Antifungal susceptibility and molecular characterization of clinical and environmental isolates of Schizophyllum commune

Schizophyllum commune is a cosmopolitan, saprophytic basidiomycete known to cause respiratory tract infections following spore inhalation. Management of such infections remained poorly defined, and antifungal susceptibility testing may provide valuable guidance for therapeutic decisions. However, existing data are limited, and protocols are not optimized for non-sporulating molds such as S. commune , requiring methodological adaptations. This study aimed to (i) perform molecular characterization of isolates and (ii) determine antifungal susceptibility profiles of a collection of S. commune isolates using both Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methods. A total of 113 fungal isolates were included, comprising environmental ( n = 31), clinical French isolates ( n = 74), and strains from fungal international collections ( n = 8). Species identification was confirmed via the large subunit (LSU) region of the ribosomal DNA sequencing and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phylogenetic analysis was conducted on a subset of 20 isolates using partial sequences of three genes: LSU, EF-1α, and RPB2. Modifications to the CLSI and EUCAST broth microdilution methods included: inoculum preparation using standardized culture slices fragmented by bead-beating, incubation at 35°C for 96 h and endpoints reading at 100% of inhibition. Phylogenetic analysis confirmed that all tested isolates were S. commune and not S. radiatum . Amphotericin B (AMB) and voriconazole (VOR) demonstrated low geometrical mean minimal inhibitory concentrations (MICs) across both methods (EUCAST: AMB = 0.39 µg/mL, VOR = 0.24 µg/mL; CLSI: AMB = 0.1 µg/mL, VOR = 0.2 µg/mL). In contrast, terbinafine exhibited high MIC values (>8 µg/mL) in both protocols. Notable discrepancies were observed for posaconazole (POS), isavuconazole (ISA), and itraconazole (ITR), with MICs approximately two dilution steps higher in EUCAST compared to CLSI (POS: 4.22 µg/mL vs 0.9 µg/mL, ISA: 3.64 µg/mL vs 0.74 µg/mL, and ITR: 3.9 µg/mL vs 0.81 µg/mL, respectively). After standardization of both methods for this non-sporulating mold, VOR and AMB have the lowest MIC values. IMPORTANCE Schizophyllum commune is a fungal pathogen increasingly associated with respiratory infections, yet therapeutic guidance remains unclear. This study provides the largest collection to date of clinical and environmental isolates (113 total) and applies standardized antifungal susceptibility testing using adapted EUCAST and CLSI protocols for this non-sporulating species. The results show that amphotericin B and voriconazole are the most active agents in vitro , while terbinafine is ineffective. These findings are critical for informing treatment decisions and interpreting susceptibility tests, especially in the absence of established guidelines. By introducing a reproducible methodology and delivering clinically relevant data, this work addresses a gap in medical mycology and supports improved management of rare fungal infections.