Clostridium difficile toxins and enterococcal translocation in vivo and invitro

Background. Clostridium difficile toxins alter permeability in cultured ent erocytes and may alter intestinal epithelial permeability to bacteria in vi vo. 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. gallina rum. E. gallinarum-enterocyte interactions were assessed by quantitative cu lture. For in vivo experiments, antibiotic-treated mice were orally inocula ted with C. difficile or saline, and all mice were orally inoculated 24 h l ater with E. gallinarum and sacrificed after another 24 h for analysis of c ecal bacteria, cecal C. difficile toxin, and enterococcal translocation. Ce cal C. difficile toxin was assayed as cytopathic effects on human foreskin fibroblasts. Results. Although neither toxin had a noticeable effect on bacterial intern alization by cultured enterocytes, C. difficile toxins were associated with increased E. gallinarum transmigration across confluent enterocyte culture s. 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 fro m the mesenteric lymph nodes of 97% of mice orally inoculated with saline f ollowed 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 entero cytes, demonstrating that C. difficile toxins facilitated bacterial migrati on across the intestinal epithelium, might have little in vivo relevance in a mouse model of antibiotic-induced C. difficile overgrowth. (C) 2001 Acad emic Press.