Concomitant occurrence of itraconazole-resistant and -susceptible strains of Aspergillus fumigatus in routine cultures

Objectives: Resistance to triazoles in Aspergillus fumigatus has emerged in several countries and is usually mediated by mutations in the cyp51A gene. We determined the presence of both itraconazole-susceptible and -resistant A. fumigatus in routine cultures isolated from environmental and clinical samples. Methods: A total of 50 environmental and 16 clinical A. fumigatus isolates obtained from single colonies were tested for itraconazole susceptibility by Etest. Serial dilution and plating for selected A. fumigatus cultures were performed to separate triazole-susceptible and -resistant phenotypes. Antifungal drug susceptibility of subcultures to itraconazole, posaconazole and voriconazole was determined by the broth microdilution method. Itraconazole resistance was determined by the presence of tandem repeats in the promoter region and other resistance-conferring mutations by PCR and/or direct DNA sequencing of cyp51A. Genotyping was performed with a panel of nine microsatellite loci. Results: PCR amplification of the promoter region identified 1 of 50 environmental and 2 of 16 clinical A. fumigatus isolates as mixed (itraconazole-resistant and -susceptible) cultures, while the remaining isolates yielded patterns that were consistent with their data for susceptibility to itraconazole. Purified subcultures yielded distinct susceptibility profiles, concomitant with genetic determinants of triazole susceptibility/resistance in cyp51A and different microsatellite patterns. Conclusions: Concomitant presence of triazole-susceptible and -resistant strains in single colonies of routine A. fumigatus cultures, obtained from environmental and clinical samples, has been conclusively demonstrated.