Jackeline Luciano¹, Kemily Nunes da Silva Moya¹, Mesaqueuri Mota Nonato¹, Adriana Dantas Gonzaga de Freitas², Ivanildes dos Santos Bastos ¹, Priscila Ferreira de Aquino¹

Introduction: Sporotrichosis, an emerging and neglected subcutaneous mycosis caused by Sporothrix brasiliensis, poses a growing threat to public and veterinary health due to zoonotic, epizootic, and environmental transmission. The rising incidence in the Amazon and the emergence of antifungal resistance highlights the urgent need for alternative therapeutic options. Materials and Methods: Fifteen fungal species from the genera Clonostachys, Penicillium, Purpureocillium, and Trichoderma, obtained from the Amazon Fungal Collection of the Instituto Leônidas e Maria Deane – Fiocruz Amazônia, were tested against S. brasiliensis (filamentous form) using paired culture assays. Mycelial growth inhibition percentage (PIRG) was calculated, antagonism was classified according to Bell’s scale, and selected interactions were examined by scanning electron microscopy (SEM) to characterize hyphal interactions in the host–pathogen interface. Fungal inocula (1×10⁶ conidia/mL) were grown in liquid BDL medium (7 days, 28 °C, 100 rpm) and subjected to liquid–liquid extraction with ethyl acetate and methanolic maceration of the mycelium, yielding ten extracts. Antifungal activity was evaluated by broth microdilution (CLSI M27-A3/M38-A2) against yeast-like and filamentous forms, determining MIC and MFC. Cytotoxicity was assessed in VERO cells (ATCC® CCL-81) using the Alamar Blue assay, with IC₅₀ values calculated and toxicity classified according to ISO 10993-5. Biofilm inhibition assays (24, 72, 120 h) were quantified by crystal violet staining, and structural effects were observed by SEM and confocal laser scanning microscopy (CLSM) with LIVE/DEAD staining. PIRG values from paired culture assays and biofilm inhibition data were statistically analyzed by ANOVA (p ≤ 0.05) using GraphPad Prism v10. Results: T. atroviride, T. asperellum, T. tawa, T. harzianum, and T. gamsii showed the highest PIRG values (35–36%), with SEM-confirmed mycoparasitism. Ethyl acetate extracts exhibited greater antifungal activity: T. tawa (MIC/MFC 6.25 mg/mL) and T. atroviride (12.5 mg/mL) showed greater antifungal activity; T. asperellum displayed morphology-dependent efficacy (12.5 mg/mL for the filamentous form and 25 mg/mL for the yeast-like form), while T. gamsii and T. harzianum required higher concentrations. Methanolic extracts of T. asperellum and T. gamsii were less cytotoxic, whereas ethyl acetate extracts of T. tawa and T. harzianum showed moderate cytotoxicity. In biofilm assays, ethyl acetate extracts of T. asperellum and T. tawa rapidly reduced biomass by >96%, while methanolic extracts of T. asperellum and T. harzianum maintained >97% inhibition of mature biofilms. SEM and CLSM revealed extracellular matrix disruption, cytoplasmic damage, reduced adhesion, and possible germination inhibition. Conclusion: Selected Amazonian fungal species demonstrated strong antagonistic effects against S. brasiliensis, producing bioactive metabolites with antifungal, antibiofilm activity and low cytotoxic profile. T. asperellum and T. tawa can be potential candidates for antifungal and antibiofilm therapy against S. brasiliensis. Future research should explore chemical characterization of active metabolites. This study reinforces the importance of bioprospecting Amazonian biodiversity as a strategic response to antifungal resistance, offering sustainable leads for innovative therapies.

Agradecimentos: The authors thanks FAPEAM (POSGRAD Program), CAPES (Funding Code 001), and PROEP/ILMD-FIOCRUZ AMAZÔNIA – LDMAIS for financial support