Antagonistic Activity Of Enterococcus Faecium WEFA23 Against Adhesion And Colonization Of Listeria Monocytogenes

Lactic acid bacteria(LAB)is the key member of probiotics,which has important physiological functions such as regulating micro-ecological balance,enhancing immunity and promoting the absorption of nutrients.Adhesion of LAB to intestinal epithelial cells contributed to promoting probiotics colonization,antagonizing the colonization of pathogens,improving the host immune system,and maintaining intestinal homeostasis.Therefore,the study of adhesion properties of LAB and its antagonistic mechanism against pathogen adhesion should be helpful for understanding the critical role of LAB in gastrointestinal tract.This study aimed to explore the properties of Enterococcus faecium WEFA23.Its antagonistics against the adhesion of pathogens was probed by cell culture model,and the effects on L.monocytogenes colonization and host immunomodulation were further tested in animal model.In chapter one,properties of LAB surface proteins,Enterococcus faecium as well as pathogenesis of Listeria monocytogenes were reviewed,which including the function of LAB surface proteins,the probiotic function of E.faecium,and the pathogenic mechanism of L.monocytogenes.Research trends of E.faecium,its surface proteins and the inhibitory effects on the adherence of pathogenic bacteria were prospected.In chapter two,adhesion properties of E.faecium WEFA23 and 5 pathogens(Escherichia coli O157:H7,Salmonella Typhimurium ATCC13311,Listeria monocytogenes CMCC54007,Staphylococcus aureus CMCC26003 and Shigella sonnei ATCC25931)were systematically evaluated,including the adhesion ability to Caco-2 cells,cell surface hydrophobicity,auto-aggregation capability,co-aggregation ability.The anti-adhesion effects of E.faecium WEFA23 against 5 pathogens by competition,exclusion and displacement were also investigated.The results indicated that 4 pathogen strains(E.coli O157:H7,S.Typhimurium,L.monocytogenes and S.sonnei)excepted S.aureus showed relatively higher hydrophobicity than 2 probiotic strains(P<0.001).However,probiotics showed much higher auto-aggregation than that of all 5 pathogens.In addition,E.faecium WEFA23 exhibited higher co-aggregation with 3 pathogens(L.monocytogenes,S.Typhimurium and S.sonnei)than LGG,and the ratio with L.monocytogenes was the highest.E.faecium WEFA23was able to compete,exclude and displace the adhesion of 5 pathogens on Caco-2cells.Among them,L.monocytogenes achieved the strongest inhibition rate in both competition(53.0%)and displacement(60.8%)assays,while in exclusion assay the highest anti-adhesion ability against S.Typhimurium(56.50%)was observed.Furthermore,the effects of external factors(temperature,growth stage and bacteria concentration)and internal factors(surface protein)on the antagonism of L.monocytogenes adherence by E.faecium WEFA23 were investigated.For L.monocytogenes,the anti-adhesion capacity was affected by heat-treatment,cell density and growth phase of E.faecium WEFA23,10~8 cfu/mL of viable cells at the stationary phase exhibited the strongest anti-adhesion activity.In addition,removal of S-layer proteins of E.faecium WEFA23 significantly(P<0.001)decreased its adhesion capacity.And the anti-adhesion ability of S-layer proteins against L.monocytogenes was dose-dependent from 50 to 200?g/mL,and the displacement reached the highest inhibition rate when compared with competition and exclusion.The results of flow cytometry indicated that both cells and S-layer proteins of E.faecium WEFA23 significantly(P<0.001)reduced the apoptosis of Caco-2 cells induced by L.monocytogenes,which was mediated by caspase-3 activation.In addition,E.faecium WEFA23 and its surface layer proteins significantly inhibited the inflammatory cytokines expression(IL-1?and TNF-?)of Caco-2 cells(P<0.05),but increased anti-inflammatory cytokines IL-10 expression(P<0.001)when compared with L.monocytogenes group.In chapter three,the colonization of E.faecium WEFA23 and L.monocytogenes in mice was analyzed;the immunomodulatory effects of E.faecium WEFA23 on mice were also investigated.The results indicated that E.faecium WEFA23 could improve mice survival rate and significantly reduced the number of L.monocytogenes in liver,spleen,ileum,cecum and colon,and the level of serum ALT and TBA.E.faecium WEFA23 could effectively colonize the jejunum,ileum and cecum.Histological analysis of stomach and liver revealed that E.faecium WEFA23 could effectively protect mice from hepatocyte necrosis and gastric mucosal damage caused by L.monocytogenes.Furthermore,E.faecium WEFA23 significantly decreased the levels of IFN-?and IL-1?(P<0.05)and increased the levels of IL-10(P<0.001)when compared with the infection group.Therefore,based on the results of cell culture in chapter two,it can be concluded that E.faecium WEFA23 regulated host immunity.In chapter four,the antagonism mechanism of pathogens adhesion and immune regulation by E.faecium were summarized,the future research directions were prospected as well.In summary,both cells and S-layer proteins of E.faecium WEFA23 could antagonize the adhesion of L.monocytogenes to intestinal epithelial cells and the apoptosis was caused by L.monocytogenes.E.faecium WEFA23 was able to antagonize the colonization of L.monocytogenes in mice organs.Histological analysis of stomach and liver revealed that E.faecium WEFA23 could protect mice from hepatocyte necrosis and gastric mucosal damage,significantly inhibited the expression of inflammatory factors,and enhanced anti-inflammatory expression.The results of this study provided a scientific basis for the use of E.faecium WEFA23 as a probiotic candidate strain.