Friday, March 23, 2012: 3:30 p.m. - 4:45 p.m.
Presentation Type: Poster Session
Objectives: This study utilized the Caenorhabditis elegans (Ce) digestive anatomy as a model to assess Aa virulence and epithelial cell colonization by fimbriated or nonfimbriated Aa. Methods: Bristol N2 Ce were maintained on NGM agar plates with E. coli OP50 as a food source prior to transfer to TSBYE plates for experiments. Synchronized Ce were allowed to feed on D7-S (rough Aa) and D7S-SA (smooth Aa) or OP50 to inoculate Ce for all experiments. Kill-Curve Experiment: Ten inoculated Ce were serially transferred every 24 hours to a consistent food source, of D7S, D7S-SA, or OP50 lawns. During every transfer, Ce were assessed for viability, counted and documented. Each condition was performed in duplicate. Fluorescent Microscopy: Prior to feeding of Ce on the bacterial cultures, D7S, D7S-SA or OP50 were labeled with Cell Tracker Red dye incorporation in broth cultures, washed, and plated on TSBYE. Ce fed on fluorescent lawns for 48 hours prior to fixation with 4% paraformaldehyde in Phosphate Buffered Saline (PBS). Ce were washed three times with PBS, mounted onto slides, and imaged with a Nikon microscope. Viable Aa Recovery: Innoculated Ce were treated with 200 µg/mL of Gentamyacin to kill external bacteria, PBS-washed extensively, and mechanically disrupted using a tissue homogenizer until no intact Ce were visible. Serial dilutions of the lysate were performed to assess the number of viable bacteria colonizing Ce internally. Results: Kill-curve experiments showed that OP50-fed Ce had a lower life-span than Aa-fed Ce. The fluorescence microscopy of Ce definitively showed ‘smooth’ Aa having a higher tendency of colonizing within the digestive tract as opposed to OP50 and ‘rough’ Aa. Viable bacterial counts from Ce were inconclusive. Conclusion: While C. elegans show great potential as an experimental model to study Aa colonization and virulence, further optimization of experimental conditions is still required.This abstract is based on research that was funded entirely or partially by an outside source: NIH/NIDCR R01 DE016715 NIH/NIDCR R01 DE016715