Study On The Key Factors Of Vibrio Parahaemolyticus Biofilm Formation

Vibrio parahaemolyticus is a gram-negative bacterium which is ubiquitous in shallow seas.It can form a biofilm on the surface of some seafood,thereby contaminating the seafood.Biofilm is a series of extracellular substances secreted by bacteria though a self-protection mechanism under unfavorable living environment conditions,and the resulting polymer wraps around the flora.Nowadays,food safety issues caused by Vibrio parahaemolyticus biofilm have attracted more and more attention.At present,the research of Vibrio parahaemolyticus biofilm mainly focuses on the influence of different external conditions on the formation and removal of the biofilm,as well as the modification of the internal genes of Vibrio parahaemolyticus to study the formation of the biofilm.However,the specific formation process of the entire biofilm is still not well understood,and further research is needed.In this project,five different strains of Vibrio parahaemolyticus were selected,and two strains with large differences in biofilm formation were selected for further analysis,Key factors for the formation of biofilms were analyzed to provide a certain theoretical basis for the prevention and control of biofilms.The main research contents and results are as follows:(1)First,the differences in the formation of biofilms between 5 species of Vibrio parahaemolyticus were explored.Secondly,through the analysis of the membrane wall characteristics of the bacteria’s flagella motility,cell self-aggregation and cell surface hydrophobicity.The results show that the strength of flagella motility can affect the contact ability between flagella and non-living media,and has a certain promotion effect on the formation of biofilm;the higher the self-aggregation between cells,the better the recognition and aggregation between cells.Conducive to the formation of biofilm;Higher self-aggregation between cells results a better recognition which can promote the formation of biofilm.Hydrophobicity of the cells can enhance the adhesion of the cell itself which can also enlarge the formation of the film.Shortly,the characteristics of the membrane wall of the bacteria themselves play an important role in the reversible adhesion of the biofilm in the early stage.(2)In order to reach a further understanding the mechanism of biofilm formation,Transcriptome sequence was carried out to analysis VP-0 and ATCC17802,whch has the largest differences between the ability of biofilm formation.The results showed that there were a total of 433 significantly different genes,of which 219 were up-regulated genes and 214 were down-regulated genes.Combining GO analysis and KEGG Pathway analysis,it is found that glycan biosynthesis and metabolism,ABC transporters,flagellar assembly,carbohydrate metabolism,catalytic activity have an important impact on the formation of biofilms.Genes related to extracellular polymer synthesis and flagella synthesis are down-regulated,which indicates that during the irreversible adhesion of biofilms,VP-0 cannot secrete a large amount of extracellular polymers and flagella cannot form normally,which has a great influence on the formation of biofilms.(3)In order to have a deeper understanding of the formation-composition mechanism of extracellular polymers,firstly observe the morphology of single colonies and biofilm formation of SEM electron microscopy of Vibrio parahaemolyticus VP-0 and ATCC17802.Secondly examine the extracellular polymers which secreted in the process of biofilm formation.Finally,RT-PCR analysis of genes related to the synthesis of capsular polysaccharides in extracellular polysaccharides.The results showed that the difference of extracellular polymer composition and the amount of extracellular polymer made the colony morphology different.The formation of biofilm has a positive correlation with the increase of extracellular polysaccharide while the extracellular protein do not has the trend.Then RT-PCR analysis of capsular polysaccharide-related genes in extracellular polysaccharides showed that the expression of capsular polysaccharide-related genes increased,and the synthesis of VP-0 capsular polysaccharides increased,which was consistent with the increase in VP-0 extracellular polysaccharides and appearance.However,this increase is not conducive to the formation of strong adhesive biofilms,and the content of extracellular polysaccharide of ATCC17802 is much higher than VP-0,which indicates that the massive secretion of extracellular polysaccharide promotes the formation of biofilm of ATCC 17802,while the specific mechanism need to be further studied.