Effects of Clostridium difficile toxin A and toxin B on bacterial penetration of the intestinal barrier in vitro and in vivo.

摘要

Clostridium difficile toxins are the widely recognized etiologic agents of a spectrum of antibiotic associated intestinal diseases. Because C. difficile toxins A and B have been associated with increased intestinal permeability in vitro, we formulated the following hypothesis: C. difficile-induced alterations in the intestinal barrier facilitates microbial penetration of the intestinal epithelium, which in turn facilitates the translocation of intestinal bacteria, a putative initial event in the pathogenesis of systemic infection in certain patient populations. This hypothesis was tested using in vitro cell culture systems, as well as in vivo mouse model.; To clarify the effects of C. difficile toxins A and B on bacterial interactions with cultured enterocytes, mature enterocytes were pretreated C. difficile toxin A or toxin B followed by incubation with cultures of various enteric bacteria. The effects of toxins A and B on enterocyte viability, cytoskeletal actin, and ultrastructural topography, were assessed. The toxins' effects on bacterial adherence and bacterial internalization by cultured enterocytes were assessed using ELISA and quantitative culture, respectively. Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracellular bacterial movement through enterocytes cultivated on permeable supports.; To clarify the in vivo effects of C. difficile toxins, antibiotic-treated mice were orally inoculated with C. difficile or saline, followed 24 hours later by Enterococcus gallinarum. Mice were sacrificed 24 hours later for analysis of intestinal permeability, cecal bacteria, cecal C. difficile toxin, and enterococcal translocation.; In vitro, neither toxin A nor toxin B had a measurable effect on the numbers of enteric bacteria internalized by cultured enterocytes; however, both toxins were associated with alterations in enterocyte actin, with decreased transepithelial electrical resistance, and with increased bacterial adherence and paracellular transmigration.; In vivo, 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.; Our original hypothesis was strongly supported by data obtained from cultured enterocytes. However, data obtained from our mouse model indicated that C. difficile toxins may have discordant in vivo and in vitro effects.