ABSTRACT Patients with Inflammatory Bowel Diseases (IBD) experience chronic intestinal inflammation as a consequence of inappropriate immune responses to the microbiota. Patients with IBD, particularly Crohn’s disease (CD), are at a greater risk of developing co-morbidities including intestinal fibrosis/stricturing and IBD-associated colorectal cancer (CRC). These diseases can be driven by specific strains of Escherichia coli. The CD and CRC microbiomes harbor high loads of mucosal E. coli described as “adherent-invasive E. coli” (AIEC). AIEC have no genomic definition, but instead are distinguished by functional attributes tested through exhaustive in vitro co-culture assays: the ability to adhere to and invade cultured epithelial cells and replicate and persist in macrophages. Our preliminary data indicate that this in vitro AIEC definition may not predict in vivo mucosal colonization, and that specific taxa expand with E. coli and contribute to inflammatory disease. The objectives of this proposal are to thoroughly interrogate the AIEC definition in vivo, define microbiome compositional changes driven by high levels of E. coli in colonic and ileal tissues, and identify candidate factors harbored by E. coli that permit colonization of the inflamed intestine. To this end, we have developed a polymicrobial colonization strategy that uses a novel barcoding technology to easily distinguish genetically similar, but functionally distinct sub-strains of clinical E. coli in a complex community. We have developed this strategy using a collection of well-characterized clinical AIEC and non-AIEC strains isolated from the intestinal mucosa of CD and healthy patients. This novel approach overcomes technological limitations associated with profiling bacterial metagenomes intimately associated with host tissues using molecular barcoded bacterial strains, gnotobiotic mouse models well-established in our lab, and barcode-targeted high-throughput sequencing and genomics. This innovative approach positions us to comprehensively interrogate the AIEC definition and identify molecular features required for colonization of the inflamed intestine. We must understand what microbial features promote mucosal colonization with high-risk E. coli strains in order to identify IBD patients at risk for a complicated disease course.