Background: To study voriconazole (VCZ) resistance in Aspergillus flavus, we selected laboratory isolates showing reduced in vitro susceptibility to VCZ. The cyp51A and B genes coding for P450 14α -sterol demethylases A (CYP51Ap) and B (CYP51Bp) were characterized to examine drug target modification dependent resistance to VCZ. Methods: DNA was isolated from three representative A. flavus isolates (Afl-VCZ1, Afl-VCZ2 and Afl-VCZ3) showing in vitro resistance to VCZ (MICs ranged from 4-16 μg/ml) and the drug-susceptible parent isolate X26728 (MIC 0.25 μg/ml). The cyp51A and B genes were cloned by PCR and gene cloning techniques, and the nucleotide (nt) sequences were determined. The CYP51Ap and CYP51Bp amino acid (aa) sequences were deduced from the nt sequences Results: The cyp51A and B genes code for 513-aa and 527-aa long polypeptides, respectively. Alignment of the nt sequence of cyp51A of A. flavus X26728 with that of A. flavus NRRL 3357 (GenBank Accession AAIHO1000692.1) showed a single nt variation at codon 214 (GAC --> AAC) resulting in D214N on CYP51Ap. Similarly, a comparison of cyp51B gene of A. flavus X26728 with that of strain NRRL 3357 (Accession AAIHO1001161.1) showed a single nt variation at codon 186 (TAC --> CAC) resulting in Y186H on the CYP51Bp. However, alignment of the deduced aa sequences of CYP51Ap and CYP51Bp of the VCZ-susceptible A. flavus X26728 with those of the VCZ-resistant isolates Afl-VCZ1, Afl-VCZ2 and Afl-VCZ3 showed no aa variation either on CYP51Ap or CYP51Bp. A comparison of the 5’-regulatory regions of cyp51A and B genes of the parent strain X26728 with those of the drug-resistant isolates showed no nt mutation. Conclusions: These results suggest that VCZ-resistant A. flavus isolates can be readily isolated in the laboratory under selection pressure. The mechanism(s) responsible for the in vitro resistance of A. flavus isolates Afl-VCZ1, Afl-VCZ2 and Afl-VCZ3 to VCZ is independent of mutational changes of the cyp51A and B genes.
Full conference title:
46th Interscience Conference on Antimicrobial Agents and Chemotherapy
- ICAAC 46th