Mo1833 Human Alpha-Defensin 6 Regulated by the Cooperation of Beta-Catenin and ATOH1, Might Be the Pathogenesis of Crohn's Disease

Abstract

Recommended therapy of bacterial infection is generally based on in vitro susceptibility of an organism to an infecting organism. Yet drug bioavailability based on solubility, protein binding and other factors along with host resistance and inflammatory-immune response importantly alter response to treatment. The objective of the study was to establish in vivo animal model to study antimicrobial therapy of C. difficile infection. C. difficile isolate with hyper-virulent factors (toxin A/B plus binary toxin and 18 base pair deletion in tcdC gene) was used in the study. The cultured C. difficile was stained with near infrared (NIR) dye. Stained C. difficilewere determined by using fluorescent microscope and confocal microscope. To monitor stained bacterial trafficking in the gut animals were imaged in real-time. The intestines from treated animals were dissected at the end of imaging study. The bacterial signal intensity and migration pattern were recorded and compared. C. difficile toxin secretion in the different segment of GI system was analyzed. Results showed the C. difficile can be stained by NIR dye. Stained bacteria can be imaged by NIR camera system to track the C. difficile traffic in the gut in real-time. Untreated control animal has different bacteria migration pattern than the animal with C. difficile infection treated with ciprofloxacin. TheNIR florescent probe can be used to visualize C. difficile trafficking in the gastrointentinal tract through culturing, staining and imaging. Fig. 1. Confocal images of C. difficile. Viable bacterial stained with NIR dye (A. Red) and dead bacterial determined with Sytox green (B. Green). Stain efficiency was determined by co-localized all cells (C. Green) and NIR (C. Red). Confocal microscope analyzed NIR dye binding at subcellular level (D). Red and green represent NIR and cell nuclei, respectively. Results demonstrate C. difficile can be stained by NIR dye. Most bacteria remain viable after the staining procedure.