Screening And Reaction Mechanism Of Probiotic Bacteria Inhibition Of Shigella Sonnei

Application of probiotics to the prevention of pathogen infection has received much attention due to safety and decreasing drugtolerance. China is rich in lactic acid bacteria (LAB) strains are abundance in China while provides the platform for research and exploition of LAB. In this thesis, effective selection methods were used to screen the probiotic lactobacilli for inhibition of Shigella sonnei growth and adhesion, and the mechanism of inhibition of S. sonnei was discussedOne hundred and ninety-seven strains were selected from traditional Chinese fermented foods and feces, using LGG as the reference strain. According to the well-diffusion assay method, 53 lactobacilli stains were screened for antimicrobial activity against E. coli, S. typhimurium and S. sonnei. In particular, 23 lactobacilli strains were found with high levels of activity against S. sonnei. Meanwhile, we analyzed the adherence of 53 lactobacilli strains on HT-29 cells and 25 of those showed effective adherence to HT-29 cells. The levels of adherence index were higher than 50 bacteria/100cells. According to the two index, 13 lactobacilli with a high degree of antagonistic activity against three pathogens and good adherence to HT-29 cells as selected(M5-L,J10-L,J21-L,Q8-L,X12-L,Q6-L,G15-L,F0421,IN3432,IN3821,IN4025,IN4024,IN3623). The 13 lactobacilli were also investigated in vitro tests for tolerance to simulated gastrointestinal conditions. The results suggested that the tolerance of lactobacilli from feces was higer than that from fermented foods. Finally, the 13 lactobacilli were identified as satisfying the criterion of the Biolog system tests.The nature of antimicrobial substances of 13 lactobacilli and LGG were investigated after 18h fermenting, the results showed that CFCS of antimicrobial activity remained active after proteinase K and heat treatment for the lactobacilli. When the CFCS was adjusted to pH 6.5, the antimicrobial activity was abolished, indicating that bacteriocins were not involved in the antimicrobial activity of the tested strains. Therefore it is believed that this activity was attributed to the production of organic acids. Time-kill assay showed that the viability of the S. sonnei was decreased by 2.7-3.9 log cfu/ml after contact with CFCS of tested strains for 2 h. However, the viability of the S. sonnei disappeared after contact for 3 h. When the pH of the CFCS was adjusted to pH 6.5, no killing effect was observed. In addition, the killing effect of the CFCS of pH 6.5 was not significantly higher than that of the control samples, which confirmed the result that antimicrobial substances produced by 13 lactobacilli and LGG were indeed organic acids. Further analysis of the organic acid composition in the CFCS revealed that the content of lactic acid ranged from 187 to 293 mmol/L.The ability of the 13 lactobacilli to exclude, compete and displace the adhesion of S.sonnei to intestinal mucosa was investigated on cultured HT-29 cells. The M5-L, J10-L, Q8-L and F0421 strains with significant inhibitory activity were screened, M5-L and F0421 strains showed well in the exclusion, competition and displacement assays all. After treatment with LiCl, metaperiodate and heat on M5-L, J10-L, Q8-L and F0421 strains, the inhibition activity decreased significantly, which indicated the S-layer protein played an important role in the inhibition activity. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant bands of approximately 45 kDa in M5-L, J10-L, Q8-L strains and 40 kDa in F0421 strains. Further analysis of S-layer proteins revealed that the hydrophobic amino acids accounted for 40.5%, 41.5%, 43.8% and 35.4% of the total amino acid for the M5-L, J10-L, Q8-L and F0421 strains, respectively. These findings suggest that S-layer proteins are involved in this adhesion inhibition. Because M5-L and F0421 strains showed well in the exclusion, competition and displacement assays, we selected two strains for further study.Finally, M5-L and F0421 strains protection of epithelial barrier function from S.sonnei infection was assayed. The result of immunohistrochemistry stain showed that F0421 protection by preventing the expression of ZO-1 and occludin proteins was induced by S.sonnei, M5-L protection by preventing the expression of occludin proteins was induced by S.sonnei. ELISA experiment of different intervention groups showed that the M5-L and F0421 strains counteracted the S.sonnei-induced increase of IL-8 production and protected the epithelial barrier damage. However, the different intervention groups did not significantly affect the production of IL-10.The M5-L and F0421 strains were futher identified as Lactobacillus paracasei subsp paracasei and Lactobacillus johnsonii by 16SrDNA, respectively.