To Screen And Evaluate The Antagonistic Activity Against Helicobacter Pylori Of Lactobacillus Separated From Human Gastric Mucosa

ObjectiveHuman gastric mucosa Lactobacillus with strong acid and bile saltresistant ability and Helicobacter pylori antagonisitic activity was screened,then evaluated its antagonistic activity against Helicobacter pylori ingnotobiotic mice stomach to provide experimental material for the furtherstudy of prevention and treatment of Helicobacter pylori infection.MethodsHomogenates of gastric mucosa tissues from patients by gastroscopedetection were cultivated on MRS plate,37℃in micro aerobic. Selectedsuspected colonies for microscopy after gram stains and pure cultured thepositive colony. Then the colonies were identified by gram stain andbiochemical reactions. Extracted the total DNA of bacteria which maybeLactobacillus, and amplified the Lactobacillus16SrDNA sequence, thenidentified by gene sequenced.The resistant ability of the isolated Lactobacillus with different pH(2.5-4.5) and bile salt (0.05%-0.2%) were detected. The research also combined with the determination of their antagonistic potential against H.pylori and the ability inhibited H. pylori adhere to gastric epithelial cells.Also detected the biofilm formation ability by crystal violet staining’sadhesion experiment.Using the screening Lactobacillus strain and H. pylori to infectgerm-free Kunming mice, then established mono-associated anddouble-associated gnotobiotic mice models. Colonized bacteria in micestomach were determined by qualitative and quantitative culture. And thepathologic manifestations were evaluated.Result1Six Lactobacillus strains were isolated from the gastric mucosa of21people who did gastroscope inspection. The numbers were L1, L2, L3,L4-1, L4-2, L4-3. L1and L2were Lactobacillus oris, L3was Lactobacilluscrispatus, L4-1was Lactobacillus salivarius, L4-2was Lactobacillusgasseri, L4-3was Lactobacillus delbrueckii.2The research screening a Lactobacillus strain L3which had thelargest acid yield and the biofilm formation ability was the best. Thegrowth rate can reach to150%when the medium pH was2.5or3.0. L3cantolerant high bile salts. The biggest inhibition zones formed when livebacteria and supernatant of strain L3reacted on H. pylori, it also made themaximum decrease in H. pylori adhered to gastric epithelial cells.3The infection rate of Lactobacillus and H. pylori to grem-free mice were100%. The glands pathological changes obviously and epithelial cellloss were observed in H. pylori-associated gnotobiotic model, also the levelof MPO was the most. The numbers of Lactobacillus and H. pylori indouble-associated gnotobiotic groups were significantly decreased(P<0.05), also the pathological changes were not obvious and the MPOlevel were significantly decreased too (P<0.05).ConclusionL3was isolated successfully from human gastric mucosa which theantagonistic activity against H. pylori was the best. The mono-associatedand double-associated gnotobiotic Kunming mice models were successfulestablished. Also Lactobacillus strain L3can reduce the colonization of H.pylori and the injury to gastric mucosa by H. pylori.