Mixed Biofilm Formation And The Interspecies Relationship Between V.parahaemolyticus And L.monocytogenes

A biofilm is defined as a microbial community composed of microorganisms adhered to a solid surface and embedded in an extracellular polymer matrix(EPS)secreted by itself.The matrix provides protection for internal cells and helps microorganisms to absorb nutrients and to adhere to surfaces.Biofilm formation is a social behavior.Cells secrete small signaling molecules to regulate cell density,and make feedback regulation by expressing initial adhesion target sites,biofilm growth,and mature cell function expression.In the food industry,biofilms are easily formed on food surfaces and equipment surfaces(food contact and non-food contact).The biofilms formed of spoilage bacteria or pathogenic bacteria can accelerate food spoilage,increase equipment loss and cause food safety problems,reducing food Quality and food safety.In addition,multi-species biofilms are the predominant form of biofilms found in most cases,and the relationship between different bacterial species and the characteristics are different.Therefore,the same set of control strategies cannot be applied.It brings great challenges and burdens to us.Therefore,it is imperative to study mixed biofilms.Vibrio parahaemolyticus(VP)is a common pathogen of aquatic origin.Humans infected with pathogenic one are likely to cause acute gastroenteritis and even death from shock.Listeria monocytogenes(LM)is widely present in various types of foods.It can cause meningitis,sepsis,miscarriage,stillbirth and other diseases after infection in humans and animals.The fatality rate is as high as 30%to 70%.Several reports in China have shown that the high detection rate of V.parahaemolyticus and the low detection rate of L.monocytogenes can be simultaneously detected in aquatic products and frozen surimi products sold in coastal areas of China.This means that while studying V.parahaemolyticus in aquatic products,Listeria monocytogenes cannot be ignored.In this study,V.parahaemolyticus(standard strain ATCC17802 and isolated strain VP24 from salmon)and L.monocytogenes(standard strain ATCC19115 and isolated strain LM5 from salmon)were used to study their characteristics and the relationship between the two bacteria.This study can provide a theoretical reference for the future formulation of strategies to prevent food safety issues in aquatic products.The main research contents and results of this article are as follows:1.Mono and mixed biofilm formation of V.parahaemolyticus and L.monocytogenes at storage temperature(4?),room temperature(25?)and human body temperature(37?)In this study,the CV assay,q PCR,laser confocal scanning microscope(CLSM)and XTT reduction were performed to analyze the biofilm formation ability,cell number,activity of mono and mixed biofilm under different temperature.It was found that the growth rate of single and mixed biofilms was affected by temperature as follows:37?>25?>4?(for V.parahaemolyticus);25?>37?>4?(for L.monocytogenes);37?>25?>4?(for mixed biofilm).When the two species are mixed and cultured to form a mixed biofilm,the biomass of the mixed biofilm is reduced and the metabolic activity is reduced at 25?and 37?,and the viable cell number ratio of V.parahaemolyticus in the mature mixed biofilm.The number of live cells of L.monocytogenes was 10~2 folds higher.However,at 4?,the formation of mixed biofilms has a similar trend as that of L.monocytogenes.In this study,CLSM method,fluorescence original hybridization(FISH),scanning electron microscopy(SEM),RT-q PCR,Raman analysis and chemical analysis were used to study the structural characteristics and gene expression analysis of mixed biofilm at room temperature.It was found that,the biological volume of the mixed biofilm is reduced by about 2 folds,the average thickness is less than 1/2 of the mono biofilm,and the porosity is increased by about 30%compared with the mono biofilm.In the mixed biofilm,most of the V.parahaemolyticus were located on L.monocytogenes,the biofilm volume formed by V.parahaemolyticus is significantly higher than that of L.monocytogenes,and the extracellular polysaccharides and proteins are significantly reduced.The genes related to the biofilm formation aph A,opa R and oxy R of V.parahaemolyticus and fla E,mot B,deg U and fla A of L monocytogenes were significantly down-regulated.It shows that the two bacteria are not conducive to the formation of mixed biofilm.3.The effect of supernatant of L.monocytogenes on the biofilm formation and cell proliferation of V.parahaemolyticusIn this study,the CV assay,Growth curve,CLSM,scanning electron microscope(SEM),XTT reduction and RT-q PCR were carried out to study the effect of L.monocytogenes metabolites in the supernatant culture on biofilm formation and cell proliferation of V.parahaemolyticus.The sterilized supernatant of L.monocytogenes and V.parahaemolyticus were used to independently culture the biofilms of the two bacteria.It was found that the supernatant of L.monocytogenes can promote the biofilm formation of V.parahaemolyticus and cell proliferation,manifested in a significant increase in the biomass,enhancing metabolic activity,small porosity,an increase in the maximum growth rate,a significant up-regulated of gene msh A related to quorum sensing.However,the supernatant of V.parahaemolyticus had an inhibitory effect on L.monocytogenes.It shows that there is an imbalance between the two species.