Screening Of Potential Lactobacillus Against Pathogens Causing Diarrhea And Its Mechanism Of Inhibition Of Diarrhea

Diarrhea is the main causes of human deaths in developing countries, which also cause world-wide infant mortality. Modern antibiotics are powerful drugs and often lifesaving, but, as with all medications, side effects may sometimes occur. The most important is that it often causes relapse so using probiotics with drugs is the future trend of development in prevention and treatment of diarrhea. In recent years, the interesting of treatment of diarrhea caused by pathogens using Lactobacillus was growing in world-wide, but its underlying mechanisms were not very clear. In this study, we screened out the Lactobacillus to inhibit pathogens growth and adhesion of intestinal cell through a reasonable screening method.In this study, five selected Lactobacillus strains isolated from human intestinal and ferment milk were preliminarily identifed by16S rDNA gene sequencing and assessed the ability to inhibit the infection of enteropathogens. Five Lactobacillus strains were screened on the basis of probiotic characteristics (i.e., resistance to low pH and bile salts, adhesion to the human gastrointestinal tract, inhibition of pathogenic strains). Using an in vitro system for simulating gastric transit, our findings indicated that the three probiotic strains had the ability to tolerate gastroenteric environment and the adhesive capacity to HT-29cells. It was demonstrated that the probiotic strains inhibited subsequent adhesion of pathogens to the HT-29cell. Among the selected strains, five selected Lactobacillus strains showed a superior probiotic potential and could be used in health-promoting food products. After analyzing the sequence of the16SrDNA regions of these three strains, five potential probiotic F0533, IN4125, G15, J5and M7were Lactobacillus rhamnosus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus casei and Lactobacillus paracasei.Here, we assessed the ability to inhibit the adhesion and to displace pathogens of five selected Lactobacillus strains using HT-29cells model through a process which may be related to specific components of the bacterial surface. Surface-layer proteins are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. Removal of S-layer proteins from the Lactobacillus (treated with5mol/L LiCl) reduced adhesion to HT-29cells, which suggested that the S-layer protein had involved in the adhesion of probiotics. The SDS-PAGE analysis confrmed that the presence of S-layer proteins with dominant bands of was approximately60kDa. Human epithelial HT-29cells were treated with S-layer protein extract after infection or pretreated with S-layer protein prior to infection to determine their importance in the inhibition of pathogen adherence. Through analysis cell cycle by flow cytometry, the HT-29cells incubated with S-layer protein and Salmonella typhimurium in S phase were30.13%compared with26.88%cells only treated by Salmonella. Moreover, we also found that Lactobacillus S-layer proteins could protect Salmonella typhimurium-induced apoptosis through reduced the caspase-3activity. This mechanism may represent a novel approach for antagonizing Salmonella typhimurium infection.We also investigated the bactericidal activity of five Lactobacillus strains against Salmonella typhimurium and illustrate the changes in the cellular composition of Salmonella typhimurium in the presence of antimicrobial compound(s). In this study, the growth of Salmonella was affected by the five probiotic strains. Especially, L. rhamnosus F0533had a strong growth-inhibiting effect against Salmonella typhimurium and induced64%loss of its viability (P<0.05). The content of intracellular ATP in Salmonella typhimurium was greatly decreased when the cells of Salmonella typhimurium was exposed to cell-free culture supernatants of L. rhamnosus F0533. Moreover, we demonstrated that when Salmonella typhimurium cells are exposed to the antimicrobial compound(s) in the Lactobacillus supernatants, both the fatty acids of the cell membrane and the polysaccharides of the cell wall are significantly affected.This study was conducted to investigate the effects of Lactobacillus in the intestinal cytoskeletal and tight junctional (TJ) protein and expression of TJ using the human colon cell line HT-29. In this study, Lactobacillus strains played an important role in increasing transepithelial electrical resistance (TER) and maintained TER in HT-29cell monolayers stable. The effect of Lactobacillus on intestinal epithelial cell resistance was accompanied by maintenance or enhancement of cytoskeletal and tight junctional protein. The Lactobacillus had more effect on TER, cytoskeletal and tight junctional protein in the competition and exclusion than the displacement. Furthermore, Lactobacillus could protect EGF from Salmonella typhimurium, which lead to the increase the mRNA expression of TJ proteins. In this study we demonstrated that Lactobacillus could protect intestinal cell from infection of Salmonella typhimurium through reduced the disruption associated with cytoskeletal and tight junctional protein. This mechanism might represent a novel approach for antagonizing Salmonella typhimurium infection.