In vitro evolution of itraconazole resistance in Aspergillus fumigatus involves multiple mechanisms of resistance

Abstract

We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions. For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole. After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations. These mutant strains had different growth rates and different levels of itraconazole resistance. Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole. The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes. Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains. Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole. Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug.

FIG. 1.

Hierarchical clustering showing the gene expression profiles of seven different A. fumigatus genes during growth in the absence (A) and in the presence (B) of itraconazole. In both experiments, the relative levels of each transcript were monitored by real-time RT-PCR of mRNA extracted after 24 h of growth. These values were log₂ transformed and submitted to a hierarchical clustering algorithm by using the default parameters of CLUSTER software, kindly provided by M. Eisen and available at http://www.microarrays.org/software.html. In both cases, as shown by the scales on the right, a color scheme was used to designate genes that were down-regulated (green) or up-regulated (red). The clustering, along with the resulting dendrogram, was displayed by using TREEVIEW software, also available at the URL mentioned above (10).