PWE-064 Increased mucosa-associated diffusely adherentE coliin Crohn's and colon cancer: possible role in pathogenesis

Abstract

<h3>Introduction</h3> Mucosa-associated adherent invasive <i>Escherichia coli</i> (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 <i>E coli</i> 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. <h3>Methods</h3> A fosmid library was constructed containing 30–60 kb DNA fragments from <i>E coli</i> 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 <i>E coli</i> isolates from IBD, CRC and healthy control patients were assessed for the presence of a gene cluster identified from sequence analysis. <h3>Results</h3> Of the 964 fosmid clones constructed, 8 displayed a strong HA+ phenotype. Sequence data demonstrated all possessed a diffuse adherence gene cluster (<i>afaA-E</i>). HA+ positive clones were diffusely adherent to Hep2 cells and showed marked ability to replicate within macrophages when compared to HA- clones (p&lt;0.01; ANOVA). In vitro data also suggest that the presence of <i>afa/dr</i> in <i>E coli</i> promotes translocation across M cells, a key portal of mucosal entry. <i>E coli</i> containing <i>afa/dr</i> were more commonly isolated from IBD and CRC patients (57% <i>afa</i> positive) than from healthy controls (28% <i>afa</i> positive, p&lt;0.001 χ² test). <h3>Conclusion</h3> <i>Afa/dr E coli</i> 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 <i>afa/dr E coli</i> 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.