Probiotic Escherichia coli inhibit adherence of Escherichia coli O157:H7 to colon explants from bovine

ETCHEVERRÍA, ANALÍA I.; ARROYO, GUILLERMO H.; 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: E. coli O157: H7 is one of the pathogens that cause hemolytic uremic syndrome (HUS). HUS is a disease endemic in Argentina with a record of 13.9 casos/100000 children under 5 years of age. It is widely demonstrated that the cattle are the main reservoirs of this pathogen being the large intestine, primarily the colon, the main site of colonization. E. coli O157:H7 is transmitted mainly through consumption of contaminated food and water, person to person transmission and direct contact with animals. There have been raised many strategies to try to lower the burden of E. coli O157: H7 in cattle. Considering the different ways in which humans can take contact with the feces of cattle, the strategies that could be implemented in the live animal before slaughter would produce the most significant reduction in human exposures to the organism. This can be achieved through the use of probiotic bacteria. The aim of this study was to test whether probiotic E. coli inhibits the adhesion of E. coli O157: H7 in bovine colon explants. We worked with samples of terminal colon obtained from cattle at slaughter. Upon collection, tissues were immersed in MEM washed with chilled sterile 0.9% NaCl solution and immersed in chilled MEM. Prior to culture, tissues were cut into 3x5mm pieces and placed mucosal side up onto sponge in six-well polystyrene tissue culture plates containing RPMI without antibiotics. Two tissues (explants) were placed on each sponge. Plates were incubated at 37 ºC in 5% CO2 on a rocker. Explants were inoculated with 25 ml of culture broth (2x107 CFU) of E. coli O157: H7-NalR and probiotic E. coli-NalR alone and in a mixture of both. Bacteria cultures were applied to the mucosal surface and incubated for 6 h. Media were changed hourly to prevent bacterial overgrowth and acidic pH. After incubation, explants were cut in two pieces, one of them used for culture and the other for inmunohistochemical assay. To determine if on the explants inoculated with both kinds of bacteria it could be possible to detect the pathogenic one, a suspension of each explant was plated onto SMAC -Nal agar. Plates were incubated at 37 ºC for 18 h. Tissues sections of 5 mm were incubated with anti-E. coli O157 serum as the primary antibody. An incubation with the 2nd Ac was then given, followed by incubations with a peroxidase anti-peroxidase complex. The explants inoculated with pathogenic bacteria showed E. coli O157-positive adherent bacteria on the surface epithelium. In the explants inoculated with both kinds of bacteria it was not possible to detect adherent bacteria to the epithelium. These results agree with our hypothesis that probiotic E. coli inhibit adherence of E. coli O157:H7 to colonic epithelium of cattle. This allows to speculate on the use of them for the control of cattle as a reservoir for E. coli O157: H7 thus reducing the arrival of these pathogens to the food chain with a concomitant decrease in the risk for humans.