Enteroaggregative Escherichia coli (EAEC) forms thick biofilms on the intestinal mucosa. Here, we show that most EAEC strains form a biofilm on glass or plastic surfaces when grown in cell culture medium with high sugar and osmolarity. Biofilm-forming ability in two prototype EAEC strains required aggregative adherence fimbriae (AAF), although many other EAEC strains that do not express AAF also developed biofilms under these conditions. Ten thousand transposon mutants of EAEC strain 042 were isolated, and 100 were found to be deficient in biofilm formation. Of these, 93 were either deficient in in vitro growth or mapped to genes known to be required for AAF/II expression. Of the seven remaining insertions, five mapped to one of two unsuspected loci. Two insertions involved the E. coli chromosomal fis gene, a DNA-binding protein that is involved in growth phase-dependent regulation. Using reverse transcription-polymerase chain reaction (RT-PCR), we determined that the effect of fis was at the level of transcription of the AAF/II activator aggR. Biofilm formation also required the product of the yafK gene, which is predicted to encode a secreted 28 kDa protein. The yafK product is required for transcription of AAF/II-encoding genes. Our data do not suggest a role for type 1 fimbriae or motility in biofilm formation. EAEC appears to form a novel biofilm, which may be mediated solely by AAF and may reflect its interactions with the intestinal mucosa.

Enteroaggregative Escherichia coli (EAEC) is an important cause of persistent diarrhea in many developing parts of the world, yet the pathogenetic mechanisms of EAEC diarrhea are unknown. Experiments with animal models suggest that EAEC strains damage the intestinal mucosa, and a putative cytotoxin has been described. To characterize the mucosal effects of EAEC, we studied strain 042, which we have shown to cause diarrhea in adult volunteers. Strain 042 was incubated in an in vitro organ culture model with biopsy-derived normal intestinal mucosa from pediatric patients. Strain 042 adhered strongly to samples of jejunal, ileal, and colonic mucosa. In addition, scanning electron microscopic examination of in vitro-infected intestinal biopsies revealed cytotoxic effects marked by exfoliation of mucosal epithelial cells. To develop an in vitro model to study these effects, we incubated 042 with polarized monolayers of the human intestinal epithelial cell lines Caco-2 and T84. Strain 042 adhered strongly to T84 cells but not to Caco-2 cells. T84 cells infected with 042 displayed marked toxic effects, most prominently in areas where bacteria were adhering. The apical membrane of damaged cells exhibited vesiculation and shedding of microvilli. The cytoplasm of affected cells displayed subnuclear vacuolization, and in some cases, nuclei of affected cells became separated from the surrounding cytoplasm. Severely affected cells ruptured, releasing their nuclei. Vacuolated remnant cells were seen throughout the monolayer. Strain 042 was not internalized by T84 cells. We concluded that EAEC strain 042 alters intestinal cell morphology, ultimately leading to cell death. Although the factor(s) required for this effect remains to be elucidated, T84 cells may serve as a valuable model in EAEC pathogenesis studies.

EAggEC are an important cause of persistent diarrhoea in the developing world, however, pathogenic mechanisms remain unclear. Animal models have suggested that EAggEC may damage the intestinal mucosa and putative toxins have been described. We have used in vitro organ culture of histologically normal human intestinal mucosa with EAggEC to assess their cytotoxic potential. Strains examined included 1) a prototype and 4 wild type EAggEC isolates from children with diarrhoea in Delhi, India, 2) two prototype and 5 wild type EAggEC strains from cases of diarrhoea in London, UK, 3) strain O42 (O44:H18), which expresses AAF/II fimbriae and causes diarrhoea in adult volunteers, in three forms — a) genetically intact, b) a TnphoA mutant which lacked AAF/II fimbrial expression, and c) an adherence-plasmid cured derivative. Group 1 strains were incubated with adult colonic mucosa, group 2 and 3 strains were incubated with small and large paediatric intestinal mucosal samples. Tissue was studied by electron microscopy following incubation. Group 1: four strains adhered to the mucosal surface and showed non-intimate attachment characteristic of fimbrially-mediated adhesion; beneath sites of adhesion vesiculation and loss of microvillus membrane were apparent. Group 2: all strains adhered to paediatric intestine — 5 bound to jejunum without morphological effects; 7 bound to ileum and induced rounding and extrusion of epithelial cells without evidence of microvillus changes; 5 adhered to colonic mucosa producing rounding-up and extrusion of epithelial cells, in addition 3 caused microvillus vesiculation, dilatation of crypt openings and increased mucus discharge from goblet cells. Group 3: strain 3a adhered to both small and large intestinal mucosa and produced similar overt changes to colonic mucosa as the latter 3 strains in group 2; strain 3b, although it did not show adherence, produced similar, but milder, mucosal changes; strain 3c did not adhere and mucosal changes were not apparent. In conclusion, EAggEC strains have a cytotoxic effect on human intestinal mucosa in vitro. This may be the basis of EAggEC induced mucosal damage.