Caenorhabditis elegans as an In Vivo Infection–Rescue Model for Early-Stage Antimicrobial Screening Against Gram-Positive and Gram-Negative Bacteria

Background/Objectives: The growing global burden of antimicrobial resistance highlights the need for whole-animal screening platforms that better reflect in vivo determinants of antimicrobial efficacy. A Caenorhabditis elegans infection–rescue model previously developed for antibiotic screening against Enterococcus faecalis was adapted to evaluate additional clinically relevant Gram-positive and Gram-negative bacteria. Methods: L4-stage AU37 worms were infected with Staphylococcus aureus 2406, E. faecalis ATCC51299, Escherichia coli CFT073, and Pseudomonas aeruginosa PAO1. Pathogen-specific infection durations were optimized using conventional antibiotic treatments, including vancomycin for S. aureus and gentamicin for E. faecalis, E. coli, and P. aeruginosa. Following antibiotic treatment, pathogen-specific mean rescue rates (MRR) were determined. Assay performance was then validated using positive- and negative-control antibiotics selected based on contrasting susceptibility profiles. Results: Optimized antibiotic-mediated rescue occurred at pathogen-specific time points following treatment with vancomycin for S. aureus and gentamicin for the remaining pathogens, 48 h for S. aureus (MRR, 92.6% ± 3.5%), 72 h for E. faecalis (89.4% ± 3.6%), 4 h for E. coli (94.5% ± 3.6%), and 72 h for P. aeruginosa (85.5% ± 9.1%) following treatment with conventional antibiotics, with all treated groups differing significantly from controls (p < 0.001). Positive-control antibiotics, including chloramphenicol, tetracycline, and amikacin, produced strong rescue at the optimized infection time points. In contrast, negative-control antibiotics, including erythromycin and chloramphenicol, produced minimal rescue. Conclusions: These data show that the C. elegans infection–rescue model can distinguish effective from ineffective antibiotic treatment across the four tested bacterial infection conditions and may serve as a useful early-stage platform for antimicrobial screening.