title: 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.
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café S/N, CEP 14040-903, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Márcia Eliana
authors.initials: ME
authors.lastname: da Silva Ferreira
authors.forename: José Luiz
authors.initials: JL
authors.lastname: Capellaro
authors.forename: Everaldo
authors.initials: E
authors.lastname: dos Reis Marques
authors.forename: Iran
authors.initials: I
authors.lastname: Malavazi
authors.forename: David
authors.initials: D
authors.lastname: Perlin
authors.forename: Steven
authors.initials: S
authors.lastname: Park
authors.forename: James B
authors.initials: JB
authors.lastname: Anderson
authors.forename: Arnaldo L
authors.initials: AL
authors.lastname: Colombo
authors.forename: Beth A
authors.initials: BA
authors.lastname: Arthington-Skaggs
authors.forename: Maria Helena S
authors.initials: MH
authors.lastname: Goldman
authors.forename: Gustavo H
authors.initials: GH
authors.lastname: Goldman
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D025141:Drug Resistance, Fungal; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D017964:Itraconazole / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013261:Sterols / Q000737:chemistry; D014170:Transformation, Genetic
chemical_list: D000935:Antifungal Agents; D018076:DNA, Complementary; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D013261:Sterols; D017964:Itraconazole; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus; C465527:cytochrome P-450 CYP51B, Aspergillus