Caenorhabditis elegans as Potential Virulence Model for Aggregatibacter actinomycetemcomitans Infections

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.