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Crohn disease (CD) is a chronic relapsing condition possibly caused by a dysbiotic microbiome. Approximately 30% to 60% of patients with CD have anti-Saccharomyces cerevisiae antibody (ASCA), but any association with gut microbiota is unexplored. We hypothesized that ASCA positivity would predict a signature microbial status and clinical phenotype.Ileocolonic mucosal biopsies were obtained from children with CD (n = 135), and controls without inflammatory bowel disease (n = 45). Comparison was made between ASCA status, microbial diversity, and clinical characteristics.ASCA was highly specific but poorly sensitive for the diagnosis of CD. In patients with CD, ASCA positivity was associated with older age (≥10 years), ileocolonic disease, and long-term risk of surgery. Microbial alpha and beta diversity were similar in patients with CD with or without ASCA, but significantly less when compared to noninflammatory bowel disease controls. Microbial richness was similar across all 3 groups. Fourteen bacterial species were associated with ASCA-positive patients with CD and 14 species with ASCA-negative patients (P < 0.05). After using a false discovery rate correction Ruminococcus torques and bacterium Yersinia enterocolitica 61 remained significantly associated with CD ASCA positivity (P = 0.0178), whereas Enterobacter cloacae and Faecalibacterium prausnitzii were significantly associated with CD ASCA negativity (P = 0.0178 and 0.0342).ASCA-positive and ASCA-negative patients with CD have significant differences in gut microbiome composition, which could possibly be influencing the phenotype of the disease.