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 of novel anti-bacterial strategies. We describe here an in vitro adhesion assay for measuring bacterial attachment to epithelial cells to be used in assessment of anti-adhesion agents. Techniques for isolating mouse epithelial cells were optimised. EDTA treatment in combination with mechanical disruption gave high yields of enterocytes (4-7x10(6) cells/ml; >80% viability). Light microscope studies revealed that maximum bacterial adherence (17.64+/-4.13 bacteria/ epithelial cell) occurred 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 with lysine and glutaraldehyde, then coated with a monolayer of epithelial cells (10(8) cells/wells) and fixed with glutaraldehyde after centrifugation to stabilise adherence to the plastic. Bovine serum albumin (0.5% in PBS) was used to block non-specific binding of bacteria. Polyclonal rabbit anti-Salmonella antiserum followed by horseradish peroxidase labelled secondary antibody was used to detect bound bacteria. A standard curve was reproducibly obtained with 1x10(4)-1x10(10) bacteria which gave optical density (O.D.) readings of 0.2-1.4. A concentration of 1x10(8) bacteria/well consistently gave O.D. readings of 1.1. This method is suitable for screening antibacterial products for their ability to inhibit Salmonella adherence to intestinal epithelial cells. (C) 1998 Elsevier Science B.V.