QUANTITATIVE ASSAY OF SALMONELLA ADHERENCE TO INTESTINAL EPITHELIAL-CELLS - A NEW METHOD FOR ASSESSING NOVEL INTERVENTION PRODUCTS

Adhesion of Salmonella spp to luminal intestinal epithelial cells, an important step in the development of disease, is currently the focus o f novel anti-bacterial strategies. We describe here an in vitro adhesi on assay for measuring bacterial attachment to epithelial cells to be used in assessment of anti-adhesion agents. Techniques for isolating m ouse epithelial cells were optimised. EDTA treatment in combination wi th mechanical disruption gave high yields of enterocytes (4-7x10(6) ce lls/ml; >80% viability). Light microscope studies revealed that maximu m bacterial adherence (17.64+/-4.13 bacteria/ epithelial cell) occurre d between 30 and 60 min post infection using 1x10(6) epithelial cells and 1x10(9) bacteria/ml. These conditions were used to devise an ELISA based bacterial adherence assay. Microtitre plates were conditioned w ith lysine and glutaraldehyde, then coated with a monolayer of epithel ial cells (10(8) cells/wells) and fixed with glutaraldehyde after cent rifugation to stabilise adherence to the plastic. Bovine serum albumin (0.5% in PBS) was used to block non-specific binding of bacteria. Pol yclonal rabbit anti-Salmonella antiserum followed by horseradish perox idase labelled secondary antibody was used to detect bound bacteria. A standard curve was reproducibly obtained with 1x10(4)-1x10(10) bacter ia which gave optical density (O.D.) readings of 0.2-1.4. A concentrat ion of 1x10(8) bacteria/well consistently gave O.D. readings of 1.1. T his method is suitable for screening antibacterial products for their ability to inhibit Salmonella adherence to intestinal epithelial cells . (C) 1998 Elsevier Science B.V.