Efficacy And Mechanism Of Inactivated Lactobacillus Reuteri Against Helicobacter Pylori

[Backgroud]Helicobacter pylori（H.pylori）infection is responsible for many stomach diseases,especially gastric cancer,which seriously affects human health.Due to the emergence of antibiotic resistant strains and the decline of compliance caused by adverse drug reactions,the eradication rate of H.pylori in traditional regimens is getting lower and lower.It has been reported that some probiotics have anti-H.pylori effect and can improve the eradication rate of H.pylori,reduce the adverse reactions,and resist the gut microbiota disorder caused by antibiotics when supplemented to traditional regimens.However,most of the studies used viable probiotics,and few clinical trials have reported the effect of inactivated probiotics used as adjuvant combining with traditional regimen on H.pylori eradication.[Objective]1.To evaluate the efficacy of inactivated Lactobacillus reuteri（L.reuteri）DSM17648 alone or combined with 14-day standard triple therapy in the treatment of H.pylori infection and its effect on gut microbiota by a clinical study.2.To explored the anti-H.pylori mechanism of inactivated L.reuteri DSM17648 by basic experiments.[Methods]1.A single centre,prospective,randomized,double-blind,placebo-controlled study was conducted.A total of 200 adult treatment-naive patients with H.pylori infection were randomly allocated into the LrDSM 17648 group and the placebo group with 1:1 ratio.Patients were given Inactivated L.reuteri DSM17648 or placebo for 4 weeks with the latter 2 weeks treated together with 14-day standard triple therapy.All patients completed the Gastrointestinal Symptom Rating Scale（GSRS）before,during and after treatment.Fecal samples were collected at baseline（wk0）,week 2（wk2）and 4 weeks after treatment（wk8）for 16S rRNA gene sequencing.H.pylori status was assessed by 13C-urea breath test（13C-UBT）4 weeks after completion of the treatment.Eradication rates were the primary outcome.Secondary outcomes were the change of GSRS scores,gut microbiota before and after treatment,symptoms improvement and new onset or aggravation,side-effects,compliance and tolerance with therapy.2.Kirby-Bauer（K-B）method was used to test the drug sensitivity of L.reuteri DSM17648 to H.pylori.The adhesion ability of inactivated L.reuteri DSM 17648 was evaluated by detecting the auto-aggregation,hydrophobicity,H.pylori copolymerization binding rate and its adhesion to gastrointestinal epithelial cells.Forty SPF BALB/c mice were prepared and divided into four groups according to the random number table,including 10 mice in blank group.The other 30 mice were used to establish H.pylori infection mouse models,and divided into model group（HP）,experimental group（LR+TP）and control group（TP）,with 10 mice in each group.The experimental group was given 2g inactivated L.reuteri DSM 17648 solid beverage（including 1×1010 inactivated L.reuteri cells,dissolved in distilled water）and triple therapy[Omeprazole 0.6mg/（kg·d）+Clarithromycin 15mg/（kg·d）+Amoxicillin 50mg/（kg·d）]twice a day for 2 weeks.The control group was gavaged with the same amount of PBS solution and triple therapy every day,while the model group was only gavaged with the same amount of PBS solution.Ten mice in the blank group were only given the same amount of PBS from beginning to the end.One week after gavage,the mice were killed,and the tissues of stomach,small intestine and large intestine and blood samples were collected for the identification of H.pylori and pathological test,as well as the detection of serum inflammatory factors and intestinal mucosal barrier.[Results]1.After 2 weeks of treatment alone,there was no significant difference in 13C-UBT values and GSRS scores between the two groups.When combined with standard triple therapy,no significant differences were observed in the eradication rates between LrDSM17648 group and placebo group,in both ITT[81.8%（95%CI:74.1%～89.5%）vs.83.7%（95%CI:76.2%～91.1%）,P=0.730]analysis and PP[86.2%（95%CI:79.1%～93.3%）vs.87.2%（95%CI:80.4%～94.1%）,P=0.830]analysis.GSRS score（1.94 ± 2.020 vs.2.65±2.824,P=0.048）after treatment was significant lower in the LrDSM17648 group than that in the placebo group.The proportion of patients with abdominal distention（5.1%vs.16.3%,P=0.010）and diarrhea（11.1%vs,23.5%,P=0.022）was significant lower in LrDSM17648 group than that in placebo group after treatment.Duiring treatment,patients in LrDSM17648 group achieved significantly higher probability of improvement referring to adominal distention（32.3%vs.19.4%,P=0.038）,diarrhea（17.2%vs.7.1%,P=0.031）and urgent need for defecation（12.1%vs.4.1%,P=0.039）,and lower probability of new onset or aggravation referring to diarrhea（8.1%vs.19.4%,P=0.021）than that in placebo group.There was no significant difference in compliance and tolerance between the two groups.Compared with wk0,after 2 weeks of treatment with L.reuteri DSM 17648 alone,the richness and a diversity of gut microbiota were significantly increased（P<0.05）,but the β diversity had no significant change.The mean relative abundance of Bacteroidota（P<0.05）and Verrucomicrobiota（P<0.01）were significantly increased at the phylum level.Bacteroides（P<0.05）,Fusicatenibacter（P<0.001）,Akkermansia muciniphila P<0.01),L.Reuteri（P<0.01）increased significantly,while Eubecterium rectale ATCC 33656（P<0.05）decreased significantly.Compared with wk0,there were no significant changes in a diversity,β diversity and species composition of gut microbiota at wk2 in placebo group.Compared with placebo group,the richnes and a diversity of gut microbiota in the LrDSM17648 group were significantly higher than those in the placebo group at wk2（P<0.01）,and the β diversity of the two groups was also significantly different（P<0.01）.Escherichia Shigellain in the placebo group was significantly higher than that in the LrDSM17648 group（P<0.05）,while the proportion of Bacteroides（P<0.05）and Fusicatenibacter（P<0.01）in the LrDSM17648 group was significantly higher than that in the placebo group.At wk8,Bifidobacterium（P<0.05）in both groups was significantly lower than that in the baseline.The richness and a diversity of LrDSM17648 group were significantly higher than that in placebo group（P<0.001）,and there was also significant difference in β diversity between the two groups（P<0.001）.In terms of species composition,Subdoligranulum（P<0.05）,uncultured Blautia（P<0.05）,Colliella（P<0.05）and Faecalibacterium prausnitzii in LrDSM17648 group were significantly higher than that in control group（P<0.01）,while Bacteroides vulgatus（P<0.05）in placebo group was significantly higher than that in LrDSM17648.DSM17648 group.Enrichment of genera Prevotella,Clostridium_sensu_stricto₁,Eubacterium_ruminantium group and Lactobacillus at baseline may predict eradication success in LrDSM17648 group.The inactivated L.reuteri DSM17648 may help to eradicate H.pylori by playing a stimulative role in the interaction of commesal beneficial bacteria in the gut.2.The results of in vitro drug sensitivity test showed that the non-viable L.reuteri DSM17648 has certain direct inhibitory effect on H.pylori in vitro,and its minimum inhibitory concentration was 1.25×108/mL.The hydrophobic rate of inactivated L.reuteri DSM17648 was 89.32±2.76,and the adhesion rates to gastric mucosal epithelial cells and small intestinal mucosal epithelial cells were 36.16±2.97 and 31.32±1.84,respectively.The auto-aggregation rate reached the highest 93.78%at 6h and 91.21%at 24h;The binding rate of copolymerization with H.pylori reached the highest 79.91%at 24h.Compared with HC group,IL-8,TNF-α and CRP in the serum of HP group increased significantly（214.54±10.58 vs.74.79±5.92,125.73±7.12 vs.84.11 ±5.50,2.76±0.51 vs.1.44±0.29,all P<0.001）;Compared with HP group,IL-8,TNF-αand CRP after treatment with triple regimen alone decreased significantly（127.69 ± 5.29 vs.214.54 ± 10.58,114.21 ± 9.88 vs.125.73±7.12,1.85±0.41 vs.2.76±0.51,all P<0.01）;The levels of IL-8 and TNF-αin the serum of mice treated with triple therapy+inactivated L.reuteri DSM17648 were significantly lower than those treated with triple therapy alone（109.25±7.03 vs.127.69±5.29,86.51 ±5.47 vs.114.21±9.88,all P<0.001）,and the level of TNF-αeven decreased to normal level.Compared with HC group,there was no significant difference in the ET and DAO in the serum of HP group;After triple therapy alone,the levels of ET and DAO were significantly higher than those in HP group（208.99±8.52 vs.132.41 ±7.75,4.33±0.18 vs.3.27±0.17,all P<0.001）;The levels of ET and DAO in the serum of mice treated with triple therapy+inactivated L.reuteri DSM17648 were significantly lower than those treated with triple therapy alone（184.37±8.91 vs.208.99±8.52,3.80±0.10 vs.4.33±0.18,all P<0.001）.[Conclusions]1.Supplemention of inactivated L.reuteri DSM17648 with standard 14-day triple therapy did not increase the eradication rate of H.pylori,but it reduced the frequencies of abdominal distention,diarrhea and urgent need for defecation and even the total score of GSRS.In addition,inactivated L.reuteri DSM17648 resisted the gut microbial disturbance caused by H.pylori eradication threapy,and was significantly better than placebo in the recovery of gut microbiota.Furthermore,inactivated L.reuteri DSM17648 has certain direct inhibitory effect on H.pylori in vitro.For the patients enriched with g_prevotella、g_Clostridium_sensu_stricto₁、g_Eubacterium_ruminantium_group、g_For Lactobacillus,it is more likely to achieve eradication success by inactivated L.reuteri DSM17648 combined with 14 day standard triple therapy.For these patients,inactivated L.reuteri DSM17648 is expected to replace bismuth combined with triple therapy for H.pylori treatment.The inactivated L.reuteri DSM17648 may help to eradicate H.pylori by playing a stimulative role in the interaction of commesal beneficial bacteria in the gut.2.In vitro experiments showed that inactivated L.reuteri DSM17648 has a certain direct inhibition of H.pylori and strong adhesion ability.In addition,in animal experiments,inactivated L.reuteri DSM17648 can reduce the inflammatory response after H.pylori infection and enhance the efficacy of triple therapy.The addition of inactivated L.reuteri DSM17648 during the eradication of H.pylori by triple therapy can reduce the damage of antibiotics to the intestinal mucosal barrier.