different foods was closely related to the gene induction differences in gut microbiota and its microbial communities function.This study investigated the differences in Vp growth,virulence factors and organ/tissue damage to mice fed six different food types.A pseudo germ-free mouse model was used to elucidate the role of host gut microbiota in Vp pathogenesis in vivo.Metagenomics16S rDNA sequencing method identified gut microbiota biomarkers associated with Vp pathogenicity in six food matrices.This study provides a new perspective and theoretical basis for Vp food safety risk assessment.