The Effect Of Clostridium Butyricum On Symptoms And Gut Microbiota In Treating Diarrhea-dominant Irritable Bowel Syndrome

Part Ⅰ:Randomized controlled trial:Clostridium butyricum in treating diarrhea-dominant irritable bowel syndromeBackground and Aims:Irritable bowel syndrome(IBS)is one of the most common functional gastrointestinal diseases worldwide and may impair social and personal functions and affect the quality of life of the patients.The main symptoms of IBS are abdominal pain,bloating or discomfort associated with defecation and change in bowel habit.IBS is classified into 4 subtypes based on the typical symptoms:diarrhea-predominant IBS(IBS-D),constipation-predominant IBS,mixed IBS and unsubtyped IBS.Recent studies observe that the imbalance of gut microbiota is an essential cause in IBS patients.The use of probiotics to improve gut microbiota is an important method in treating IBS.Several researches prove that probiotics can promote IBS symptoms and quality of life and it is useful of probiotics in treating IBS.Clostridium butyricum(CB)is a butyric acid-producing Gram-positive anaerobe which exists in the intestine of humans.The metabolite of Clostridium butyricum,butyric acid,may play an important role in gut function.Present studies confirm that Clostridium butyricum is useful in treating some gastrointestinal diseases such as inflammatory bowel disease(IBD)and antimicrobial-associated diarrhea.Therefore,we propose that the protective function of Clostridium butyricum may exist in treating IBS-D.In this study,we aim to assess the improvement of clinical symptoms,quality of life,stool consistency and frequency of Clostridium utyricum in IBS-D patients in a multi-centre,randomized,double-blind,placebo-controlled trial and provide a new method in clinically treating IBS.Methods:This was a prospective,multi-centre,randomized,double-blind,placebo-controlled trial.The IBS-D patients diagnosed by Rome Ⅲ who fulfilled the inclusion criteria but did not fulfilled the exclusion criteria from Qilu Hospital of Shandong University,Taian City Central Hospital and Linyi People’s Hospital were randomized into Clostridium butyricum and placebo groups.All of the patients in Clostridium butyricum and placebo groups took 3 capsules 3 times a day for 4 weeks and recorded their basic information,symptoms,IBS symptom severity scale(IBS-SSS),IBS quality of life(IBS-QOL)score,stool consistency(Bristol stool scale)and frequency from questionnaires before and after treating with Clostridium butyricum or placebo.The primary endpoint was the difference in change of IBS symptoms between the two groups as measured by the IBS-SSS from baseline to week 4.Secondary endpoints included the difference in changes of IBS-QOL scores,Bristol stool scale and stool frequency from baseline to week 4.Results:A total of 200 patients met inclusion criteria and were randomized to receive either placebo(N=95)or Clostridium butyricum(N=105)treatment from December 2015 and November 2016.166 patients finished the study,with 81 in placebo group and 85 in Clostridium butyricum group,respectively.There was a significant reduction of IBS-SSS from baseline to week 4 in the Clostridium butyricum group compared with that in the placebo group(-62.12±74.00 vs.-40 74±63.67,P=0.038).A higher overall responder rate was found in the Clostridium butyricum group compared with that in the placebo group(44.76%vs.30.53%,P=0.0422)by an ITT analysis.Significant improvement in change of overall IBS-QOL score was observed in the Clostridium butyricum group compared with the placebo group(7.232±14.06 vs.3.159±11.73,P=0.032).No significant difference was found in change of Bristol stool scale from baseline to week 4 between the Clostridium butyricum group and the placebo group(-1.012±1.078 vs.-0.9000±1.296,P=0.259).However,the improvement of stool frequency from baseline to week 4 was significantly superior in the Clostridium butyricum group than in the placebo group(-1.602±1.416 vs.-1.086±1.644,P=0.035).No severe adverse events have been recorded in either group.Conclusions:Compared with the placebo group,4-week use of Clostridium butyricum is effective in treating IBS-D and may improve the overall symptoms and quality of life and reduce the stool frequency.However,the improvement in stool consistency by Clostridium butyricum is limited.Significance:Before this study,no multi-centre,randomized,placebo-controlled trials about the effect of Clostridium butyricum in treating IBS are reported internationally.In the present study,we find that Clostridium butyricum can improve the symptoms of IBS-D patients,which indicates that Clostridium butyricum may be used as a potential medication in treating IBS-D clinically and provide a new method in clinically treating IBS.Part Ⅱ:The alteration on gut microbiota of Clostridium butyricum in treating diarrhea-dominant irritable bowel syndromeBackground and Aims:Irritable bowel syndrome(IBS)is one of the most common gastrointestinal disorders characterized by abdominal pain,bloating or discomfort associated with defecation and change in bowel habit.The concise etiology and pathophysiology of IBS remain unknown,while some factors are involved in this process such as visceral hypersensitivity,gastrointestinal motility,psychosocial factors,brain-gut axis regulation,infection,immune activation and also intestinal microbiota alteration.It is reported that an increase of Firmicutes-associated taxa,a depletion of Bacteroidetes-related taxa and a significantly lower biodiversity of microbes happen in the intestinal microbiota of IBS patients.Probiotics are important medications in treating IBS that can supplement the intestinal microbiota,improve microbiota characteristics and recover the bowel functions.Clostridium butyricum is clinically used widely,which can inhibit the proliferation of some harmful bacteria and promote the proliferation of some beneficial bacteria to improve the gut microbiota.In this study,we aim to investigate the alteration of gut microbiota after treating with Clostridium butyricum in IBS-D patients and predict the function and relative pathways in this process to reveal the possible mechanism of Clostridium butyricum in treating IBS.Methods:The stool samples were obtained from IBS-D patients before and after the 4-week treatment with Clostridium butyricum or placebo.All stool samples were collected for a further analysis using 16s rRNA pyrosequencing and metagenome sequencing analysis.The alteration of gut microbiota before and after treating with Clostridium butyricum was assessed by diversity analysis,cluster analysis,principal co-ordinates analysis(PCoA)and OTU difference analysis.EggNOG and KEGG databases were used to perform metagenome sequencing data.The LEfSe analysis,STAMP analysis and iPath pathway analysis were used to predict the function and possible pathways of Clostridium butyricum in treating IBS-D.Results:A total of 200 stool samples from baseline and week 4 of 100 patients(42 in the placebo group,58 in the Clostridium butyricum group)were analyzed in this study using 16s rRNA pyrosequencing.The sequencing data showed no significant differences in microbial diversity before and after treatment between Clostridium butyricum and placebo groups.Prevotella,Faecalibacterium,Lachnospiraceae,Veillonellaceae and Bacteroides were the most abundant OTUs in most stool samples.The cluster analysis showed that two clusters were found based on the abundance of all of the variable taxa and there were no significant differences between two clusters in both baseline and week 4.The PcoA analysis showed that the microbial community was similar in two groups at baseline but a significant difference was found at week 4(X2=7.006,df=1,P=0.008).Firmicutes and Actinobacteria were increased and Bacteroidetes was decreased after treated with Clostridium butyricum.45 significantly changed OTUs from baseline to week 4 were found between Clostridium butyricum and placebo group and a significant reduction of clostridium sensu stricto was existed in Clostridium butyricum group(-0.0007532±0.005059 vs.0.01950±0.007487,P=0.023).Fifty-two stool samples from baseline and week 4 of 26 patients(13 per group)were analyzed via metagenome sequencing.No significant differences were observed between placebo group and Clostridium butyricum group in baseline and week 4 for eggNOG and KEGG analysis.The LEfSe analysis,STAMP analysis and iPath pathway analysis showed that the Clostridium butyricum group was associated with mRNA surveillance pathway and thiamine metabolism and the placebo group was more correlated with pathways such as fatty acid metabolism,beta-alanine metabolism,tryptophan metabolism and bacterial invasion of epithelial cells,etc.Conclusions:In the present study,we observe the alteration of gut microbiota after treating with Clostridium butyricum in IBS-D patients.Treatment of Clostridium butyricum cannot influence the microbiota diversity but alter microbiota community.Firmicutes and Actinobacteria were increased,while Bacteroidetes and clostridium sensu stricto were decreased after treated with Clostridium butyricum.Some pathways,such as fatty acid metabolism,beta-alanine metabolism and tryptophan metabolism,may be involved in Clostridium butyricum treating IBS-D.The metagenome sequencing indicates the possible mechanism of Clostridium butyricum in treating IBS.Significance:In this study,we reveal the significance of gut microbiota in Clostridium butyricum treating IBS and provide strong evidences for clinical use of Clostridium butyricum.Our predictions about mechanism pathways of Clostridium butyricum in treating IBS provide targets for future basic medicine researches.