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Bacteria have been suspected in the etiopathogenesis of Crohn’s disease (CD). Over 80% of intestinal microbial flora represent unidentified species for which plasmid-mediated virulence factors exist. Molecular biology allows detection of virulence-associated genes independent of species. Because of transmural inflammation in CD, we hypothesize that submucosal bacterial populations are more relevant to chronic inflammatory disease as opposed to mucosal or luminal populations. The purpose is to determine prevalence of virulent genes and/or infectious agents in submucosal tissues from patients with disease and controls using genomic markers in a comparative assay. Submucosal tissues were obtained from fresh surgical specimens by manual excision and digestion of mucosal layers. DNA was extracted by a modification of the Human Microbiome Project protocol and assayed for 30 virulence genes and/or unique genomic sequences representing 16 distinct bacterial species using quantitative real-time PCR (qPCR). All positive results were repeated, and all qPCR products were verified by gel electrophoresis and sequencing of the PCR amplicon product. Non-Inflammatory Bowel Disease (nIBD) controls represented negative margins of colon cancer patients. A positive result was assigned only if results were reproducible and the PCR product was at least 97% homologous to the known sequence. Tissues from 11 patients with Crohn’s disease and 7 controls have been examined. The intestinal intimin (eaeA) invasion gene of enteropathogenic E. coli and the InvA invasion gene of Salmonella were detected predominately in Crohn’s disease (6/11 - 55%). The absence of other sequences suggests these plasmid-mediated invasion genes may not be associated with either E. coli or Salmonella. In the absence of eaeA and InvA invasion genes, M. paratuberculosis associated sequences were detected in 4/11 (36%) of CD submucosal tissues. Virulence-associated genes were not identified in one suspected Crohn’s patient (1/11 , 9%). The eaeA invasion gene was detected in only 1/7 nIBD controls (14%). Other virulence-associated genes and/or infectious agents sought in our assay system were randomly detected in both study populations. This study examines and reports on the bacterial populations within submucosal tissues as opposed to the mucosal and/or luminal microbiome. Preliminary data suggests the existence of a submucosal microbiome in both normal and diseased intestinal tissue. CD may be divided into 2 distinct populations based on presence/absence of adhesion/invasion genes or the presence of M. paratuberculosis-associated sequences. Future efforts focus on confirming these findings in populations from various geographical locations. Confirmation of these findings could have ramifications to the care of CD by the implementation of targeted therapy based on the submucosal microbiome-type.