Inhibition Of Vibrio Mimicus Biofilm And Virulence By Tea Polyphenol EGCG

Vibrio mimicus is a foodborne pathogen that may contaminate fish,shrimp,crab,shellfish and other aquatic products.Vibrio mimicus may carry virulence fectors such as adhesin entero toxin and hemolysin,thus causing diseases.Bio film is a structure composed of bacteria and the extracellular matrix they produce including polysaccharide,protein and DNA.Vibrio mimicus is capable of forming biofilm.The existence of bio film increases the risk of Vibrio mimicus contamination.Epigallocatechin gallate(EGCG)is the most effective and abundant ingredient in tea polyphenols.It has significant antibacterial effects on Gram-positive and Gram-negative pathogens.In recent years,the application of EGCG in dental health and food preservation has been popular,but the inhibition effects of EGCG against Vibrio mimicus remain unkown.Therefore,in this paper,a Vibrio mimicus strain SNJ isolated from silver caqp was used.The effects of EGCG on the bio film formation and virulence of Vibrio mimicus were studied and the influences of EGCG on the cell membrane were investigated to elucidate the potential mechanism of action.The main results of this paper were listed as belows:identification of Vibrio mimicus SNJ and characterization of its biofilm formation.A strain named SNJ was isolated from silver carp.The strain SNJ was further identified by morphobgical characteristics,physioehemical properties and 16SrDNA sequence analysis.The results showed that the strain SNJ was identified as Vibrio mimicus.Vibrio mimicus SNJ was cultured in different mediums at 28? for 5 days,and the amounts of biofilm were determined by crystal violet staining every 24 hours.The results showed that Vibrio mimicus SNJ could form strong bio films in LB,BHI,NB,TSB,1/2LB and MH medium,and the most strongest biofilm was formed in LB medium at 28? on the third day.The composition of Vibrio mimicus SNJ biofilm was determined.The results showed that the extracellular natrix of Vibrio mimicus biofilm was eomposed of protein,polysaccharide and DNA.The dry weight of biofilm consisted of 4.295%protein,0.441%polysaccharide and 0.04%DNA.2.Sub-MIC EGCG inhibited biofilm formation by Vibrio mimicus SNJ.The minimal inhibitory concentration(MIC)of EGCG against Vibrio mimicus SNJ was determined to be 256?g/mL by the standard broth microdilution method.Vibrio mimicus SNJ was cultured on polystyrene microtiter plate to develop biofilm.Biofilms were grown at 15? for 11 days,28? for 6 days and 37? for 5 days.The amounts of bbfilm were measured every 24 hours.The results of crystal violet staining showed that EGCG at the sub-MIC(64 ?g/mL,128 ?g/mL)and the MIC(256 ?g/mL)could significantly inhibit the bio film formation of Vibrio mimicus SNJ.Vibrio mimicus SNJ was grown on LB agar(0.3%)containing different concentrations of EGCG Plates were incubated at 15? for 72 h,28? for 24 h and 37? for 20 h.The results showed that EGCG at sub-MIC(64 ?g/mL,128 ?g/mL)and MIC(256?g/mL)could significantly inhibit the swimming ability of Vibrio mimicus SNJ.Vibrio mimicus SNJ was cultured on 0.5%agar medium containing EGCG at 28?for 24 h.The results showed that sub-MICs of EGCG(64?g/mL,128 ?g/mL)had no effects on swarming motility.Vibrio mimicus SNJ was incubated with EGCG at 37 ?for 3 days in 8-well chanber slide.After staining with PI and FITC-ConA,the bio film was observed by confocal laser scanning microscopy.The results showed that EGCG at sub-MIC(32?g/mL)could inhibit biofilm formation and reduce biofilm thickness.3.Effect of sub-MIC EGCG on virulence of Vibrio mimicus SNJ.PCR assays were performed to amplify the enterotoxin genes ctx,st,zot and hemolysin gene vmh from Vibrio mimicus SNJ.The results showed that Vibrio mimicus SNJ did not carry enterotoxin gene,but had hemolysin gene.EGCG and Vibrio mimicus SNJ were incubated together for 24 hours.After centrifuged and washed,the bacteria cells were collected and treated with polymyxin B.The supernatants were used to induce mice macrophage RAW264.7.Compared to the control group {Vibrio mimicus without EGCG trreatment),the bacteria treated with EGCG(64 ?g/mL and 256 ?g/mL)significantly inhibited the apoptosis rate of mice macrophage,indicating that EGCG could reduce the virulence of Vibrio mimicus SNJ.4.Effect of EGCG on oxidative stress of Vibrio mimicus SNJ.After treated with different concentrations of EGCG,the levels of ROS,H2O2 and SOD of Vibrio mimicus SNJ were measured.The results showed that low concentrations of EGCG(64?g/mL,128?g/mL,256 ?g/mL)and high concentration of EGCG(512 ?g/mL)could significantly increase the levels of ROS and H2O2,but significantly inhibit the SOD activity,resulting in the oxidative stress reaction of Vibrio mimicus SNJ.5.Effect of EGCG on cell membrane of Vibrio mimicus SNJ.Vibrio mimicus SNJ was exposed to different concentrations of EGCG,The integrity and cell membrane potential of the bacterial membrane were checked by flow cytometry using two different fluorescent probes,namely PI and DiBCA4(3),respectively.The results showed that 64 ?g/mL,128 ?g/mL and 256 ?g/mL EGCG could damage the cell membrane integrity and change the cell membrane potential The outer membrane permeability and inner membrane permeability were detected by NPN and ONPG,respectively The results showed that 1MIC EGCG increased inner membrane permeability of Vibrio mimicus SNJ.In summary,it was found that sub-MIC of EGCG could significantly inhibit the biofilm formation and reduce the virulence of Vibrio mimicus SNJ.EGCG may damage the cell membrane structure by inducing oxidative stress,change the crosslinking state between outer membrane and peptidoglycan,and then change the secretion of outer membrane vesicles,leading to inhibition of biofili formation and virulence of Vibrio mimicus SNJ.These results indicate that EGCG can be used as a natural preservative in food preservation to control biofilm formation of Vibrio mimicus SNJ.Further research is needed to conduct to clarify the mechanism of action of EGCG.