The Antibacterial Mechanism Of Mode Of Epigallocatechin Gallate Against Listeria Monocytogenes

Listeria monocytogenes is an important food-borne pathogen that may contaminate meat,dairy product,egg,sea food and vegetables.Biofilm is a structure composed of bacteria and their own extracellular matrix,such as polysaccharides,proteins and DNA.Listeria monocytogenes is capable of developing biofilms.Biofilm formation enhances the risks of Listeria monocytogenes contamination in foods.Listeria monocytogenes could produce listeriolysin O?LLO?and phosphatidylinositol-specific Phospholipase C?PI-PLC?,invades host cells and disrupts barriers,therefore causes diseases.Tea extract contains various polyphenolic compounds.Among tea polyphenols,epigallocatechin-gallate?EGCG?has the highest amount and the greatest antibacterial effects against a number of food-borne pathogens such as Listeria monocytogenes.Though the application of EGCG in food industry is promising,the mechanisms underlying the antibacterial properties of EGCG are still not well documented.Therefore,in this paper,Listeria monocytogenes type strain ATCC 19114 was used and the inhibitory effects of low concentrations of EGCG on the biofilm formation and virulence were investigated.The main results obtained in the paper were listed as belows.1,Assay of EGCG influence on biofilm formation.The minimum inhibitory concentration?MIC?of EGCG for Listeria monocytogenes ATCC 19114 was determined to be 200?g/mL by the standard broth microdilution method.And the minimum bactericidal concentrantion?MBC?of EGCG against Listeria monocytogenes ATCC 19114 was3.2mg/mL.Listeria monocytogenes ATCC 19114 was grown on polystyrene microtiter plate to develop biofilms.The incubation times were 6 days for 15?-grown biofilms and72h for 30?-and 37?-grown biofilms.Crystal violet staining and conoly plate counting showed that sub-MIC?20?g/mL and 40?g/mL?and MIC of EGCG could significantly reduce the biofilm formation by Listeria monocytogenes ATCC 19114.Listeria monocytogenes ATCC 19114 was grown on BHI agar?0.3%?containing different concentrations of EGCG.The result showed that sub-MIC?20?g/mL and 40?g/mL?of EGCG could significantly reduce its swimming motility.Reverse transcription real time PCR showed that EGCG?40?g/mL and 200?g/mL?down-regulated agrA expression.EGCG might reduce biofilm formation by suppressing quorum-sensing system.2,Assay of EGCG influence on virulence.Listeria monocytogenes ATCC 19114 was grown in BHI broth containing different concentrations of EGCG until the culture reached stationary phase.Then the supernatant was used to measure the lysis ratio of sheep red blood cells?SRBC?.EGCG?40?g/mL and 200?g/mL?reduced the lysis ratio of SRBC compared with control,indicating that EGCG could inhibit the hemolytic activity of Listeria monocytogenes.PI-PLC activity was measured by using egg yolk lecithin hydrolysis assay.EGCG?40?g/mL and 200?g/mL?inhibited the degradation rate of egg yolk lecithin compared with control,indicating that EGCG inhibited PI-PLC activity of Listeria monocytogenes.EGCG and Listeria monocytogenes ATCC 19114 were incubated together for 18h,centrifuged and washed.The bacterial cells were then collected and treated with polymyxin B for 30min.The supernatants were used to induce mice macrophage RAW264.7.Compared to control,EGCG?40?g/mL,200?g/mL and 1mg/mL?inhibited the cell apoptosis of mice macrophage.Therefore,EGCG could inhibit the virulence of Listeria monocytogenes.EGCG?40?g/mL and 200?g/mL?also down-regulated listeriolysin O encoding gene hly,phospholipases encoding gene plcA and invasion protein encoding gene inlA.3,Assay of EGCG influence on oxidative stress.Listeria monocytogenes ATCC 19114was treated by different concentrations of EGCG?20?g/mL,40?g/mL,200?g/mL and1mg/mL?,respectively.The levels of intracelluar ROS,H₂O₂ and SOD were measured.Sub-MIC of EGCG?20?g/mL and 40?g/mL?significantly reduced the levels of intracelluar ROS and H2O2,but it could not affect SOD activity.High concentration of EGCG?1mg/mL?significantly increased the levels of ROS and H₂O₂,while decreased SOD activity.Sub-MIC of EGCG decreases Listeria monocytogenes ROS,while high concentration of EGCG may activate oxidative stress,thus inhibiting bacteria growth.The gene recA is associated with SOS response and yneA is involved in oxidative stress.Low concentrations of EGCG?40?g/mL and 200?g/mL?down-regulated recA and yneA expression.Taken together,sub-MIC of EGCG significantly inhibited the biofilm formation.Low concentrations of EGCG inhibited the virulence of Listeria monocytogenes by reducing PI-PLC activity and hemolytic activity.In conclusion,EGCG is potential to be used in food industry to control Listeria monocytogenes growth and biofilm formation.