SS310 - Microbial Characteristics of Non-healing Perianal Fistulas

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Purpose/Background: Bacterial infection in the intersphincteric space is believed to be crucial for anal fistula formation, as suggested by Park’s cryptoglandular hypothesis. In our preliminary work, we showed that cryptoglandular fistulas have distinct microbial community structures related to treatment outcomes. To validate our previous work, we enrolled additional patients to further characterize the cryptoglandular fistula and correlate the microbiome with clinical outcomes.

Methods/Interventions: A prospective single-institution observational study was conducted from 2021 to 2024, collecting fistula specimens from patients undergoing definitive surgical management for cryptoglandular perianal fistula disease. Video-Assisted Anal Fistula Treatment (VAAFT) was used to obtain biopsy samples near the internal opening, where the most relevant bacteria and the least contaminants were expected. Stool samples were collected at the time of surgery. Deep sequencing of 16S rRNA gene amplicons was employed to analyze the microbial community structure of the fistulas. Patients were classified as healers or non-healers at the 3-month postoperative visit. Differences in microbial community structure between groups were assessed through alpha and beta diversity comparisons, as well as taxon-by-taxon differential abundance analysis.

Results/Outcomes: A total of thirty-seven patients were included in our analysis, consisting of 25 healers, 10 non-healers, and 2 lost to follow-up. There were no significant differences in mean age, BMI, or smoking status between the healer and non-healer groups. However, a higher prevalence of diabetes was observed in the healer group compared to non-healers (4 vs. 0, p = 0.002). Nineteen patients underwent fistulotomy, eleven were treated with Video-Assisted Anal Fistula Treatment and fibrin glue, six received ligation of the fistula tract, and one had an endorectal advancement flap.
The most common phylum across all samples was Firmicutes. A trending difference in alpha diversity (Shannon index) was noted between the healer and non-healer groups, with higher diversity in the healers (p=0.36). Beta diversity analysis (PERMANOVA) did not reveal significant community-level differences. Despite considerable within-group variation, the class Verrucomicrobiae was significantly underexpressed in healers (log2 FC = -3.04, p = 0.03), as was the family Oxalobacteraceae (log2 FC = -0.87, p = 0.02). Conversely, four bacterial genera were significantly overexpressed in the healer group: Megasphaera (log2 FC = 3.95, p = 0.03), Erysipelotrichaceae UCG-003 (log2 FC = 2.39, p = 0.05), Acidaminococcus (log2FC = 2.51, p = 0.05), and Fenollaria (log2 FC = 3.76, p = 0.03). Additionally, there were significant correlations between bacteria found in fistula and stool samples at all taxon levels.

Conclusion/Discussion: Healing and non-healing cryptoglandular fistulas appear to differ in their microbial community structures, with an increasing trend in alpha diversity among the healers. The bacterial genera that were overexpressed in healers have been shown to have anti-inflammatory properties. Future research should focus on characterizing the perianal fistula microbiome and its association with surgical outcomes.