Study On The Heterogeneity Of Vibrio Parahaemolyticus Under Bile Salts Stress

Vibrio parahaemolyticus is ubiquitous in aquatic products.Once humans eat aquatic products containing V.parahaemolyticus,it will cause clinical symptoms such as vomiting,diarrhea,and fever.If it is more serious,it will cause reactive arthritis and heart disease.This poses a huge threat to human life,health and property safety.Scholars have established risk assessment models to predict and reduce the harm of V.parahaemolyticus to humans.However,V.parahaemolyticus from different sources will show significant heterogeneity in different growth environments,which will reduce the accuracy of risk assessment and increase the risk of disease in human.V.parahaemolyticus generally makes people sick by entering the human gastrointestinal tract.After eating,bile salts will produce enterohepatic circulation 2-3 times.Bile salts have antibacterial activity,which can damage cell membranes and slowing or killing bacterial growth.V.parahaemolyticus from different sources inevitably come into contact with bile salts after entering the human small intestine.Under bile salts stress,the growth characteristics,membrane formation and virulence of V.parahaemolyticus will affect the accuracy of pathogenicity and risk assessment.Therefore,in order to understand the heterogeneity of V.parahaemolyticus under bile salts stress,further improve the risk assessment system of V.parahaemolyticus,and improve the accuracy of risk assessment,the following research work was carried out in this paper.Firstly,the growth heterogeneity of 50 V.parahaemolyticus strains from different sources under bile salts stress was studied.Secondly,on the basis of the growth heterogeneity,appropriate strains were selected to study the heterogeneity of biofilm formation under bile salts stress.Finally,the virulence heterogeneity of V.parahaemolyticus strains under bile salts stress was explored.The main contents and results of this study are as follows:1.Growth heterogeneity of V.parahaemolyticus under bile salts stressV.parahaemolyticus is an important foodborne disease pathogen in aquatic products,and it generally makes people sick by entering the human gastrointestinal tract.In this study,the growth curves of 50 strains of V.parahaemolyticus（clinical and environmental）were simulated in TSB with different concentrations of bile salts（0,0.03,0.10,0.20,0.30 g/100 m L bile salts in tryptone soybean broth（TSB））,and the growth heterogeneity was compared.The growth kinetic parameters(maximum specific growth rate(μ_max)and lag time（LT）)were calculated using the modified Gompertz model.The results showed that the strain could not grow when the bile salts concentration was 0.30 g/100 m L,and the cell wall and membrane of the bacteria were severely damaged by Scanning Electron Microscope.With the increase of bile salts concentration,theμ_max of V.parahaemolyticus showed a decreasing trend.When the bile salts concentration was 0.03 g/100 m L,some strains were not significantly affected.The environmental strain could better adapt to the stress of lower bile salts concentration than the clinical strain,and the clinical strain could better adapt to the environment with higher bile salts concentration（0.20 g/100 m L）than the environmental strain.The demurrage of both environmental and clinical strains was gradually prolonged with the increase of bile salts concentration,but when bile salts concentration reached 0.20 g/100 m L,the demurrage of clinical strains was lower than that of environmental strains.Analysis of different genotypes showed that the coefficient of variation（CV）value of tlh⁺/tdh⁺/trh⁺strains was significantly lower than that of other genotypes under the stress of different concentrations of bile salts.The CV value of tlh⁺/tdh^-/trh⁺strains increased significantly with the increase of bile salts concentration and reached the maximum value when bile salts concentration reached 0.20 g/100 m L,showing greater growth heterogeneity.These results provide theoretical support for accurate risk assessment of V.parahaemolyticus.2.Biofilm heterogeneity of V.parahaemolyticus under bile salts stressIn response to the antimicrobial effects of bile salts,foodborne pathogens must evolve strategies to deal with their destructive effects in order to colonize the intestine and exert their pathogenicity.In human gastrointestinal tract,pathogenic bacteria can adapt to bile salts environment by reducing the permeability of cell membrane,and also form biofilm to prevent bile salts from entering cells.In this study,the dynamic membrane formation process of V.parahaemolyticus under different concentrations of bile salts,as well as the heterogeneity of time of membrane formation,maximum biomass and cell activity of clinical and environmental strains were analyzed in detail,and the membrane structure was analyzed by confocal laser scanning microscopy.The results showed that the biofilm formation of V.parahaemolyticus was inhibited under different concentrations of bile salts stress,and the formation time of the maximum biofilm was heterogeneous.The biofilm-forming ability of clinical strains was greater than that of environmental strains under higher concentration of bile salts stress,and the biofilm-forming ability of environmental strains was greater than that of clinical strains under lower concentration of bile salts stress.Observation by confocal scanning laser microscopy showed that when the concentration of bile salts reached 0.20 g/100 m L,V.parahaemolyticus hardly formed a biofilm,and high concentrations of bile salts significantly inhibited the formation of V.parahaemolyticus biofilm.These results provide reference for the control of V.parahaemolyticus biofilm and further provide theoretical support for risk assessment.3.Virulence heterogeneity of V.parahaemolyticus under bile salts stressUnder the stress of external environment,pathogenic bacteria can adapt to the adverse environment by changing their biological characteristics.The virulence heterogeneity of foodborne pathogenic bacteria strains is mainly related to the strain itself,growth environment and population.In this study,the expression of virulence genes of V.parahaemolyticus from different sources under different concentrations of bile salts stress was analyzed in detail,and the change of hydrophobicity of V.parahaemolyticus from different sources under different concentrations of bile salts stress was analyzed by using microbial adhesion carbonyl compound method.The results showed that the relative expression levels of virulence genes tdh and tox R increased significantly with the increase of bile salts concentration.The relative expression levels of virulence genes trh and tlh in clinical strains increased significantly when the bile salts concentration was low,and then decreased significantly with the bile salts concentration increasing.The relative expression levels of virulence genes tolc and vp A0450 in both environmental and clinical strains were inhibited by bile salts to varying degrees.The hydrophobicity of both environmental strains and clinical strains did not change significantly under bile salts stress at lower concentration,but decreased significantly when bile salts concentration reached 0.20 g/100 m L.Higher concentration of bile salts can reduce the adhesion of V.parahaemolyticus.These results further provide theoretical basis for risk assessment from the perspective of hazard characterization.