Heterogeneity Of Vibrio Parahaemolyticus Under Artificial Gastric Fluids

Vibrio parahaemolyticus is ubiquitous in the estuary environment and is common in seafood products.Vibrio parahaemolyticus causes people to produce clinical symptoms such as diarrhea,vomiting,and fever.More severe cases can cause reactive arthritis and heart disease.It is related to the consumption of contaminated seafood,especially unprocessed food such as oysters and salmon.It poses a huge threat to human health.In order to cope with these diseases,especially in the 1990s,several immunomolecular detection methods have been developed,which can be used to rapidly detect and quantify Vibrio parahaemolyticus.Use molecular biology methods to distinguish between clinical and environmental strains of Vibrio parahaemolyticus,accurate identification and characterization of Vibrio parahaemolyticus in seafood and the detection of virulence factors for pathogenic and non-pathogenic strains.Control food safety caused by Vibrio parahaemolyticus for risk assessment.The research mainly uses animal infection models and in vitro digestive system simulation to explore the pathogenic mechanism of infection,and human gastrointestinal tract model to simulate its tolerance and pathogenicity in the gastrointestinal environment;in order to solve foodborne pathogenic.The food safety issues caused by bacteria provide a scientific basis.Therefore,this article aims to:1)use the in vitro static digestion model to study the heterogeneity of the growth of 50 strains of Vibrio parahaemolyticus from different sources in artificial simulated gastric fluids;The variation of growth coefficient of variation CV value and its impact on growth heterogeneity,3)further studied the effect of different genotypes on phenotypic growth heterogeneity from a genetic perspective,4)using flow cytometry to detect pathogenic vice the survival status of Vibrio hemolyticus.The research content and experimental results of this subject are as follows:1.Study on the growth heterogeneity of Vibrio parahaemolyticus under artificial gastric fluidsThe current research focus is less on growth heterogeneity differences in simulated gastric fluid environments of different pathogenic VP.In addition,most of the?about 95%?Vibrio parahaemolyticus in our environment are non-pathogenic,while a smaller portion of about 5%are pathogenic strains isolated from clinical cases The tlh gene is present in pathogenic and non-pathogenic Vibrio parahaemolyticus,while the tdh and trh genes are only present in pathogenic strains.First,simulated human gastric fluids was used to explore the growth variability of 50 strains of Vibrio parahaemolyticus at 37�C.Mathematically modified the Gompertz model to fit the strain growth curve,and calculated the maximum specific growth rate(?_max),lag period?LT?and its coefficient of variation?CV?values,comparing pathogenic and non-pathogenic Vibrio parahaemolyticus response to stress in simulated human gastric fluids environment.The results showed that after treatment with simulated gastric fluids,the?_max of pathogenic strains increased significantly?P<0.01?,the lag period was significantly shortened?P<0.05?,and the?_max of nonpathogenic strains increased significantly?P<0.01?,the lag period was significantly increased Prolonged?P<0.05?.At the same time,the CV value of the genotype?tlh⁺/tdh⁺/trh^-?increased significantly?P<0.05?,indicating that the pathogenic genotype?tlh⁺/tdh⁺/trh^-?strain had strong activity on simulated gastric fluids.The results show that the pathogenic Vibrio parahaemolyticus strains show great resistance and growth heterogeneity to the simulated gastric fluids environment,which will provide new insights into the effective control of food-borne pathogens.2.Heterogeneity of Vibrio parahaemolyticus biofilms under artificial gastric fluidsUnder simulated gastric fluids environment,six pathogenic and non-pathogenic paralytic strains VPC40,VPC89,VPC90,VPE05,VPE37,VPE49 were studied.Observe the biofilm formation of each strain at 2h,8h,12h and 24h.Six strains of different pathogenic Vibrio parahaemolyticus were used to measure the dynamics of biofilm formation by crystal violet staining.The results showed that after artificial gastric fluids treatment,the biofilms of VPC40,VPC89,VPC90,VPE05,VPE37,and VPE49 increased significantly compared with the pure culture?P<0.05?.The growth rate of biofilms of the six strains was slow when cultured for 2 hours,and the growth rate of biofilms increased significantly when cultured for 8 hours?P<0.05?.The increase in the amount of pathogenic Vibrio parahaemolyticus biofilm is a rapid response of the strain to the harsh external environment,which poses a great threat to human health.3.Detection and application of Vibrio parahaemolyticus in artificial gastric fluidsUsing flow cytometry to study the changes in viable counts of pathogenic Vibrio parahaemolyticus in artificial gastric fluids at 2h,6h,and 12h,compared with the traditional plate counting method.The results showed that the flow cytometry had a significant correlation with the plate counting method,and could well distinguish the viable bacterial state of Vibrio parahaemolyticus in artificial gastric fluids during the culture process.The viability gradually increased during the 2 h culture.Gastric fluids-treated strains are live strains,so large areas of negative red fluorescent PI appear.With the increase of culture time,the number of dead bacteria in the remaining bacterial suspension gradually increased,the red fluorescent PI-negative area gradually changed,and the area of the red fluorescent?SYTO?-positive area increased.Flow cytometry detection is completed within 2 hours,which greatly shortens the detection time.Therefore,this method can be used to provide a scientific and effective method for rapid detection of bacterial activity in food-borne foods.This paper studies the difference in the maximum specific growth rate of pathogenic and non-pathogenic Vibrio parahaemolyticus of four different genotypes in simulated gastric fluids environment.The results showed that after treatment with simulated gastric fluids,the?_max of 87%of pathogenic strains increased significantly,and that of non-pathogenic strains decreased by 89%.At the same time,the CV value of genotype?tlh⁺/tdh⁺/trh^-?is significantly higher than other genotypes?tlh⁺/tdh^-/trh^-,tlh⁺/tdh^-/trh⁺and tlh⁺/tdh⁺/trh⁺?CV value?P<0.05?.Therefore,more attention should be paid to clinical genotypes related to virulence factors?tlh⁺/tdh⁺/trh^-?.The biofilm of pathogenic strains is significantly higher than that of non-pathogenic strains,and the coefficient of variation CV reaches a peak value of18.1 at 12 hours of cultivation.It provides effective methods and theoretical basis for predicting microbiology and food risk assessment.