Preliminary Mechanism Of Temperature On Biofilm Formation By Vibrio Parahaemolyticus

Vibrio parahaemolyticus(V.parahaemolyticus)is a major food-borne pathogen in aquatic products,which has been the first place of microbial food poisoning in China.V.parahaemolyticus is easily to adhere to the surface of most food and food contact materials,embedded within a matrix of extracellular polymeric substances(including exopolysaccharides,proteins and extracellular DNA)and form a complex three-dimensional membrane structure,called biofilm.Biofilm cells are more resistant to various external environmental factors(disinfectants,antibacterial agents,low temperature,pressure,etc.)than their planktonic form,so it cause serious burden on food processing.In nature,the growth of V.parahaemolyticus is highly correlated with sea surface temperature and seasonal temperature fluctuation.In the process of food processing,V.parahaemolyticus has to to undergo a series of temperature changes and fluctuation.Therefore,it is of great significance for the development of food safety and food industry to study the dynamic formation process of V.parahaemolyticus biofilm and the mechanism of temperature on biofilm formation.V.parahaemolyticus isolated from shrimp and clinical were used in this study.First of all,we analyzed the dynamic processof biofilm formation at different temperatures(15 oC,25 oC,37 oC).Next,the changes of biofilm formation characteristics was investigated after low temperature(4 oC,10 oC)shock of preformed biofilm.Finally,the preliminary mechanism of temperature on biofilm formation was explored using Raman spectroscopy and reverse transcription fluorescence quantitative PCR(RT-qPCR),to provide a theoretical reference for the effective control of biofilm formation.The main content of this study were summarized as follows: 1.Dynamic process of biofilm formation by Vibrio parahaemolyticus at different temperaturesIn this study,crystal violet staining was used to analyze the dynamic forming process of food isolated V.parahaemolyticus at different temperatures,meanwhile,we compared the biofilm-forming ability between clinical isolates and food isolates,and the variation of biofilm architecture during biofilm formation was investigated using confocal laser scanning microscopy.The results show that biofilm biomass continuously increased over the period of 48 h incubation at 15 oC.While biofilm production enhanced gradually and reached a peak at 24 h when cultured at 25 oC,and meanwhile strains showed the strongest biofilm forming capacity.At 37 oC,no significant increase of biofilm production was observed.Besides,the biofilm forming capacity of pathogenicity strains were stronger than that of non-pathogenic strains.In addition,the variation of the strain increased with the increasing of culture temperature,and the variation reached the maximum when the culture time was 12 h.The analysis of the ability of biofilm formation of V.parahaemolyticus from different sources indicated that biofilm forming capability of clinical isolates was significantly higher(p < 0.05)than that of food isolates.2.Effect of low temperature stimulation on the formation of preformed biofilm by Vibrio parahaemolyticusIn this paper,the effects of low temperature(4 oC,10 oC)stimulation on the formation characteristics of preoformed V.parahaemolyticus biofilm and viable count in biofilm after cold shock(4 oC,10 oC)were analyzed.The results showed that the formation rate of biofilm after cold shock was lower in comparison with that at constant temperature incubation of 37 oC,but with the extension of incubation time,the matrix amount of biofilm increased gradually to the same as that of constant temperature culture.EPS analysis showed that the extracellular polysaccharide and extracellular proteins still increased gradually after low temperature stimulation,and reached the maximum at 48 h.Plate counting results indicated that after 60 h cultivation,the number of viable bacteria in biofilm which endure cold shock were significantly higher(p < 0.05)than that of the number without low temperature stimulation.The above results showed that low temperature stimulation could only slow down the speed of biofilm formation,once it adapted to the cold environment,it will growth rapidly and form more biofilm,cold shock can also effectively protect the bacteria,thereby endangering food safety.3.Preliminary mechanism of temperature on biofilm formation of Vibrio parahaemolyticusIn this study,the morphological structure of biofilm at different temperatures was observed by scanning electron microscopy,raman spectroscopy and RT-qPCR were used to explore the mechanism of temperature on V.parahaemolyticus biofilm from the aspect of metabolites and gene level.The results showed that the most tightly structure of biofilm was observed at 25 oC,and the amount of extracellular polysaccharide and extracellular protein was the largest at this temperature,followed by 37 oC,then is 15 oC,and 4 oC is the weakest.Raman spectroscopy analysis indicated that the number and intensity of characteristic peaks at different temperatures had significant differences,the maximum number and the strongest intensity of feature peaks was obtained at 25 oC,4 oC is the weakest.Discrepant feature peaks at various temperarues corresponding to the main substances of carbohydrate,protein and carotenoid.RT-qPCR analysis showed that the temperature mainly influenced biofilm formation by altering the motility and q uorum sensing of bacteria.