Flow cytometric analysis of Clostridium difficile adherence to human intestinal epithelial cells

Clostridium difficile is the most common cause of diarrhoea in hospitalised patients. Bacterial adherence to gut epithelial cells is a likely prerequisite to infection and toxin production. A novel flow cytometric method was developed for detecting adherence of C. difficile to human colonic and small intestinal epithelial cells (EC) and human intestinal cell lines. Small intestinal and colonic EC were isolated from biopsy specimens with mucolytic and chelating agents. Adherence of fluorochrome-labelled C. difficile to EC was measured by flow cytometry and was calculated as increase in median fluorescent intensity (ΔMFI). Cells with bacteria attached could be distinguished easily from cells alone or cells with unlabelled bacteria attached. Toxin-positive C. difficile adhered to colonic and small intestinal EC (ΔMFI mean 21.2 SD 16.7, n=33 and 16.5 SD 20.7, n=19 respectively). The toxin-negative strain also adhered to both epithelial cell types (ΔMFI 26.1 SD 32.5, n=17 and 18.3 SD 31.3, n=16). Adherence of toxin-positive C. difficile to the intestinal cell lines Caco-2 (ΔMFI 9.4 SD 4.4, n=14) and HT29 (ΔMFI 8.1 SD 3.1, n=12) was quantifiable, although at a significantly lower level than with primary colonic epithelial cells. Adherence of the toxin-negative strain was slightly lower, ΔMFI 6.5 SD 1.8, n=9 with Caco-2 cells and ΔMFI 6.0 SD 2.0, n=10 with HT29 cells. Adherence of C. difficile to epithelial cell lines was blocked with C. difficile antiserum, confirming specificity of adherence. In conclusion, flow cytometry is a useful approach to quantifying adherence of C. difficile to human colonic and small intestinal epithelial cells. Binding of toxin-negative as well as toxin-positive bacteria was detectable by this approach. Analysis of C. difficile adherence to target cells may have important implications for the understanding of the pathogenesis of C. difficile-related disease.