Distinct Microbial and Resistome Signatures Differentiate Healthy and Inflamed Ileal Pouches in Ulcerative Colitis Patients

Background Fecal matter transplantation (FMT) from donors with a healthy functioning colon has been investigated for its potential use in treating chronic pouchitis, with limited effect. However, what constitutes a normal functioning ileal pouch-anal anastomosis (IPAA) (healthy) pouch microbiome is unknown. We investigated the microbial characteristics of a healthy IPAA (HealthyPouch) compared with chronic pouchitis (SickPouch), ulcerative colitis (UC), and a normal gut microbiome (NormalGut). Furthermore, we investigated how repeated antibiotic use influences antimicrobial resistance on a genome level. Methods Fecal samples from 191 participants were collected; HealthyPouch (n=38), SickPouch (n=38), UC (n=23), and NormalGut (n=92) and sequenced with Illumina and taxonomically profiled with MetaPhlAn4. Samples from HealthyPouch, SickPouch, and NormalGut were also long-read sequenced using Oxford Nanopore Technologies for metagenome-assembled genome (MAG) reconstruction. Findings The HealthyPouch microbiome is distinct from both the UC and NormalGut microbiome while significantly more diverse compared to the SickPouch. E. coli MAGs in the SickPouch microbiome had significantly higher prevalence of gyrA and parC mutations conferring resistance in the SickPouch (n=22) microbiome compared to both HealthyPouch (n=25) and NormalGut (n=41) (p<0.0001). Lastly, DNA methylation pattern was used to associate three plasmids to E. coli with extended-spectrum beta lactamase (ESBL) genes. Interpretation HealthyPouch microbiota differs from NormalGut microbiota, and could therefore be more appropriate for FMT treatment of pouchitis. Repeated antibiotic consumption drives shifts in community structure and enriches gyrA/parC resistance mutations in E. coli in the SickPouch group. Using DNA methylation, ESBL carrying plasmids could be associated with E. coli MAGs. These results emphasize the pouch as a unique microbial niche and underscore the need for careful antibiotic use and resistance monitoring.