Clostridioides difficile infection is a significant public health concern, being the primary cause of healthcare-associated diarrhea worldwide. It can cause severe diarrhea to life-threatening pseudomembranous colitis [1]. Gut microbiota dysbiosis, especially due to antibiotic use, can lead to C. difficile proliferation. The clinical manifestation of C. difficile infection is primarily attributed to the production of toxins A and B. However, other factors can enhance C. difficile virulence, such as the production of a third toxin called binary toxin (CDT), modifications to the regulators of toxins A and B, and other virulence factors, including proteins involved in adhesion, invasion, and motility. Moreover, other less-known virulence factors may also be related to the increase in the pathogenesis, and we need in vivo models to study these interactions. Establishing correlations between genomic features and in vivo virulence is a crucial approach to better understanding the mechanisms underlying C. difficile pathogenicity and to further treatment studies as well [[1], [2], [3], [4]].

In order to study in vivo aspects of C. difficile infection, a variety of animal models have been used, such as large animals, small mammals, or even non-mammalian animals. Since mammalian models require a more structured and expensive environment, studies with non-mammalian models have been increasing recently. The zebrafish (Danio rerio) model offers several advantages, including embryo transparency for observing internal responses, rapid development that allows for real-time tracking of all stages, and a genome with 70 % similarity to the human genome, which shares several physiological similarities, such as immune responses [5]. In the last years, the zebrafish model has become increasingly used for analyzing bacterial infections (mostly aerobic) and antimicrobial treatments, focusing on survival rates and cardiotoxicity [[6], [7], [8], [9], [10], [11], [12]].

There are still a few studies using zebrafish with C. difficile, and most of them use the extracted toxins, while only two use the bacterial strain. Studies using the purified toxins already demonstrated observable effects in zebrafish embryos, with survival rate and cardiotoxicity evaluation; however, the literature lacks studies with these analyses using live C. difficile [[13], [14], [15], [16], [17]].

A 2018 study applied the zebrafish model to determine the ability of three human gastrointestinal microorganisms in colonize the D. rerio gastrointestinal tract and C. difficile was the most persistent species in the larvae [18]; another study conducted in 2020 analyzed the zebrafish innate immune response to the microorganism using fluorescence and was able to visualize the immune response only when the inoculum was given by microinjection [19]. While their conclusions on the viability of this model differ, the studies used different methodologies and had distinct goals. Also, a limitation of both studies was that they used only one C. difficile strain.

To our knowledge, this is the first study to evaluate C. difficile virulence using different clinical strains with a zebrafish embryo in vivo model. This study aims to comprehend the viability of the zebrafish model as an alternative to evaluate C. difficile virulence using mortality rates and cardiotoxicity effects as parameters.