Phenotypic and Molecular Characterization of Three Clinical Voriconazole-Resistant Aspergillus fumigatus Isolates Harboring the cyp51A TR46/Y121F/T289A Genotype

Azole-resistant Aspergillus fumigatus poses an increasing clinical challenge. In this study, 22 clinical filamentous fungal isolates from southwestern China were preliminarily identified as A. fumigatus based on morphological characteristics, and their antifungal susceptibility was determined using broth microdilution. Three isolates (AF268, AF275 and AF277) exhibiting high-level voriconazole resistance were selected for further phenotypic and molecular characterization. Compared with AF293, the three resistant isolates showed no significant differences in biofilm metabolic activity or biomass, but all exhibited significantly reduced conidiation at 72 h. In a Galleria mellonella infection model, these isolates exhibited attenuated virulence, as indicated by increased larval survival and reduced fungal burdens. All three isolates harbored the cyp51A TR46/Y121F/T289A genotype and shared Hmg1 S212P/Y564F variants, while no Cyp51B substitutions were detected. RT-qPCR analysis further indicated transcriptional dysregulation of ergosterol biosynthesis- and efflux pump-associated genes. These findings show that the three clinical isolates carrying the canonical cyp51A TR46/Y121F/T289A resistance genotype showed differences in biological traits and transcriptional responses relative to the reference strain AF293. The high voriconazole MICs are consistent with the shared cyp51A genotype, whereas the functional significance of the Hmg1 variants and transcriptional changes requires further genetic validation.