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Brad Allen Feltis, MD; Adam S. Kim, BA; Karen M. Kinneberg, MS; David L. Lyerly, PhD; Tracy D. Wilkins, PhD; Stanley L. Erlandsen, PhD; Carol L. Wells, PhD

Arch Surg. 1999;134:1235-1242.

Background  Clostridium difficile can be recovered from many high-risk hospitalized patients receiving broad-spectrum antibiotic therapy. Clostridium difficile toxins A and B have been associated with increased intestinal permeability in vitro and there is growing evidence that increased intestinal permeability may be a common mechanism whereby enteric bacteria penetrate the intestinal epithelium.

Hypothesis  Clostridium difficile–induced alterations in the intestinal barrier facilitate microbial penetration of the intestinal epithelium, which in turn facilitates the translocation of intestinal bacteria.

Design  Mature Caco-2 enterocytes were pretreated with varying concentrations of toxin A or toxin B followed by 1 hour of incubation with pure cultures 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 toxins' effects on bacterial adherence and bacterial internalization by cultured enterocytes were assessed using enzyme-linked immunosorbent assay and quantitative culture, respectively. Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracellular 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 toxins were associated with alterations in enterocyte actin, decreased transepithelial electrical resistance, and increased bacterial adherence and paracellular transmigration.

Conclusion  Clostridium difficile toxins A or B may facilitate bacterial adherence and penetration of the intestinal epithelial barrier.

From the Departments of Surgery (Drs Feltis and Wells and Mr Kim), Laboratory Medicine and Pathology (Dr Wells and Ms Kinneberg), and Genetics, Cell Biology and Development (Dr Erlandsen), University of Minnesota, Minneapolis; and the Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg (Drs Lyerly and Wilkins). Drs Wilkins and Lyerly are co-owners of TechLab Inc, Blacksburg, Va. This company manufactures and sells intestinal diagnostic assays. One of these products is an enzyme-linked immunosorbent assay that detects Clostridium difficile toxins A and B in stool samples.

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