BACKGROUND: An increased prevalence of phylogenetic B2 group Escherichia coli (E. coli) has been associated with active disease in IBD patients (IBDEC). Many of the B2 group strains harbor virulence genes present in extraintestinal pathogenic E.coli including hemolysin (hly) and cytotoxic necrotizing factor type 1 (CNF1) the loci of which is often linked. Here we investigated the potential of B2 strains of E.coli isolated from IBD patients to damage the integrity of the intestinal epithelium and the potential contribution of Hly and CNF1. METHODS: E coli strains isolated from 35 patients with IBD were tested for virulence (Petersen et al 2009) and of those isolate C691-04A showed high cytotoxicity on dendritic cells ( submitted) and a rapidly decrease of the trans-epithelial electric resistance (TEER) of a monolayer of intestinal epithelial cell lines. Genome sequencing of C691-04A confirmed the presence of hly cluster. It is known that some E coli have two hly-clusters. In order to investigate the role of hly and CNF1 in disruption of the epithelial integrity the following mutants were created in strain C691-04A; 1: CAS965, hly cluster II upstream of CNF1; 2: CAS954, hly cluster I mutant; 3: CAS956 is a knock out of both loci of hly; 4: CAS964, CNF1 mutant; 5: CAS966, clbA mutant. Caco-2 cells were grown to confluence in 24-well trans-well devices for 14 days to form a polarized cell monolayer and TER measured constantly during exposure to E. coli strains at MOI 10. The wild type and commensal E. coli were included as controls. RESULTS: E. coli C691-04A WT as well as CNF1, clbA and single loci of hly mutants from cluster I and II were all able to damage Caco-2 cell tight junctions in less than 6 hrs co-culture (see figure). Confocal microscopy of occludin stained trans-well sections revealed loss of occludin from the tight junction within 3 hours. However, this phenotype was lost in a mutant with knock out of both hly loci (p<0.001). Commensal E. coli isolated from healthy controls also did not cause loss of TER in 6 h. CONCLUSION: IBD-associated E. coli such as C691-04A can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect is abolished in a mutant with knock out of both loci of the hly genes and diminished in one of the single knockout mutants. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.