Study On The Mechanism Of Extracellular DNA In Biofilm Formation Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus?VP?has been considered as the main human pathogen in marine bacteria because of the increasing number of outbreaks and infections,and it has the ability to form biofilm?BF?on the surface of food processing facilities and aquatic products.Biofilms are complex communities of microorganisms that are embedded in a self-produced extracellular polymeric substances?EPS?comprised of polysaccharides,proteins,and extracellular DNA?e DNA?.Although EPS provide structural support to maintain the biofilm stability of V.parahaemolyticus,their respective functional role is still unclear.Therefore,this study aimed to illuminate the role of key chemical components e DNA of EPS in biofilm formation of V.parahaemolyticus.This study may provide a further understanding of biofilm formation mechanisms of V.parahaemolyticus,and also give novel insight to establish environmentally friendly cleaning methods to eliminate the biofilms in food industry.In this study,we initially focus on the difference analysis of biofilm formation of V.parahaemolyticus from different sources and genotypes.Secondly,V.parahaemolyticus ATCC17802 was selected as a model organism to investigate the role of e DNA in the biofilm development.The results showed that there might be other extracellular matrix components to maintain the structural stability of the mature biofilm,or e DNA and other extracellular matrix components to construct the skeleton structure of the mature biofilm together.Finally,further explore whether other extracellular matrix components work in the biofilm formation of V.parahaemolyticus and the relationship with e DNA.The main research contents and results are as follows:1.The difference analysis of biofilm formation of V.parahaemolyticus from different sources and genotypesIn this study,crystal violet staining was used to analyze the dynamic formation process of VP biofilm from different sources?clinical and food?and genotypes?pathogenic and non-pathogenic?,and to compare the BF formation ability of VP strains isolated from clinical and food,as well as that of pathogenic and non-pathogenic VP strains.The results showed that the biofilm formation ability of clinical isolated and pathogenic VP strains was higher than that of food isolated and non-pathogenic VP strains.The maturation cycle of clinical isolated and pathogenic VP strains BF is 24hours and has a small coefficient of variation,while the food isolated and non-pathogenic VP strains BF has a maturity cycle of 48 hours and a large coefficient of variation.In addition,the maximum specific growth rate(?_max)of VP was also determined and Spearman correlation analysis showed that there was no correlation between the?_max of VP strains and the source and genotype?r_p=0.095,P>0.05,r_p=-0.235,P>0.05?.2.The role of e DNA in the biofilm formation of V.parahaemolyticusFirst,the crystal violet staining method,Confocal laser scanning microscopy?CLSM?,ISA software analysis and scanning electron microscopy?SEM?were used to observe the growth of V.parahaemolyticus ATCC17802 biofilm.The results showed that ATCC17802 strain formed the mature biofilm after 24 hours of culture.Then we used the crystal violet staining method to evaluate the effect of DNase I enzyme treatment and the addition of exogenous DNA on the biofilm formation of Vibrio parahaemolyticus.The results showed that e DNA mediated the initial adhesion of VP and promoted the early development of biofilm.Besides,DNase I enzyme treatment experiment also found that the early biofilm was very sensitive to enzyme treatment,but the mature BF could not be significantly damaged by DNase I enzyme,which indicated that there might be other extracellular matrix components involved in the construction of VP mature biofilm.3.The effect of e DNA and other extracellular matrix on the structural stability of V.parahaemolyticus biofilmIn this study,the changes of e DNA,extracellular proteins and exopolysaccharides content during V.parahaemolyticus biofilm formation was analyzed by chemical method.Pearson correlation analysis showed that the content of e DNA and extracellular proteins were positively correlated with the biofilm formation of V.parahaemolyticus?P<0.01?,but the exopolysaccharides content showed no correlation.The CLSM combined with ISA software analyzed and evaluated the effect of DNase I enzyme,proteinase K and their combination?DNase I-proteinase K?on the biofilm.The results showed that individual DNase I or protease K treatment didn't induce significant dispersion of mature biofilms,while the DNase I-protease K treatment induced the obvious dispersion of the mature biofilms through the concurrent destruction of e DNA and extracellular proteins,indicating that e DNA and extracellular proteins are the skeleton structure of the mature VP biofilm,and both of them maintained the structural stability of biofilm.Raman spectroscopy?RM?analysis showed that the collapse of biofilms was mainly attributed to the great damage of the loop-configuration of e DNA and the secondary structure of proteins caused by the enzyme treatment.