Clostridium difficile toxins may augment bacterial penetration of intestinal epithelium

Background: Clostridium difficile can be recovered from many high-risk hosp italized patients receiving broad-spectrum antibiotic therapy. Clostridium difficile toxins A and B have been associated with increased intestinal per meability in vitro and there is growing evidence that increased intestinal permeability may be a common mechanism whereby enteric bacteria penetrate,t he intestinal epithelium. Hypothesis: Clostridium difficile-induced alterations in the intestinal bar rier facilitate microbial penetration of the intestinal epithelium, which i n turn facilitates the translocation of intestinal bacteria. Design: Mature Caco-2 enterocytes were pretreated with varying concentratio ns of toxin A or toxin B followed by 1 hour of incubation with pure culture s of either Salmonella typhimurium, Escherichia coli, or Proteus mirabilis. The effects of toxins A and B on enterocyte viability, cytoskeletal actin, and ultrastructural topography were assessed using vital dyes, fluorescein -labeled phalloidin, and scanning electron microscopy, respectively. The to xins' effects on bacterial adherence and bacterial internalization by cultu red enterocytes were assessed using enzyme-linked immunosorbent assay and q uantitative culture, respectively. Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracel lular bacterial movement through Caco-2 enterocytes cultivated on permeable supports. Results: Neither toxin A nor toxin B had a measurable effect on the numbers of enteric bacteria internalized by Caco-2 enterocytes; however, both toxi ns were associated with alterations in enterocyte actin, decreased transepi thelial electrical resistance, and increased bacterial adherence and parace llular transmigration. Conclusion: Clostridium difficile toxins A or B may facilitate bacterial ad herence and penetration of the intestinal epithelial barrier.