Zebrafish as an in vivo model to study the pathogenicity of Clostridioides difficile clinical strains

Objectives

Clostridioides difficile is a major cause of nosocomial diarrhea, and its virulence is typically attributed to the production of toxins. However, other genomic factors may contribute to its pathogenicity. To study the in vivo aspects of C. difficile infection, various animal models have been employed. Zebrafish (Danio rerio) offers several advantages over mammalian models, but there are still few studies using it to evaluate C. difficile infection. Here, we aimed to explore in vivo virulence differences among clinical strains by employing the zebrafish embryo model using eight sequenced C. difficile isolates with distinct genomic profiles.

Methods

Embryos were microinjected with bacterial suspensions, and mortality and cardiac edema were monitored over 96 h. Survival and cardiotoxicity were assessed and correlated with whole-genome data and clinical outcomes.

Results

Two strains exhibited distinct pathogenic effects: HC48 (ST42) caused significantly increased mortality (p < 0.0001), and HC132 (ST669) induced cardiotoxicity in 20 % of embryos. Surprisingly, the hypervirulent control strain NAP1/027 did not produce enhanced virulence in this model.

Conclusion

While zebrafish embryos showed promise for distinguishing strain-specific virulence, limitations such as colonization capacity and host–microbe interactions suggest that further research is needed to validate this model for C. difficile virulence testing.