Phenotypic and Molecular Characterization of Three Clinical Voriconazole- Resistant Aspergillus fumigatus Isolates Harboring TR46/M143L Mutations in cyp51A

Azole-resistant Aspergillus fumigatus poses an increasing clinical challenge. In this study, 22 clinical A. fumigatus isolates from southwestern China were identified by morphological and molecular approaches, and their azole susceptibility was determined using broth microdilution and disk diffusion assays. Three isolates exhibiting high-level voriconazole resistance were selected for further phenotypic and molecular characterization. Compared with the reference strain AF293, the resistant isolates showed heterogeneous conidiation, while biofilm metabolic activity and biomass were not significantly altered. In a Galleria mellonella infection model, these isolates exhibited attenuated virulence, as indicated by increased larval survival and reduced fungal burdens. Sequencing revealed shared cyp51A TR46/M143L alterations in all three isolates, together with additional variants in cyp51B Y411D and/or hmg1 A266T/S291N. qPCR analysis further indicated transcriptional dysregulation of ergosterol biosynthesis- and efflux pump–associated genes. Collectively, these findings provide a detailed characterization of three clinical voriconazole-resistant A. fumigatus isolates carrying cyp51A TR46/M143L alterations and suggest that target-site alterations, additional genetic variants, and pathway-level transcriptional changes may contribute to resistance in these isolates. Studies involving larger isolate collections are needed to determine whether these features are conserved among broader TR46/M143L-harboring populations.