To Örebro University

Introduction: Inflammatory bowel diseases are becoming increasingly common. The underlying mechanisms are not entirely known but the gut microbiota seem to be involved in the pathogenesis. 

Aim: The aim of this thesis was to characterise gut microbiota related to diagnosis, disease course and response to biological treatment, taking aspects of the source of biological material into account. 

Materials and methods: Patients and healthy individuals from several different cohorts in Sweden and Europe were invited. Faecal samples and mucosal biopsies were analysed using different sequencing platforms to investigate the gut microbiota. In Study I the faecal microbiota was correlated to different inflammatory bowel diseases. In Study II we compared the microbiota in faeces to the microbiota in mucosal biopsies. In StudyIII we related the faecal microbiota to the outcome of biological treatment. In Study IV we investigated the diagnostic and prognostic properties of the GAmap^TM Dysbiosis Test.

Results: The faecal microbiota in collagenous colitis resembles the faecalmicrobiota in inflammatory bowel disease. The faecal microbiota differs from the mucosal microbiota. Faecal microbiota at initiation of biological treatment among patients with Crohn’s disease differ between responders and non-responders. The GAmap^TM Dysbiosis Test discriminates patients with inflammatory bowel disease from healthy individuals.

Conclusion: Collagenous colitis may share microbial underpinnings with other inflammatory bowel diseases. Conclusions about mucosal interactions with the gut microbiota should be made with caution when usingfaecal samples to characterise the microbiota. In Crohn’s disease, the faecal microbiota may be included in a model to predict the outcome of biological treatment. The GAmap Dysbiosis Test does not seem to be superior to other current diagnostic tools in clinical decision-making. 

Method: We examined faecal samples, using the GA-map (TM) Dysbiosis Test, to associate gut microbiota composition with Crohn's disease (CD) and ulcerative colitis (UC) and to identify markers for future biomarker identification. We conducted a prospective case-control study (EU-ref. no. 305676) in an inception cohort of 324 individuals (64 CD, 84 UC, 116 symptomatic non-IBD controls and 44 healthy controls) across five European centres and examined 54 predetermined bacterial markers. We categorized patients according to the Montreal Classification and calculated the dysbiosis index (DI). Non-parametric tests were used to compare groups and the Bonferroni correction to adjust for multiple comparisons.

Results: The fluorescent signals (FSSs) for Firmicutes and Eubacterium hallii were lower in inflammatory bowel disease (IBD) vs. symptomatic controls (p<.05). FSS for Firmicutes, Lachnospiraceae, Eubacterium hallii and Ruminococcus albus/bromii were lower, whereas the signal fo rBacteroides Fragilis was higher in UC vs. symptomatic controls (p<.05). FSS was higher for Bifidobacterium spp., Eubacterium hallii, Actinobacteria and Firmicutes among patients with ulcerative proctitis, compared to extensive colitis (p<.05). In CD, we observed no association with disease location. The DI correlated with faecal-calprotectin in both CD and in UC (p<.001). In terms of treatment escalation and anti-TNF response, differences were observed for some bacterial markers, but none of these associations were statistically significant.

Conclusion: Our data reveal that the GA-map (TM) Dysbiosis Test holds the potential to characterize the faecal microbiota composition and to assess the degree of dysbiosis in new-onset IBD. On the other hand, our results cannot demonstrate any proven diagnostic or predictive value of this method to support clinical decision making.

INTRODUCTION: In inflammatory bowel disease (IBD), an aberrant immune response to gut microbiota is important, but the role of the microbiota in collagenous colitis (CC) is largely unknown. We aimed to characterize the microbiota of patients with CC compared with that of healthy control and patients with IBD.

METHODS: Fecal samples were collected from patients with CC (n = 29), age- and sex-matched healthy controls (n = 29), patients with Crohn's disease (n = 32), and patients with ulcerative colitis (n = 32). Sequence data were obtained by 454 sequencing of 16S rRNA gene amplicons, and the obtained sequences were subsequently taxonomically classified.

RESULTS: Analysis of similarity statistics showed a segregation between patients with CC and healthy controls with increasing taxonomic resolution, becoming significant comparing operational taxonomic unit data (P = 0.006). CC had a lower abundance of 10 different taxa. Taxa-specific analyses revealed a consistent lower abundance of several operational taxonomic units belonging to the Ruminococcaceae family in patients with CC, q < 0.05 after false discovery rate correction. Loss of these taxa was seen in patients with CC with active disease and/or corticosteroid treatment only and resembled the findings in patients with IBD.

DISCUSSION: CC is associated with a specific fecal microbiome seen primarily in patients with active disease or ongoing corticosteroid treatment, whereas the microbiome of CC patients in remission resembled that of healthy controls. Notably, the shift in key taxa, including the Ruminococcaceae family, was also observed in IBD. There may be common mechanisms in the pathogenesis of CC and IBD.

BACKGROUND: Faecal samples are often used to characterise gut microbiota, since they are easily collected. However, whether or not the faecal microbiota differ from the mucosa-associated microbiota remains largely unknown. This may be specifically relevant in conditions that are characterised by complex mucosal microbe-host interactions, such as Crohn's disease. We aimed to determine the degree of agreement between faecal and mucosal microbiota profiles in healthy individuals, using two commonly used collection procedures.

MATERIAL AND METHODS: The gut microbiota composition of faecal samples (sent at ambient temperature before storage at -70°C) and of colonic biopsies (obtained at endoscopy and immediately stored at -70°C) was determined by sequencing the 16S rRNA gene. Thirty-one randomly selected healthy individuals from the population-based colonoscopy (Popcol) study were included.

RESULTS: Faecal samples were characterised by a reduced degree of richness (p<0.0001) and diversity (p=0.016), and also differences in several phyla, including a lower relative abundance of Proteobacteria (p<0.0001) and Verrucomicrobia (p=0.008) than in biopsies. Only 3 of 30 individuals had a similar faecal and mucosal microbiota profile, based on weighted UniFrac analysis. A difference in Crohn's disease dysbiosis-associated bacteria was observed, including a lower relative abundance of Faecalibacterium (p=0.004) and a higher relative abundance of Ruminococcus (p=0.001) in faeces than in biopsies.

CONCLUSIONS: Analysis of faecal samples that have been transported at ambient temperature does not adequately reflect the colonic mucosa-associated microbiota in healthy individuals. These findings have implications for the interpretation of the previous literature, and may be specifically relevant to studies on Crohn's disease.