The Inhibition Mechanism Of Epigallocatechin Gallate Against Escherichia Coli O157:H7

Epigallocatechin gallate(EGCG)is the most effective and abundant ingredient in tea polyphenols,which has significant antimicrobial effects on Gram-negative and Gram-positive pathogens.Escherichia coli O157:H7 is a food-borne pathogen,which produces Shiga toxin(Stx)and causes many diseases.Shiga toxins are divided into Stx1 and Stx2,which are encoded by the stx1 and stx2.stx1 and stx2 are located on the lysogenic bacteriophages which are integrated into the chromosome of E.coli O157:H7.The activation and release of Stx are induced by bacterial SOS response.In addition to the pathogens themselves,the toxins are also a threat to the food industry and patient.In recent years,the application of EGCG in food preservation has been popular,but its antibacterial mechanism remains unclear.In this study,the antibacterial and antitoxin effects of EGCG against E.coli O157:H7 and the underlying mechanism were investigated.The main results of the study were as follows:1.Inhibitory effects of EGCG on the growth of E.coli O157:H7.The minimum inhibitory concentrations(MIC)of EGCG and citric acid against E.coli O157:H7 EDL933 were determined by microdilution method,respectively.The MIC of EGCG was 1.024mg/mL and the MIC of citric acid was 4mg/mL.E.coli O157:H7 EDL933 was incubated in broth or inoculated on the surface of chilled meat artificially.Then the two samples were treated with 1mg/mL EGCG,5mg/mL EGCG,4mg/mL citric acid,and 1mg/mL EGCG combined with 4mg/mL citric acid,respectively.The viable cell number of E.coli O157:H7 EDL933 was measured by plate colony counting.Compared with PBS control,both citric and EGCG could reduce the cell number of E.coli O157:H7 EDL933.5mg/mL EGCG had the strongest inhibitory effect,followed by 1mg/mL EGCG combined with 4mg/mL citric acid,4mg/mL citric acid alone and 1mg/mL EGCG alone.2.Effects of EGCG on the gene and protein expression of E.coli O157:H7.E.coli O157:H7 EDL933 was incubated to stationary phase or exponential phase,then treated with 1mg/mL EGCG,5mg/mL EGCG,4mg/mL citric acid,and 1mg/mL EGCG combined with 4mg/mL citric acid,respectively.The gene expression of E.coli O157:H7 EDL933 was detected by quantitative PCR.Genes used in Q-PCR analyses were shiga toxin genes stx1 and stx2,Q(late antitermination gene of Stx-producing prophage),recA(SOS response inducer),porin encoding gene ompC and genes participating in stress responses,such as rpoS and oxyR.The expression of RecA protein in E.coli O157:H7 EDL933 was further detected by Western blot.The transcription levels of stx1,Q and recA genes could be reduced by EGCG alone and citric acid alone.However,1mg/mL EGCG combined with 4mg/mL citric acid can induced the expression of stx1,stx2,Q and recA genes.1mg/mL EGCG suppressed ompC expression,while 5 mg/mL EGCG increased ompC expression.Both EGCG and citric acid induced the expression of oxyR.The results of Western blot showed that the expression of RecA protein decreased after treated with 1mg/mL EGCG,5mg/mL EGCG and 4mg/mL citric acid in vitro.3.Effects of EGCG on the biofilm formation of E.coli O157:H7.Sub-MIC of EGCG(0.125mg/mL,0.25mg/mL and 0.5mg/mL)could inhibit the biofilm formation and swimming ability of E.coli O157:H7 EDL933.4.Effects of EGCG on the cell membrane of E.coli O157:H7.E.coli O157:H7 EDL933 was treated with EGCG at various concentrations for 1hour in vitro,then its morphology was observed under a fluorescence microscope after stained with PI dye.EGCG at concentrations of 1mg/mL,2.5mg/mL and 5mg/mL could destroy the cell membrane,thus leading to the death of E.coli O157:H7 EDL933.The higher the concentration of EGCG tested,the more of the cell death.E.coli O157:H7 EDL933 was treated with EGCG,then the outer membrane permeability and the inner membrane permeability were detected by NPN and ONPG,respectively.The results showed that EGCG at low concentrations(0.125mg/mL,0.25mg/mL,0.5mg/mL,and 1mg/mL)had little impacts on the outer membrane permeability and the inner membrane permeability of E.coli O157:H7 EDL933.5.Effect of EGCG on intracellular ROS of E.coli O157:H7.EGCG caused an increase in intracellular ROS levels in E.coli O157:H7 EDL933,thus inducing the oxidative stress.6.The result of drug susceptibility assay showed that 1mg/mL EGCG treatment did not affect the sensitivity of E.coli O157:H7 EDL933 to antibiotics.In this paper,it was found that the transcriptional responses of E.coli O157:H7 EDL933 to EGCG and citric acid were different in exponential phase and stationary phase.In addition,it was shown that tea polyphenol EGCG reduced the expression of stx1 by inhibiting the SOS reaction in exponential phase,but it induced the expression of stx2 by producing oxidative stress.EGCG at sub-MIC concentrations inhibited the biofilm formation of E.coli O157:H7 EDL933.EGCG combined with citric acid treatment could enhance the antibacterial effect,while it could activate the bacterial SOS reaction.These results revealed that tea polyphenol EGCG could be used as an excellent natural preservative in food production.Further studies need to conduct to clarify the mechanism by which bacteria and Shiga toxin are suppressed by EGCG.