Dysbiosis in metabolic genes of the gut microbiomes of patients with an ileo-anal pouch resembles that observed in Crohn’s Disease

Background Crohn’s disease (CD), ulcerative colitis (UC) and pouchitis are multifactorial and chronic inflammatory diseases of the gastrointestinal tract, termed together as inflammatory bowel diseases (IBD). Pouchitis develops in former patients with UC after total proctocolectomy and ileal pouch-anal anastomosis (“pouch surgery”) and is characterized by inflammation of the previously normal small intestine comprising the pouch. It has been extensively shown that broad taxonomic and functional alterations (“dysbiosis”) occur in the gut microbiome of patients with IBD. However, the extent to which microbial dysbiosis in pouchitis resembles that of CD or UC has not been investigated in-depth, and the pathogenesis of pouchitis largely remains unknown. Results In this study we collected 250 fecal metagenomes from 75 patients with a pouch, including both non-inflamed (normal pouch) and inflamed (pouchitis) phenotypes, and compared them to publicly available metagenomes of patients with CD (n=88), and UC (n=76), as well as healthy controls (n=56). Patients with pouchitis presented the highest level of dysbiosis compared to other IBD phenotypes based on species, metabolic pathways and enzyme profiles, and their level of dysbiosis was correlated with intestinal inflammation. In patients with pouchitis, the microbiome mucin degradation potential was lower, but was accompanied by an enrichment of Ruminococcus gnavus strains encoding specific mucin-degrading glycoside hydrolases, which might be pro-inflammatory. Butyrate and secondary bile acids producers were decreased in all IBD phenotypes and were especially low in pouchitis. Butyrate synthesis genes were positively correlated with total dietary fiber intake. Patients with pouchitis harbored more facultative anaerobic bacteria encoding enzymes involved in oxidative stress response, suggesting high oxidative stress during pouch inflammation. Finally, we have developed enzymes-based classifiers that can distinguish between patients with a normal pouch and pouchitis with an area under the curve of 0.91. Conclusions We propose that the non-inflamed pouch is already dysbiotic (function- and species-wise) and microbially is more similar to CD than to UC. Our study reveals microbial functions that underlie the pathogenesis of pouchitis and suggests bacterial groups and functions that could be targeted for nutritional intervention to attenuate or prevent small intestinal inflammation present in pouchitis and CD. ### Competing Interest Statement Iris Dotan: Consultation/advisory boards for Pfizer, Janssen, Abbvie, Takeda, Roche/Genentech, Celltrion, Celgene, Medtronic/Given Imaging, Rafa Laboratories, Neopharm, Sublimity, Arena, Gilead. Speaking/teaching: Pfizer, Janssen, Abbvie, Takeda, Roche/Genentech, Celltrion, Celgene, Falk Pharma, Ferring. Grant support: Pfizer, Altman Research. The remaining authors declare no competing interests ### Clinical Trial This is a retrospective-observational study and not a clinical trial, but this study is registered in the National Institutes of Health with this ID: [NCT01266538][1] ### Funding Statement This work was supported by a generous grant from the Leona M. and Harry B. Helmsley Charitable Trust. V.D. was partially supported by a fellowship from the Edmond J. Safra Center for Bioinformatics at Tel-Aviv University. U.G. was also supported by the Israeli Ministry of Science and Technology. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval for the study was given by Rabin Medical Center (Petah-Tikva, Israel) IRB, ethical approval number: 0298-17. All patients signed informed consent before inclusion to the study. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All the metagenomic sequence data generated and used in this study (patients with a pouch) have been deposited in NCBI SRA and is available under BioProject number PRJNA637365. This study also used our previously published sequence data from PRJNA524170. Metagenomes from patients with UC, CD and healthy controls were obtained from PRJNA400072. Analysis scripts with code for the machine learning classifiers are available in the GitHub repository (). * IBD : inflammatory bowel diseases CD : Crohn’s disease UC : ulcerative colitis SCFA : short-chain fatty acid CLDP : Crohn’s like disease of the pouch EC : Enzyme Commission number GH : glycoside hydrolases GBT : gradient boosting trees AUC : under the curve DCA : deoxycholic acid LCA : lithocholic acid RPKM : reads per kilobase per million mapped reads cpm : copies per million [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01266538&atom=%2Fmedrxiv%2Fearly%2F2020%2F09%2F25%2F2020.09.23.20199315.atom