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 prerequi site to infection and toxin production. A novel flow cytometric method was developed for detecting adherence of C, difficile to human colonic and smal l intestinal epithelial cells (EC) and human intestinal cell lines. Small i ntestinal and colonic EC were isolated from biopsy specimens with mucolytic and chelating agents. Adherence of fluorochrome-labelled C, difficile to E C was measured by flow cytometry and was calculated as increase in median f luorescent intensity (Delta MFI). Cells with bacteria attached could be dis tinguished easily from cells alone or cells with unlabelled bacteria attach ed. Toxin-positive C, difficile adhered to colonic and small intestinal EC (Delta MFI 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 (Del ta MFI 26.1 SD 32.5, n = 17 and 18.3 SD 31.3, n = 16), Adherence of toxin-p ositive C, difficile to the intestinal cell lines Caco-2. (Delta MFI 9.4 SD 4.4, n = 14) and HT29 (Delta MFI 8.1 SD 3.1, n = 12) was quantifiable, alt hough at a significantly lower level than with primary colonic epithelial c ells. Adherence of the toxin-negative strain was slightly lower, Delta MFI 6.5 SD 1.8, n = 9 with Caco-2 cells and Delta MFI 6.0 SD 2.0, n = 10 with H T29 cells. Adherence of C, difficile to epithelial cell lines was blocked w ith C, difficile antiserum, confirming specificity of adherence, In conclus ion, flow cytometry is a useful approach to quantifying adherence of C, dif ficile to human colonic and small intestinal epithelial cells. Binding of t oxin-negative as well as toxin-positive bacteria was detectable by this app roach. Analysis of C, difficile adherence to target cells may have importan t implications for the understanding of the pathogenesis of C, difficile-re lated disease.