Sporotrichosis is the most prevalent subcutaneous mycosis globally, and it is typically caused by direct inoculation of the soil saprophytic fungus Sporothrix spp. into the patients’ skin. However, sporotrichosis has an important zoonotic transmission route between cats and humans in hot-spot endemic areas such as Brazil. Antifungal itraconazole is the first-line treatment; however, it is frequently associated with recurrence after withdrawal, mainly on cats. Biofilms are important resistance structures related to the environmental persistence of most microorganisms. In the present work, we evaluated Sporothrix yeasts’ ability to form biofilms in an ex vivo model of infected claws of cats. Using scanning electron microscopy, we demonstrated the presence of fungal biofilms in the claws of cats diagnosed with sporotrichosis confirmed by isolation of Sporothrix spp. in culture. We present here evidence of antibiofilm activity of miltefosine and suggest its use off-label as an antifungal as a putative alternative to itraconazole against Sporothrix biofilms. Claw contamination could sustain infections through a continuous inoculation cycle between open lesions and cat claws. Our results further support the off-label use of miltefosine as a promising alternative, especially for mycosis refractory to conventional treatment.

The increase in the prevalence and severity of fungal infections and the resistance to available antifungals highlights the imperative need for novel therapeutics and the search for new targets. High-content screening of libraries containing hundreds of compounds is a powerful strategy for searching for new drug candidates. In this study, we screened the Pandemic Response Box library (Medicines for Malaria Venture) of 400 diverse molecules against the Sporothrix pathogenic species. The initial screen identified twenty-four candidates that inhibited the growth of Sporothrix brasiliensis by more than 80%. Some of these compounds are known to display antifungal activity, including olorofim (MMV1782354), a new antifungal drug. Olorofim inhibited and killed the yeasts of S. brasiliensis, S. schenckii, and S. globosa at concentrations lower than itraconazole, and it also showed antibiofilm activity. According to the results obtained by fluorimetry, electron microscopy, and particle characterization analyses, we observed that olorofim induced profound alterations on the cell surface and cell cycle arrest in S. brasiliensis yeasts. We also verified that these morphophysiological alterations impaired their ability to adhere to keratinocytes in vitro. Our results indicate that olorofim is a promising new antifungal against sporotrichosis agents.