Molecular characterisation of cyp51A and cyp51B genes coding for P450 14α-lanosterol demethylases A (CYP51Ap) and B (CYP51Bp) from voriconazole-resistant laboratory isolates of Aspergillus flavus

Aspergillus flavus is the second most common Aspergillus spp. causing invasive infections in immunocompromised patients. Extensive prophylactic use of voriconazole (VCZ) in immunocompromised patients may enhance the selection of VCZ-resistant clinical isolates of A. flavus, compromising the effectiveness of this antifungal drug against A. flavus infection. To study triazole resistance, we selected A. flavus isolates in the laboratory showing reduced in vitro susceptibility to VCZ. The cyp51A and cyp51B genes coding for P450 14α-sterol demethylases A (CYP51Ap) and B (CYP51Bp) were characterised to examine possible drug target modification-dependent resistance to VCZ in this fungus. High-molecular-weight DNA was isolated from 10 A. flavus isolates showing in vitro resistance to VCZ (minimum inhibitory concentration (MIC) range 4-32 μg/mL) as well as from the drug-susceptible parent isolate X26728 (MIC = 1 μg/mL). The cyp51A and cyp51B genes were cloned and the nucleotide sequences were determined. A comparison of the deduced amino acid sequences of CYP51Ap from 10 VCZ-resistant isolates with that of the drug-susceptible parent showed no amino acid variation in six of the ten isolates. CYP51Ap from isolates Afl-VCZ6 and Afl-VCZ46 showed a K197N change, CYP51Ap from isolate Afl-VCZ114 showed Y132N and T469S changes, whereas that from isolate Afl-VCZ45 showed K197N, D282E and M288L changes. These results suggest that VCZ-resistant A. flavus isolates can be readily isolated in the laboratory under selection pressure. Multiple mechanisms, including drug target modification, may be responsible for the in vitro resistance of A. flavus to VCZ.