A Novel Combination of Mutations Confers Pan-Azole Resistance in Aspergillus fumigatus.

The treatment of invasive and chronic aspergillosis involves triazole drugs. Its intensive use has resulted in the selection of resistant isolates, and at present, azole resistance in is considered an emerging threat to public health worldwide. The aim of this work is to uncover the molecular mechanism implicated in the azole resistance phenotype of three clinical strains isolated from an Argentinian cystic fibrosis patient under long-term triazole treatment. Strain susceptibilities were assessed, and gene sequences were analyzed. Two of the studied strains harbored the TR34-L98H allele. These strains showed high MIC values for all tested triazoles (>16.00 μg/ml, 1.00 μg/ml, 1.00 μg/ml, and 2.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The third strain had a novel amino acid change (R65K) combined with the TR34-L98H mutations. This new mutation combination induces a pan-azole MIC augment compared with TR34-L98H mutants (>16 μg/ml, 4.00 μg/ml, 4.00 μg/ml, and 8.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The strain harboring the TR34-R65K-L98H allele showed no inhibition halo when voriconazole susceptibility was evaluated by disk diffusion. The effect of these mutations in the azole-resistant phenotype was confirmed by gene replacement experiments. Transformants harboring the TR34-L98H and TR34-R65K-L98H alleles mimicked the azole-resistant phenotype of the clinical isolates, while the incorporation of the TR34-R65K and R65K alleles did not significantly increase azole MIC values. This is the first report of the TR34-L98H allele in Argentina. Moreover, a novel allele (TR34-R65K-L98H) that induces a pan-azole MIC augment is described.