Bridging the gap in AMR research: validation of a standardized Galleria mellonella model to evaluate Clostridioides difficile pathogenesis and treatment efficacy.

BACKGROUND

Clostridioides difficile infection (CDI) is a significant global health burden. Galleria mellonella offers an ethical, cost-effective invertebrate alternative to mammalian pre-clinical models. This study evaluated the feasibility of using Galleria mellonella to study CDI pathogenesis and treatment.

METHODS

We optimized larval storage, temperature, and inoculum size. The model was validated by assessing survival following infection with diverse C. difficile ribotypes (RT) and vancomycin treatment. Bacterial burden was quantified via qPCR, and survival was analyzed using Kaplan-Meier curves.

RESULTS

An inoculum of 1 × 107 CFU/mL in fresh larvae (<7 days since receipt) at 37 °C proved optimal. Fresh larvae exhibited significantly higher survival compared to older larvae (P = 0.003). Infection with toxigenic strains resulted in significantly lower survival (35%) compared to uninfected controls (90%; P < 0.001), with virulence varying significantly among RTs. Vancomycin treatment improved survival to 90% in a dose-dependent manner compared to untreated controls (P < 0.001), with a corresponding reduction in bacterial burden confirmed by qPCR.

CONCLUSION

We conclude that this optimized oral G. mellonella model provides a robust and ethically sound high-throughput platform for screening novel anti-C. difficile compounds and for comparative virulence studies of diverse ribotypes, effectively bridging in vitro assays and mammalian studies for future drug development.