Screening And Culture Method Researching Of Probiotics With Antagonistic Activity Against Helicobacter Pylori From Human Stomach

Objective: Helicobacter pylori(Hp)is a common gastrointestinal pathogens closely associated with the incidence of chronic gastritis,peptic ulcers,gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer.Classical treatment regimens for eradicating the pathogen is decreasing due to a gradual increase in antibiotic resistance rates.Currently,some studies suggested that probiotics have an inhibitory action on Hp,but most of the probiotics are from intestinal and dairy products,and the anti-Hp activities are not ideal.In this study,we isolated and identified gastric mucosal bacteria by selective isolation technology to Lactobacillus and agarose gel microdrops technique,searched for potential anti-Hp probiotics,and explored the probiotic characteristics and potential mechanism against Hp.Methods: The bacteria were isolated and cultured under anaerobic conditions by Lactobacillus selective isolation technology.The strains were was identified based on typical colony appearances,Gram staining,sequence analyses of 16 S r RNA gene and building a phylogenetic tree.Lactobacillus gasseri was cultured at p H 2 for for 30 min,60min and120 min respectively in vitro to evaluate its viability at low p H.Genome wide sequencing of some Lactobacillus gasseri was carried out,and the general characteristics of the genome and the correlation between the bacteriocin gene and Hp were analyzed.The agarose gel microdrops technique was used to encapsulate a single bacteria cell by filtration,fluorescence counting and with an embedding ratio of 1 to 10,then cultured in Brian Heart Infusion(BHI)medium for 48 h under aerobic conditions.The strain was identified by sequence analyses of bacterial genome DNA and the 16 S r RNA gene,BLAST alignment and building a phylogenetic tree.Results: A total of 108 Lactobacillus strains were isolated and identified from 404 human gastric mucosa specimens,including 11 strains of Lactobacillus gasseri.The detection rate was 2.7%(11/404),accounting for 10.2%(11/108)of all Lactobacillus.The detection rate of Hp in colonized Lactobacillus gasseri patients decreased significantly compared with the control group(9.1% vs 38.7%,P<0.05).The result suggested that naturally colonized Lactobacillus gasseri in the stomach has a potential inhibition growth effect on Hp.Lactobacillus gasseri isolates from human stomach had strong acid resistance,among which LG4,LG2 and LG11 had the strongest acid-tolerant ability.Ninty(90%)of the strains could grow in medium at p H 2.0 for 30 minutes.They could be used as candidates for anti-Hp probiotics.Four of the six strains of Lactobacillus gasseri genome sequenced expressed bacteriocin,and none of the corresponding gastric mucosa specimens was infected with Hp.The result suggested that the growth inhibition of Lactobacillus gasseri may be mediated by bacteriocin.A total of 37 strains were isolated which belong to 12 species.Among them,Brevibacillus agri,Bacillus flexus,Micrococcus antarcticus and Microbacterium oleivorans were isolated from gastric mucosa specimens for the first time.According to 12 bacteria cultured by Micro-embedding technology,we searched the literature about it from Pub Med.It was found that Staphylococcus hominis could produce bacteriocins,and it has the characteristics of acid resistance,bile resistance,antioxidant activity and adhesion to epithelial cells,suggesting that it may have potential anti-Hp effect.Conclusions: 1.Lactobacillus gasseri isolated from human gastric mucosa has potential anti-Hp effect.According to acid resistance characteristics,general genomic characteristics and sequence analysis of bacteriocin gene,LG2 has strong acid resistance and can produce antimicrobial peptides,which can be a good candidate for future anti-Hp probiotics.2.Brevibacillus agri,Bacillus flexus,Micrococcus antarcticus and Microbacterium oleivorans were isolated from gastric mucosa specimens for the first time.The results confirmed that Micro-embedding technology can be used for gastric mucosal flora cultivation.It would provide theoretical and experimental basis for further optimization of standard culture conditions and isolation of more species.3.Studies have showned that Staphylococcus hominis has characteristics of acid-resistant,bile-resistant,antioxidant and adherent to epithelial cells.It also can produce bacteriocins to inhibit the growth of other bacterias.Therefore,Staphylococcus hominis has the potential to become a probiotic against Hp,which needs further study.