W1826 Increased Mucosa-Associated Diffusely Adherent E.Coli in Crohn's and Colon Cancer: Possible Role in Pathogenesis

Abstract

Introduction Mucosa-associated adherent invasive Escherichia coli (AIEC) are found in colorectal cancer (CRC) and Crohn9s disease (IBD).1 They invade intestinal epithelial cells, replicate in macrophages and interact with epithelial cells in vitro to promote proinflammatory cytokine release and NFkB activation.2 The adherent-invasive phenotype strongly correlates with the ability to agglutinate red blood cells (RBCs). We suspect that mucosa-associated E coli may also play a role in CRC pathogenesis by activation of key cellular pathways. Our aim here was to identify and characterise adherence mechanisms for our colonic AIEC isolates. Methods A fosmid library was constructed containing 30–60 kb DNA fragments from E coli HM358, a CRC mucosa-associated isolate. Clones were screened for the ability to haemagglutinate RBCs. Agglutinating positive (HA+) clones were further assessed for their ability to adhere to epithelial Hep2 cells, to replicate within macrophages and for their ability to translocate across M-cells. They were then subjected to 454 DNA sequencing. Subsequently, 280 E coli isolates from IBD, CRC and healthy control patients were assessed for the presence of a gene cluster identified from sequence analysis. Results Of the 964 fosmid clones constructed, 8 displayed a strong HA+ phenotype. Sequence data demonstrated all possessed a diffuse adherence gene cluster ( afaA-E ). HA+ positive clones were diffusely adherent to Hep2 cells and showed marked ability to replicate within macrophages when compared to HA- clones (p afa/dr in E coli promotes translocation across M cells, a key portal of mucosal entry. E coli containing afa/dr were more commonly isolated from IBD and CRC patients (57% afa positive) than from healthy controls (28% afa positive, p 2 test). Conclusion Afa/dr E coli are more commonly isolated from IBD and CRC patients than from healthy controls and have properties, including epithelial invasion and replication in macrophages, that overlap with AIEC. Other studies have shown that adherent afa/dr E coli play a key role in the upregulation of angiogenic vascular endothelial growth factor (VEGF) in the intestinal epithelium3 and they may thus have a role in pathogenesis of CRC as well as IBD.