7.1. Paper I: Mucosal 5-aminosalicylic acid concentration, drug formulation and mucosal microbiome in patients with quiescent ulcerative colitis We measured the mucosal 5-ASA concentration at three different locations (10, 25 and 40 cm proximal of the anal verge) in the distal colon of 42 patients with UC in remission using three different oral 5-ASA formulations; Mezavant, Asacol and Pentasa, in dosages 4.0-4.8g once daily. Disease activity was assessed by Mayo score and histologically by Geboes score. All patients were genotyped for NAT1 and NAT2, which encode the enzymes metabolising 5-ASA in the intestinal mucosa and liver. Faecal and mucosa-associated bacterial microbiota were assessed by 16S rRNA sequencing. We found large inter-individual variations in mucosal 5-ASA concentrations. Patients using Mezavant had significantly higher mucosal 5-ASA concentrations in the distal colon and rectum than patients using Pentasa, while there was no difference in mucosal 5-ASA concentration between patients using Mezavant and Asacol. We found no correlation between NAT genotypes and mucosal 5-ASA concentration, and different NAT genotypes could not explain the large inter-individual variations in 5-ASA concentration.
Mucosal 5-ASA concentration was associated with alterations in the mucosa-associated bacterial microbiota. High 5-ASA concentration was associated with high bacterial diversity, decreased abundances of Proteobacteria and increased abundances of Firmicutes and Bacteroidetes phyla in addition to increased abundance of 10 bacterial families and 18 bacterial genera and decreased abundance of six bacterial families and one bacterial genus. High mucosal 5-ASA was associated with a presumed favourable bacterial microbiota composition. Mucosal 5-ASA concentration was not associated with alterations in the faecal bacterial microbiota.
7.2. Paper II: Bacterial mucosa-associated microbiome in inflamed and proximal non-inflamed ileum of patients with Crohn’s disease
CD patients (n=51) and HC (n=40) scheduled for endoscopic examination were recruited.
Paired mucosal pinch biopsies were sampled approximately 5 cm and 15 cm orally of the ileocecal valve or ileocolic anastomosis. CD patients where the 5-cm location was endoscopically inflamed, and the 15-cm location was normal were termed terminal ileitis. CD patients with endoscopic inflammation at both 5-cm and 15-cm location were termed active disease. In CD patients in remission and HC, both 5-cm and 15-cm location were endoscopically normal. In CD patients with ileal stenosis, biopsies were only sampled on 5-cm location as the stenosis prevented further intubation into the ileum.
The mucosa-associated microbiota in CD patients was characterised by reduced a-diversity and clear separation from HC on b-diversity plots. CD patients displayed increased abundances of Proteobacteria, Tyzzerella 4, Escherichia shigella and decreased abundances of Ruminiclostridium 5, Ruminiclostridium 6, Eisenbergiella and Faecalibacterium. The abundance of Tyzzerella 4 was profoundly increased. Comparison of the inflamed and proximal non-inflamed mucosa in 20 CD patients with terminal ileitis and no history of upper CD involvement revealed no difference in a-diversity and no separation according to b-diversity. Also, differential expression analyses did not identify any taxa to be differentially expressed between inflamed and proximal non-inflamed mucosa in these patients. a-diversity and microbiota composition did not differ between 5-cm and 15-cm location within all CD patients. a- and b-diversities were also similar regardless of endoscopic inflammation. Biopsies characterised as histologically inflamed had lower a-diversity, but b-diversity did not differ according to histological inflammation. Patients with ileal stenosis clustered further away from HC than CD patients with terminal ileitis and CD patients in remission on b-diversity plots.
In addition, the presumed favourable species Bacteroides massiliensis B84634 and unidentified species of Sutterella and Akkermansia were underrepresented in stricturing CD. Patients who had undergone ICR had lower a-diversity, but the bacterial composition in ICR patients did not differ from non-ICR CD patients. Summarised, our
findings suggested that the altered ileal mucosa-associated microbiota in CD patients is present across different locations in the ileum and is independent of inflammation status at biopsy location.
7.3. Paper III: Fungal microbiota in the ileal mucosa of patients with Crohn’s disease
CD patients and HC scheduled for endoscopic examination were recruited (same patient cohort as paper II). Paired mucosal pinch biopsies from approximately 5 cm and 15 cm proximal of the ileocecal valve or ileocolic anastomosis were collected. The study comprised of 44 CD patients of which 22 had terminal ileitis with endoscopic inflammation at 5-cm location and normal endoscopic appearance at 15-cm location, 10 with endoscopic inflammation at both 5- and 15-cm location and 12 with normal endoscopic appearance at both 5- and 15-cm location. Forty HC were also included. CD associated mycobiota were characterised by reduced fungal evenness, increased Basidiomycota-to-Ascomycota ratio and altered b-diversity and fungal composition compared to HC. Also, an expansion of Malassezia and a depletion of Saccharomyces, as well as increased abundances of Candida albicans and Malassezia restricta were found in CD patients compared to HC. We separately analysed the inflamed and proximal non-inflamed mucosa of 20 CD patients with terminal ileitis without a history of upper CD involvement. There was no difference in a-diversity, but the inflamed and proximal non-inflamed mucosa separated on b-diversity plots, with the inflamed mucosa harbouring a more dysbiotic mycobiota composition compared to HC and non-inflamed mucosa. Several fungal taxa were found to be differentially abundant between inflamed and the proximal non-inflamed mucosa. Candida sake was increased in the inflamed mucosa, whereas Exophiala equina and Debaryomyces hansenii were increased in the proximal non-inflamed mucosa. In the whole CD cohort, neither inflammation (endoscopic and histologic) nor sub-location (5- or 15 cm) influenced fungal a- or b-diversity. Summarised, this study confirmed CD specific alterations in the mucosa-associated mycobiota and described structural alterations in
the mycobiota composition between the inflamed and proximal non-inflamed ileal mucosa within the same CD patients.