Study On Formation Characteristics Of Vibrio Parahaemolyticus Biofilm

Foodborne pathogenic bacteria could form biofilm on the surface of food or food processing, transportation and storage equipments. The bacteria of biofilm formation would pollute the total production environment randomly through the way of remnanting, contacting, spreading microbes or aerosols pollution, which can be easy to be ignored. Therefore, foodborne pathogenic bacteria that formed biofilm on the surface of the food contact materials could trigger food safety risk. And these put forward a serious challenge for the food quality safety and people’s life and seriously hindered the development of related industries, which caused a certain degree of negative impact to the national economy. Generally, the mechanism study of biofilm formation on the surface of common food contact materials has important application value.In this paper, the primary foodborne pathogenic bacteria Vibrio parahaemolyticus was chosed as the research object. The crystal violet staining method, combining with scanning electron microscope and laser confocal Raman spectroscopy were applied to ananlyze biofilm formation on food contact materials at different temperature conditions. Finally, proteomics was used to further study preliminary mechanism of biofilm formation on three contacts were analyzed. The main content of this study were summarized as follows:(1) Crystal violet staining method was applied to quantify biofilm-forming ability of V. parahaemolyticus(n = 39) on different contact materials(polystyrene, glass and stainless steel) with regard to various temperatures(4, 10, 15, 25 and 37 oC). The results showed that V. parahaemolyticus adhered better at high temperatures(25 and 37 oC) than that at low temperatures(4- 15 oC). Meanwhile, 25 oC was the optimal temperature for biofilm formation of V. parahaemolyticus. Compared with the other two materials, the level of bacterial biofilm formation was highest on glass. Based on coefficient of variation(CV) analysis, it displayed that the increase in strain variability of biofilm formation caused by temperature was much greater than that caused by material. Further analysis of the results revealed that the pathogenic bacteria can adhered significantly(p < 0.05) better than no-pathogenic bacteria.(2) Raman spectroscopy technology was used to do qualitative and quantitative analysis of secretion chemical composition by the VP-S36 strain during the process of biofilm formation. VP-S36 strain experienced four stages, adhesion period(0- 12 h), development period(12- 36 h), maturation period(48 h) and degradation period(48 h later). Biofilm architecture of VP-S36 strain was the dominant honeycomb-like morphotype. In the process of biofilm formation, VP-S36 strain will continue to secrete extracellular material and cell can elongate, up to about 12 microns. Sixty-five advantage raman spectral characteristic peaks were discovered on extracellular polymeric substance. The results showed that the main components are carbohydrates, protein, lipid, nucleic acid, carotenoids, and so on. Among them, there are 36, 21, 18, 9, 2 characteristic peaks on behalf of protein, polysaccharides, nucleic acid, lipid and other components, respectivelly. This study found 18 marker peaks and provided a theoretical basis for structure and function associated with extracellular polymer and the further study of complex biofilm systems.(3) Based on the view of proteomics,differential proteins of the biofilm by VP-S36 strains on polystyrene, glass and stainless steel material surfaces were studied. Bacteria can adhered significantly(p < 0.05) better on glass surface. The maximum amount of biofilm extracellular protein and polysaccharide were also presented on glass. And the genes expression level of biofilm related genes(pilA, mshA, opaR and aphA) were highest on glass surface, then polystyrene. Additionally, on these three kinds of food contact materials, 156 differential protein spots of biofilm were revealed, including 123 spots on polystyrene, 135 spots on glass and 57 spots on stainless steel surface. The results of the Gene ontology(GO) enrichment analysis displayed that small molecule metabolic process and single-organism process play a leading role in biological process. When V. parahaemolyticus formed discrepant biofilm on three contact surfaces, cell components analysis mainly were reflected on cytosol and cytoplasmic part, and metal ion binding and protein binding were the mainly molecular function. Because of these, bacteria coulde undergo dramatic elongation, secretion of extracellular polymeric substances and bind metal positive ion around for attaching to different contact surfaces. KEGG pathway analysis showed that the biosynthesis of secondary metabolites pathway plays a more important role on differential biofilm formation on three surfaces. Finally, protein interaction analysis displayed that chaperone protein DnaK and outer membrane protein TolC may regulate the process of biofilm formation by V. parahaemolyticus on various contact surfaces. This study provides basic data for subsequent research of different biofilm formation by Vibrio parahaemolyticus on contact materials, meanwhile provides meaningful goals for further analysis of biofilm formation mechanism.