João Sabino

A decrease of the butyrate-producing species Roseburia hominis and Faecalibacterium prausnitzii defines dysbiosis in patients with ulcerative colitis

Objective Bacteria play an important role in the onset and perpetuation of intestinal inflammation in inflammatory bowel disease (IBD). Unlike in Crohns disease (CD), in which dysbiosis has been better characterised, in ulcerative colitis (UC), only small cohorts have been studied and showed conflicting data. Therefore, we evaluated in a large cohort if the microbial signature described in CD is also present in UC, and if we could characterise predominant dysbiosis in UC. To assess the functional impact of dysbiosis, we quantified the bacterial metabolites. Design The predominant microbiota from 127 UC patients and 87 age and sex-matched controls was analysed using denaturing gradient gel electrophoresis (DGGE) analysis. Differences were quantitatively validated using real-time PCR. Metabolites were quantified using gas chromatography–mass spectrometry. Results Based on DGGE analysis, the microbial signature previously described in CD was not present in UC. Real-time PCR analysis revealed a lower abundance of Roseburia hominis (p<0.0001) and Faecalibacterium prausnitzii (p<0.0001) in UC patients compared to controls. Both species showed an inverse correlation with disease activity. Short-chain fatty acids (SCFA) were reduced in UC patients (p=0.014), but no direct correlation between SCFA and the identified bacteria was found. Conclusions The composition of the fecal microbiota of UC patients differs from that of healthy individuals: we found a reduction in R hominis and F prausnitzii, both well-known butyrate-producing bacteria of the Firmicutes phylum. These results underscore the importance of dysbiosis in IBD but suggest that different bacterial species contribute to the pathogenesis of UC and CD.

Donor Species Richness Determines Faecal Microbiota Transplantation Success in Inflammatory Bowel Disease

BACKGROUND AND AIMS Faecal microbiota transplantation is a successful therapy for patients with refractory Clostridium difficile infections. It has also been suggested as a treatment option for inflammatory bowel disease, given the role of the intestinal microbiota in this disease. We assessed the impact of faecal microbiota transplantation in patients with inflammatory bowel disease and studied predictors of clinical (non-)response in microbial profiles of donors and patients. METHODS Fourteen refractory patients (8 with ulcerative colitis and 6 with Crohns disease) underwent ileocolonoscopy with faecal microbiota transplantation through a nasojejunal (n = 9) or rectal (n = 5) tube. Efficacy was assessed by endoscopic healing at week 8, clinical activity scores and C-reactive protein measurement. Faecal microbiota was analysed by 16S rDNA pyrosequencing. RESULTS There was no significant improvement among the 6 patients with Crohns disease at week 8 following faecal microbiota transplantation. One patient experienced temporary clinical remission for 6 weeks. In contrast, 2/8 patients with ulcerative colitis had endoscopic remission at week 8, and of the 6 remaining patients with ulcerative colitis, 1 reported temporary remission for 6 weeks. The donor microbiota richness and the number of transferred phylotypes were associated with treatment success. Persistent increased C-reactive protein 2 weeks after transplantation was predictive of failure of response. CONCLUSION Faecal microbiota transplantation led to endoscopic and long-term (>2 years) remission in 2 out of 8 ulcerative colitis patients. Higher donor richness was associated with successful transplant. Therefore, faecal microbiota transplantation with donor prescreening could be a treatment option for selected refractory ulcerative colitis patients.

Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD

Objective Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease often leading to end-stage liver disease. Its pathogenesis remains largely unknown, although frequent concomitant IBD hints towards common factors underlying gut and bile duct inflammation. Considering the mounting evidence on the involvement of the intestinal microbiota in initiating and determining IBD phenotype, we investigated intestinal microbiota composition in patients with PSC. Design Stool samples were collected from 147 individuals (52 patients with PSC, 52 age, gender and body mass index-matched healthy volunteers, 13 UC and 30 patients with Crohns disease). An independent validation cohort of 14 PSC and 14 matched controls was recruited. 16S rDNA sequencing of faecal DNA was performed (Illumina MiSeq). Results The microbiota of patients with PSC was characterised by decreased microbiota diversity, and a significant overrepresentation of Enterococcus (p=3.76e-05), Fusobacterium (p=3.76e-05) and Lactobacillus (p=0.0002) genera. This dysbiosis was present in patients with PSC with and without concomitant IBD and was distinct from IBD, and independent of treatment with ursodeoxycholic acid. A decision tree based on abundances of these three genera allowed reliable classification in the validation cohort. In particular, one operational taxonomic unit belonging to the Enterococcus genus was associated with increased levels of serum alkaline phosphatase (p=0.048), a marker of disease severity. Conclusions We here present the first report of PSC-associated faecal dysbiosis, independent from IBD signatures, suggesting the intestinal microbiota could be a contributing factor in PSC pathogenesis. Further studies are needed to confirm these findings and assess causality.

Quantitative microbiome profiling links gut community variation to microbial load

Current sequencing-based analyses of faecal microbiota quantify microbial taxa and metabolic pathways as fractions of the sample sequence library generated by each analysis. Although these relative approaches permit detection of disease-associated microbiome variation, they are limited in their ability to reveal the interplay between microbiota and host health. Comparative analyses of relative microbiome data cannot provide information about the extent or directionality of changes in taxa abundance or metabolic potential. If microbial load varies substantially between samples, relative profiling will hamper attempts to link microbiome features to quantitative data such as physiological parameters or metabolite concentrations. Saliently, relative approaches ignore the possibility that altered overall microbiota abundance itself could be a key identifier of a disease-associated ecosystem configuration. To enable genuine characterization of host–microbiota interactions, microbiome research must exchange ratios for counts. Here we build a workflow for the quantitative microbiome profiling of faecal material, through parallelization of amplicon sequencing and flow cytometric enumeration of microbial cells. We observe up to tenfold differences in the microbial loads of healthy individuals and relate this variation to enterotype differentiation. We show how microbial abundances underpin both microbiota variation between individuals and covariation with host phenotype. Quantitative profiling bypasses compositionality effects in the reconstruction of gut microbiota interaction networks and reveals that the taxonomic trade-off between Bacteroides and Prevotella is an artefact of relative microbiome analyses. Finally, we identify microbial load as a key driver of observed microbiota alterations in a cohort of patients with Crohn’s disease, here associated with a low-cell-count Bacteroides enterotype (as defined through relative profiling).

Specific members of the predominant gut microbiota predict pouchitis following colectomy and IPAA in UC

Objective Pouchitis is the most common complication after colectomy with ileal pouch-anal anastomosis (IPAA) for UC and the risk is the highest within the 1st year after surgery. The pathogenesis is not completely understood but clinical response to antibiotics suggests a role for gut microbiota. We hypothesised that the risk for pouchitis can be predicted based on the faecal microbial composition before colectomy. Design Faecal samples from 21 patients with UC undergoing IPAA were prospectively collected before colectomy and at predefined clinical visits at 1 month, 3 months, 6 months and 12 months after IPAA. The predominant microbiota was analysed using community profiling with denaturing gradient gel electrophoresis followed by quantitative real-time PCR validation. Results Cluster analysis before colectomy distinguished patients with pouchitis from those with normal pouch during the 1st year of follow-up. In patients developing pouchitis, an increase of Ruminococcus gnavus (p<0.001), Bacteroides vulgatus (p=0.043), Clostridium perfringens (p=0.011) and a reduction of two Lachnospiraceae genera (Blautia (p=0.04), Roseburia (p=0.008)) was observed. A score combining these five bacterial risk factors was calculated and presence of at least two risk factors showed a sensitivity and specificity of 100% and 63.6%, respectively. Conclusions Presence of R. gnavus, B. vulgatus and C. perfringens and absence of Blautia and Roseburia in faecal samples of patients with UC before surgery is associated with a higher risk of pouchitis after IPAA. Our findings suggest new predictive and therapeutic strategies in patients undergoing colectomy with IPAA.

Mucosal Healing and Long-term Outcomes of Patients With Inflammatory Bowel Diseases Receiving Clinic-Based vs Trough Concentration-Based Dosing of Infliximab

BACKGROUND & AIMS The Trough Concentration Adapted Infliximab Treatment (TAXIT) trial demonstrated that maintaining infliximab trough concentrations at 3 to 7 &mgr;g/mL is most effective at inducing remission in patients with inflammatory bowel diseases (IBDs), with fewer flares than clinic‐based dosing. We performed a follow‐up analysis of study participants to explore the correlation between trough dosing strategy and mucosal healing, continued infliximab use, and rates of hospitalization, surgery, and steroid use. METHODS This was a retrospective single‐center study of 226 patients with IBD who completed the maintenance phase of TAXIT, performed at the University Hospitals of Leuven in Belgium. Baseline patient characteristics, laboratory test results, and endoscopic data were obtained at the end of that study between June 2012 and December 2013 (n = 125). Long‐term outcome data (IBD‐related hospitalization, abdominal surgery, and systemic steroid use) were collected from the time of the last TAXIT study visit (August 2012–April 2013) until April 1, 2016. We also collected data on continued use of infliximab and trough concentrations. RESULTS At baseline, 91% of patients in the clinic‐based dosing group and 90% of patients in the trough concentration‐based dosing group had mucosal healing. After a median follow‐up time of 41 months (interquartile range, 39–42 mo), infliximab treatment was continued by 81 of 108 patients (75%) from the clinic‐based dosing group and 86 of 107 (80%) from the trough concentration‐based dosing group. However, within 1 year, infliximab was discontinued by 10 of 27 patients (37%) from the clinic‐based dosing group and 2 of 21 patients (10%) from the trough concentration‐based dosing group (P = .04). The rates of hospitalization, surgery, and steroid use were below 15% in both groups. CONCLUSIONS At the end of a trial of clinic‐based dosing vs trough concentration‐based dosing of infliximab in patients with IBD, most patients had mucosal healing. Most patients (≥75%) in both groups continued taking infliximab for more than 3 years after the trial, but a significantly higher proportion of patients in the clinic‐based dosing group discontinued infliximab in the first year after the end of the trial. Both groups had low rates of hospitalization, surgery, and steroid use.

Comparisons of gut microbiota profiles in wild-type and gelatinase B/matrix metalloproteinase-9-deficient mice in acute DSS-induced colitis

Gut microbiota help to educate the immune system and a number of involved immune cells were recently characterized. However, specific molecular determinants in these processes are not known, and, reciprocally, little information exists about single host determinants that alter the microbiota. Gelatinase B/matrix metalloproteinase-9 (MMP-9), an innate immune regulator and effector, has been suggested as such a host determinant. In this study, acute colitis was induced in co-housed MMP-9-/- mice (n = 10) and their wild-type (WT) littermates (n = 10) via oral administration of 3% dextran sodium sulfate (DSS) for 7 days followed by 2 days of regular drinking water. Control mice (10 WT and 10 MMP-9-/-) received normal drinking water. Fecal samples were collected at time of sacrifice and immediately frozen at −80 °C. Microbiota analysis was performed using 16S rRNA amplicon sequencing on Illumina MiSeq and taxonomic annotation was performed using the Ribosomal Database Project as reference. Statistical analysis correcting for multiple testing was done using R. No significant differences in clinical or histopathological parameters were found between both genotypes with DSS-induced colitis. Observed microbial richness at genus level and microbiota composition were not significantly influenced by host genotype. In contrast, weight loss, disease activity index, cage, and phenotype did significantly influence the intestinal microbiota composition. After multivariate analysis, cage and phenotype were identified as the sole drivers of microbiota composition variability. In conclusion, changes in fecal microbiota composition were not significantly altered in MMP-9-deficient mice compared to wild-type littermates, but instead were mainly driven by DSS-induced colonic inflammation.Colitis: a protein cleared of involvementA protein that regulates aspects of the immune system has been proposed to influence gut microbial populations implicated in the inflammatory conditions known as colitis, but new evidence suggests the protein has no such effect. Ghislain Opdenakker and colleagues at the Rega Institute for Medical Research in Belgium examined the issue in mice with chemically induced colitis. The gut microbes of normal “wild-type” animals were compared with those in animals lacking the gene for the protein, “gelatinase B/matrix metalloproteinase-9”. The absence of the gene, and therefore of the protein it codes for, caused no significant alteration in the gut microbial population. The presence of colitis, however, did alter the gut microbial population relative to mice with no colitis. The results will assist work to understand the networks of cause and effect linking gut microbes and colitis.

P767 The Fit trial: anti-inflammatory dietary intervention effects on the intestinal microbiota

Background: The intestinal microbiota is implicated in the pathogenesis of several immune-mediated disorders including inflammatory bowel diseases and has subsequently been the target of different therapeutic interventions. We designed the Food influence on the Intestinal microbioTa (FIT) trial to s

P459 Predominant dysbiosis in patients with ulcerative colitis is different from Crohn's disease patients

P459 Predominant dysbiosis in patients with ulcerative colitis is different from Crohn’s disease patients K. Machiels1 *, M. Joossens1,2,3, J. Sabino1, V. De Preter1, I. Arijs1, V. Ballet1, K. Claes1, J. Verhaegen1, G. Van Assche1, P. Rutgeerts1, S. Vermeire1. 1University Hospital Gasthuisberg, Leuven, Belgium, 2Research group of Bioinformatics and (eco-)systems biology, Department of Structural Biology, VIB, Brussels, Belgium, 3Research group of Bioinformatics and (eco-)systems biology, Microbiology Unit (MICR), Department of Applied Biological Sciences (DBIT), Vrije Universiteit Brussel, Brussels, Belgium