Screening And Clinical Functional Evaluation Of Lactobacillus Strains For Their Anti-Helicobacter Pylori Effects

Helicobacter pylori(H.pylori)infection is associated with the pathogenesis of many digestive tract diseases,including peptic ulcers,lymphoma and gastric cancer.The eradication of H.pylori by multi-antibiotics is an effective way to prevent gastrointestinal diseases.Owing to the frequent use of antibiotics,the increased resistance of H.pylori to antibiotics and adverse effects patients suffered during the consecutive treatment period affected the effectiveness of therapy.To date,a large number of cell and animal studies have proved the ability of Lactobacillus strains to inhibit the growth and the colonization of H.pylori both in vitro and in vivo.However,limited number of relevant clinical studies were perfomed and most of them were focused on the efficiency of commercial strains such as L.rhamnosus GG.Therefore,this study intends to screen Lactobacillus with antagonistic activities against H.pylori,and then evaluate their clinical efficacy,aiming to develop Lactobacillus strains with independent intellectual property rights for the intervention and treatment of H.pylori infection.Firstly,98 Lactobacillus strains were tested for their ability to inhibit the growth of H.pylori as well as to reduce the adhesion of H.pylori to gastric epithelial cell.The results indicated that 66 Lactobacillus strains showed antibacterial activity(the diameter of the inhibition zone was between 9.41-13.88 mm),and 9 strains of Lactobacillus significantly reduced H.pylori adhesion(the reduction in adhesion rate ranged from 24.76% to 31.26%).7 Lactobacillus strains with relatively superior properties in both aspects were selected for further evaluation of their colonization ability in mice stomach with 16 S rRNA sequencing and quantitative real-time PCR technology.The results showed that stomach flora diversity in mice increased after the gavage of Lactobacilli,and the abundance of Lactobacillus,Proteus and Enterococcus were significantly affected after Lactobacilli treatment.6 Lactobacillus strains could colonize in stomach for more than one day,and 4 of them have a colonization period for more than 3 days(L.crispatus G14-5M,L.rhamnosus JS-SZ-2-1,L.helveticus M2-09-R02-S146 and L.plantarum CCFM8610).Therefore,these 4 strains were selected for clinical trial evaluation.In total,97 H.pylori-positive patients were recruited and randomly divided into 5 groups: placebo group and 4 diffrent Lactobacillus strain groups.Lactobacillus strain powder was given twice a day to volunteers for one month,and then the indicators were tested to evaluate the effect of Lactobacillus strains against H.pylori infection.The results showed that all 4 Lactobacillus strains can alleviative H pylori infection by reducing value of 14C-Urea breath test(?UBT 71.58-120.40 dpm/mmol)and gastrointestinal symptom rating scale(?GSRS 3.62-5.00).Among them,L.crispatus G14-5M and L.helveticus M2-09-R02-S146 decreased the level of Interleukin-8(IL-8)in the serum of subjects.Besides,there was no significant alteration of the structure of intestinal flora and no adverse reactions occurred in the process of probiotic intervention.Further correlation analysis was conducted to analyze the in vitro and clinical data of Lactobacillus against H.pylori.The results showed that the reduced colonization density of H.pylori in clinical subjects had the strongest correlation with the inhibition of H.pylori adhesion rate,with a correlation coefficient of 0.96.Finally,the safety of the 4 Lactobacillus strains was preliminarily evaluated by analyzing the genomic properties and antibiotic phenotypes of the 4 strains.The results showed that all the 4 strains contained CRISPR-Cas system to resist the invasion of substances,and no potentially harmful virulence factors were detected.All 4 strains carried resistance genes,but the genes were not on the prophage(except CCFM8610).This means low possibility of transferring of resistence gene among these Lactobacillus strains.Besides,L.plantarum CCFM8610 showed multiple resistance to antibiotics while the other 3 strains showed resistance to only one or two antibiotics and were considered to be relatively safe.