Study On The Formation Of Heterogeneous Biofilms On Abiotic Surfaces Of Several Foodborne Pathogens

Biofilm formation on the surface was a viable survival strategy for almost all bacteria.As a remarkable phenomenon exhibited in the interaction between organism and surface,using biofilm as an intuitive entry point to analyze the biotic-abiotic mechanism interaction was a new and appropriate research model to exploring this basic scientific problem.Bacterial biofilm is a complex three-dimensional aggregate of microorganisms that encapsulated by the secretion of protective and adhesive extracellular polymers(EPS)such as polysaccharides,proteins,nucleic acids and lipids.It has the characteristics of difficult removal and high environmental resistance after attached to the surface of medical devices,water system and food processing equipment,easily leading to adverse effects on the utilization of social resources and human life and health[].Consequently,understanding the interactions between bacteria and surfaces is important for controlling biofilm infection in industrial applications.In this study,Vibrio parahaemolyticus(VP),Escherichia coli(EC)?Staphylococcus aureus(SA),Listeria monocytogenes(LM)was used as biotic objects,and the interfaces of three common materials(polystyrene(PS),glass(GS)and stainless steel(SS))were used as abiotic objects,biofilms development of these bacteria on different materials surfaces was monitored under liquid-solid culture.The effects of different surfaces on the formation of BF and the relationship between the physicochemical properties of biofilm and the surface properties of contact materials were studied,Through proteomics technology,the survival adaptation mechanism of the biofilm of Vibrio parahaemolyticus on the surface of different abiotic contact materials was explored.The main contents and results of this study are as follows:(1)In this project,AFM and contact angle analyzer were used to characterize the surface morphology and hydrophobicity of the materials.The auto-aggregate rate(AGG),the biomass of biofilm and the hydrophobicity of biofilm cells of four common food borne pathogenic bacteria,Vibrio parahaemolyticus,Listeriamonocytogenes,Escherichia coli and Staphylococcus aureus on different contact surfaces were also measured.The relationship between the surface roughness,hydrophobicity and the physical properties of bacterial biofilm and the behavior of biofilm formation were explored.Three groups of materials were used in the experiment: GS sheets with hydrophilic and smooth surface(Rq = 2.5 nm,contact angle = 11.25°);SS sheets with hydrophobic and smooth surface(Rq = 4.5 nm,contact angle = 56.90°)and PS sheets with hydrophobic and rough surface(Rq = 48 nm,contact angle = 73.23°).The BATH determination results showed that VP and EC were hydrophilic strains(CSH = 19.87%,CSH = 10.25%),SA and LM were hydrophobic strains(CSH = 30.39%,CSH = 45.67%).Among them,the hydrophilicity of VP cells increased(CSH = 15.13%)after BF was formed on GS surface,while the hydrophilic VP cells became hydrophobic(CSH = 36.30%,CSH =31.87%)after BF was formed on SS and PS surface.However,the CSH values of PB(CSH = 8.08%)and GB(CSH = 7.00%)adhered to PS and GS surfaces increased respectively.The BF cells formed by hydrophobic strain SA on the three surfaces became hydrophilic,while the BF cells formed by hydrophobic strain LM on the SS and PS surfaces were hydrophilic(CSH = 6.22%,CSH = 21.09%,respectively),while they became more hydrophobic on the GS surface(CSH = 59.11%).The results showed that the AAG of bacteria on different contact surfaces gradually increased with time from 1 h to 10 h.Among them,the AAG of hydrophilic strains VP and EC were GS > PS > SS;there were significant differences in AGG of hydrophobic strains SA and LM on different surfaces,which were SS > GS > PS and PS > SS > GS,respectively.The biomass analysis showed that the hydrophilic strains VP and EC could form more BF on the surface of GS,but less BF on the surface of SS;the trend of hydrophobic strains SA and LM to form BF on different surfaces was consistent with the trend of their AGG results.The above results showed that the physical properties of BF cells were significantly affected by different abiotic contact surfaces,and these results varies with the species.(2)In order to further explore the interaction between abiotic contact surface and bacteria,on the basis of previous research in the laboratory,Vibrio parahaemolyticus(VP)was selected as the object of in-depth study.Laser confocal scanning microscope(LSCM)was used to analyze the influence of different contact surfaces on the structure of BF,and the changes of main components(polysaccharide,protein,polysaccharide e DNA)of BF extracellular matrix were measured to study theeffect of abiotic contact surface properties on the production of extracellular substances in BF.LSCM observation showed that the biomass of SB on SS surface were less,its structure was sparse and its distribution was uneven.However,PB on PS surface has compact structure,more cell aggregates and higher biomass.In addition,BF showed a more compact structure on the surface of GS(GB).BF structure analysis showed that the bio-volume(BV)and mean thickness(MT)of GB were significantly larger than those of PB and SB;the porosity(AP)of the three BFs was similar,while the average diffusion distance(ADD)of PB was significantly larger than that of GB and SB(p < 0.001),indicating that the metabolic pressure of BF cells on PS surface might increase;compared with the other two BFs,GB had higher structure entropy(TE),indicating that the structure of GB was more heterogeneous and complex.The analysis of the key chemical components of BF extracellular matrix showed that the content of extracellular protein and e DNA in VP biofilm on different surfaces showed a trend of GS > PS > SS;the content of extracellular polysaccharide showed a significant difference,in turn,PS > SS > GS.Pearson correlation analysis showed that the hydrophobicity of the contact surface was negatively correlated with the production of e DNA,extracellular protein and biofilm biomass,while it was positively correlated with extracellular polysaccharide.The hydrophobicity of VP biofilm cells also showed a similar trend,indicating that the contact surface can indirectly affect the physical properties of the biofilm cells surface by influencing the production of extracellular substances.The results showed that the chemical properties of biofilm of Vibrio parahaemolyticus were significantly affected by different abiotic contact surfaces.(3)In order to explore the physiological response and survival adaptation mechanism of the biofilm cells of Vibrio parahaemolyticus on different abiotic surfaces,the proteomic changes between biofilm adhered on these three surfaces were identified by 2-DE and MALDI-TOF-MS.The results showed that the proteins expressed by Vibrio parahaemolyticus biofilm cells constructed on the surface of SS were mainly involved in the physiological activities of alleviating the stress and toxicity of metal ions;the proteins up-regulated in the biofilm cells formed on the surface of GS were mainly involved in the biological processes such as sugar uptake,protein synthesis and bacterial chemotaxis;the proteins significantly expressed in the biofilm cells formed on the surface of PS were mainly involved in the metabolism of aromatic compounds,osmotic stress,nutrient transport and other cell physiologicalactivities.