Azole resistance of Aspergillus fumigatus is a global problem. The major resistance mechanism is through cytochrome P450 14-α sterol demethylase Cyp51A alterations such as a mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerance and resistance mechanisms, such as the hmg1 (a 3-hydroxy-3-methylglutaryl coenzyme-A reductase gene) mutation, are known, few reports have described studies elucidating non-Cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in A. fumigatus by in vitro mutant selection with tebuconazole, an azole fungicide. After three rounds of selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the isolates obtained and the parental strain revealed a nonsynonymous mutation in MfsD, a major facilitator superfamily protein (Afu1g11820; R337L mutation [a change of R to L at position 337]), in all isolates. Furthermore, nonsynonymous mutations in AgcA, a mitochondrial inner membrane aspartate/glutamate transporter (Afu7g05220; E535Stop mutation), UbcD, a ubiquitin-conjugating enzyme E2 (Afu3g06030; T98K mutation), AbcJ, an ABC transporter (Afu3g12220; G297E mutation), and RttA, a putative protein
Keywords: Aspergillus fumigatus; azoles; drug resistance.