Clostridium difficile toxins and enterococcal translocation in vivo and in vitro

Background. Clostridium difficile toxins alter permeability in cultured enterocytes and may alter intestinal epithelial permeability to bacteria in vivo. Experiments were designed to test the effects of C. difficile toxins on in vitro interactions of Enterococcus gallinarum with cultured enterocytes, as well as on translocation of E. gallinarum in mice. Materials and methods. Mature Caco-2 and HT-29 enterocytes were pretreated with C. difficile toxin A or toxin B followed by incubation with E. gallinarum. E. gallinarum-enterocyte interactions were assessed by quantitative culture. For in vivo experiments, antibiotic-treated mice were orally inoculated with C. difficile or saline, and all mice were orally inoculated 24 h later with E. gallinarum and sacrificed after another 24 h for analysis of cecal bacteria, cecal C. difficile toxin, and enterococcal translocation. Cecal C. difficile toxin was assayed as cytopathic effects on human foreskin fibroblasts. Results. Although neither toxin had a noticeable effect on bacterial internalization by cultured enterocytes, C. difficile toxins were associated with increased E. gallinarum transmigration across confluent enterocyte cultures. Mice orally inoculated with saline rather than C. difficile (n = 29) had no detectable cecal toxin, while mice orally inoculated with C. difficile (n = 30) had detectable cecal toxin. Viable E. gallinarum was recovered from the mesenteric lymph nodes of 97% of mice orally inoculated with saline followed by oral E. gallinarum, but only 37% of mice orally inoculated with C. difficile followed by oral E. gallinarum (P < 0.01). Conclusions. These results suggested that observations with cultured enterocytes, demonstrating that C. difficile toxins facilitated bacterial migration across the intestinal epithelium, might have little in vivo relevance in a mouse model of antibiotic-induced C. difficile overgrowth.