Research Of Mixed Biofilms Of Vibrio Parahaemolyticus Of Different Virulence Genotypes

Contamination of food,especially aquatic products,by Vibrio parahaemolyticus poses a serious risk to human health and safety worldwide.Food poisoning caused by V.parahaemolyticus persists worldwide.Most bacteria exist in the form of biofilms,which are also associated with bacterial infections in people’s lives.The formation of biofilms is a complex and dynamic process governed by a variety of factors and has a more stable structure than the planktonic state,making it difficult to eliminate.Whether in the food sector or in nature,biofilms are often in the form of mixed biofilms.Compared to monobacterial biofilms,there are more complex interactions in mixed biofilms that affect the structure,development and function of biofilms.Whereas most of the relevant studies at this stage have focused on monobacterial biofilms,which do not accurately model the real risk of biofilms,mixed biofilms are of greater interest in order to develop strategies to control or even eliminate them.Most of the current studies on mixed biofilms of V.parahaemolyticus have been conducted by mixing VP with other harmful foodborne pathogens,and very few studies have been conducted on mixed biofilms formed by bacteria of the same genus.This paper therefore investigates the characteristics of biofilms formed by clinical strains of VP carrying different virulence genes.The main elements and results of this study are as follows:1.Expression of mixed biofilms of V.parahaemolyticusIn general,biofilms function through collective behaviour and coordinated activities,thus contributing to the survival of constituent cells in stressful environments.Therefore,this experiment investigated the mixed biofilm formed by clinical strains of V.parahaemolyticus carrying different virulence genes.The formation of biofilms was quantified using crystalline violet and the biofilm formation process was analysed dynamically.It was found that VP containing tlh~+/tdh~+/trh~-genes had a higher biofilm formation capacity,followed by VP containing tlh~+/tdh~-/trh~+genes,and that mixtures with other strains that had VP containing tlh~+/tdh~+/trh~-genes had a The mixed biofilm formation capacity was also higher when mixed with other strains containing tlh~+/tdh~+/trh~-genes.The biofilm structure was observed by laser confocal microscopy and scanning electron microscopy,and the differences in the structure of single and mixed biofilms were analysed.ISA analysis showed that the biofilm structure of V.parahaemolyticus clinical strains was mainly dense and smooth,or loose and rough,and the smalller the size of the biofilm,the more stable the structure.This study illustrates the heterogeneity between single and mixed biofilms,and provides a new idea for the control of V.parahaemolyticus biofilms.2.Expression of mixed biofilms of V.parahaemolyticus under bile salt stressV.parahaemolyticus is an important foodborne pathogen in aquatic products and usually causes disease when it enters the human gastrointestinal tract.It has been shown that bile salts play a key role in reconstituting and promoting the absorption of lipids and fat-soluble vitamins and can also induce the formation of biofilms in vivo in some bacterial biofilms.In this study,we investigated the mixed biofilms formed by clinical strains of V.parahaemolyticus carrying different virulence genes under bile salt stress using bile salts(0.10 g/100 m L mass concentration)to simulate the human gastrointestinal environment.The results showed that the clinical strains of V.parahaemolyticus carrying tlh~+/tdh~+/trh~-were the most competent and inhibited the formation of biofilm under bile salt stress;the structure of the biofilm of single and mixed organisms was traced by laser confocal microscopy and scanning electron microscopy,and analysed The results showed that bile salts affected the structure of V.parahaemolyticus biofilm,and the structure of VP biofilm was more compact and stable under bile salt stress.The present study simulated V.parahaemolyticus in the perithelial state in the human gastrointestinal tract and provided a new approach to control the V.parahaemolyticus biofilm.3.Transcriptomics-based analysis of mixed biofilm heterogeneity in V.parahaemolyticusThe genome of V.parahaemolyticus has been extensively studied and it has been found that oxy R regulates biofilm formation;aph A,lus R and opa R are population-sensing related genes that are associated with biofilm and the expression of aph A gene is required for biofilm formation in V.parahaemolyticus.In this study,VPM 1 and VPH,which have a high and stable capacity for biofilm formation,were selected for transcriptome sequencing with VPC 32,VPC 54 and VPC 94 in the periplasmic state to uncover the differentially expressed genes between single and mixed biofilms.It was found that 133genes were up-regulated and 75 genes were down-regulated in the periplasmic VPM 1compared to the periplasmic VPC 32,and 379 genes were up-regulated and 217 genes were down-regulated in the periplasmic VPC 54.VPH in the periplasmic state had 45 up-regulated and 114 down-regulated genes compared to VPC 32 in the periplasmic state,580 up-regulated and 539 down-regulated genes compared to VPC 54 in the periplasmic state,and 600 up-regulated and 367 down-regulated genes compared to VPC 94 in the periplasmic state.These genes were the most pronounced in the cellular fractions,especially in the organic fraction that makes up the membrane,suggesting that these genes are very closely related to the formation of the biofilm.This study provides a theoretical basis for investigating the regulatory mechanisms involved in the formation of the VP biofilm and the mechanisms of bacterial drug resistance.