Comparison of adhesion and invasion of VTEC (verocytotoxigenic Escherichia coli) O157:H7 and O91:H21 in two lines of cultured cells

ETCHEVERRÍA, ANALÍA I.; ARROYO, GUILLERMO H.; PADOLA, NORA L.; PARMA, ALBERTO E.

Ciudad de Buenos Aires. Argentina

Simposio; 7th International Symposium on Shiga Toxin (Verocytotoxin)?producing Escherichia coli infections (VTEC 2009); 2009

Asociación Argentina de Microbiología

Resumen: Most of VTEC that cause disease in children are carriers of locus of enterocyte effacement (LEE). The products of translation of LEE constitute a type III secretion system of protein translocation and a system of adhesion which consists of a protein called outer membrane intimin, encoded by the eae gene, and its translocated receptor (Tir). The Tir and intimin are critical factors in the intimate attachment and the formation of the characteristic lesion of attaching and effacing. However, some VTEC isolated from patients with severe disease do not harbor LEE but possess a protein called autoagglutinating adhesin Saa coded by the bacterial plasmid. The aim of this study was to compare the adhesion of two strains, a LEE-negative (O91:H21, vt2, saa, ehxA) and a LEE- positive (O157:H7, vt2, eae, ehxA) and its ability to invade cell lines HEp-2 and CHO-K1. Both strains were isolated from cattle in our laboratory but O91:H21 was more resistant to stress conditions in vitro than O157:H7. The cell lines were cultured in MEM at 37ºC with 5% CO2. We performed the inoculation of cells with 105 cfu / ml of VTEC O157:H7 and O91:H21 in MEM without antibiotics. The plates were incubated at the same conditions for 3 h and washed with PBS. To assess adherence topcoat was recovered with trypsin and the suspension obtained was cultured in SMAC plates. To appreciate the internalization, after 3 h of incubation plates were washed with PBS and incubated in MEM with gentamicin for 1 h to kill the bacteria attached to the membrane for the recovery of internalized bacteria. This assay was conducted on duplicate in two different events. Both VTEC strains adhered to both cell lines. E. coli O157:H7 showed greater capacity of adhesion than O91:H21. However, there were differences in internalization. VTEC O157:H7 internalized more on the cell line CHO-K1 than on HEp-2 while O91:H21 internalized equally in both cell lines. However, the percentage of internalization of O91:H21 was greater than those of O157:H7. In this study, O91:H21 was more invasive than O157:H7 although it lacks LEE. Our results suggest that VTEC strains may have multiple mechanisms of adhesion that would allow them to internalize cell lines. This may contribute to understand the mechanism of intestinal phatogenesis.