Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species

Abstract

The incidence of azole resistance in Aspergillus species has increased over the past years, most importantly for Aspergillus fumigatus. This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant A. fumigatus strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of cyp51 genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.

Keywords: Aspergillus flavus; Aspergillus fumigatus; Aspergillus niger; Aspergillus terreus; azole drug resistance; cyp51A; cyp51B; cyp51C; diagnostics; efflux; tandem repeats.

FIG 1

Chemical structures of clinically used azole antifungals. Azoles are characterized by five-atom heterocycles which contain at least one nitrogen atom (red). Compounds containing moieties with two nitrogen atoms are called diazoles, and those with three nitrogen atoms are called triazoles. Recently marketed antifungals contain one (B and D) or more (A, C, E, and F) triazole moieties and a benzene ring substituted with fluorine (A to C and F) rather than chlorine (D and E). Triazole antifungals are derivatives of either fluconazole (A to C) or ketoconazole (D to F) as the lead compound. This correlates with cross-resistance phenotypes observed in clinical and environmental isolates (see the text).

FIG 2

Azole antifungal drug resistance mechanisms in fungal cells. (A) A. fumigatus, including the cell wall (CW) and the cytoplasmic membrane (CM), in the absence of azoles. Cyp51 activity is required for the biosynthesis of the membrane compound ergosterol (green). The abcA, cdr1B, and mdr1 genes encode efflux pumps, which are localized in the plasma membrane. These genes are regulated by different transcription factors (TF), such as AtrR. (B) In the presence of azoles, some drug molecules can be pumped out through efflux pumps, but intracellular levels are sufficient to inhibit Cyp51A, resulting in decreased amounts of ergosterol in the membrane. (C) Mutations in cyp51A (asterisk) can reduce target binding of the antifungal drug, therefore conferring resistance. (D) Overexpression of the drug target Cyp51A can be mediated by mutations in the HAP complex (HAP*) or by different kinds of tandem repeats within the promoter region of cyp51A. (E) Increased expression of efflux pumps, such as AbcA, Cdr1B, and Mdr1, increases the drug tolerance of A. fumigatus. Possible mechanisms for increased transcription may be gain-of-function mutations in the regulating transcription factors (TF*, AtrR*).

FIG 3

Potential sampling workflow for ARAf screening studies. No standardized scheme for conducting screening studies is established yet, but combining several approaches proposed in the literature gives rise to an efficient workflow that eliminates false-positive results and yields robust numbers on the prevalence and phylogenetic cohesion of resistant isolates.

FIG 4

Mutations in A. fumigatus cyp51A and its promoter region. (A) Alignment of the TR₃₄ (taken from isolate 168 [16]), TR₄₆ (taken from an environmental isolate [39]), and TR₅₃ (sequence taken from a Columbian isolate [; P. LePape, personal communication]) promoter alleles with the wild-type sequences from A. fumigatus (A. fu), A. fischeri (A. fish), and A. oerlinghausenensis (A. oerl) (119). Green, TR₃₄ repeat unit; blue or red, 5′ or 3′ region and respective repeat sequences probably stemming from there. Black uppercase residues indicate sequence divergence in the repeat region, lowercase residues indicate adjacent residues, and black boxes indicate TR₃₄, TR₄₆, and TR₅₃ repeats. (B) In sequencing data, these three different types of A. fumigatus can easily be differentiated by the 5′ upstream sequence preceding the static repeat unit: sequences different from the wild-type sequenc are indicated by underscored nucleotides. (C) Known amino acid substitutions in Cyp51A. Red, known resistance-conferring substitutions; black, substitutions present in the population, probably without an effect on drug susceptibility. (D) cyp51A gene features visualized by use of sequence analysis software (Geneious R10). The data are available as a .gb annotated file or as a Word document in a community-editable form from https://github.com/oliverbader/Aspergillus_fumigatus_cyp51A.

FIG 5

Amino acid substitutions in Cyp51C proteins. Numbers and asterisks show the reference positions of residues discussed in the text (blue). Vertical dashes, sequence identity between A. flavus NRRL3357 Cyp51C (accession number XP_002383931) and A. fumigatus Cyp51A (accession number AF338659); dots, residues identical to those in A. flavus NRRL3357 Cyp51C. Accession numbers for the other reference sequences are as follows: A. flavus ATCC MYA-384/AF70, KOC15064; A. oryzae RIB40, AB514682; A. nomius, XP_015411243; A. lentulus, AEB77687; Trichophyton interdigitale, EZF31978; Trichophyton tonsurans, EGD95049; Trichophyton rubrum, XP_003235929; Trichophyton soudanense, EZF72647; Microsporum canis, XP_002845046; Histoplasma capsulatum, EER42982; and Blastomyces dermatitidis, EGE84227.

FIG 6

Interpretation of AluI-digested A. fumigatus cyp51A PCR product surrounding the L98 position. (A) In isolates carrying the L98H substitution, the AluI restriction site at this codon is abolished (167). (B) A PCR product amplified with the P5-P7 primer pair (16) encompasses both the L98 site and the promoter repeat region. When digested with AluI, the wild-type product is cleaved into four fragments, and the length of fragment A is characteristic of the length of the TR region. In L98H isolates, fragments B and C are not cleaved apart, resulting in a visible size shift. (C) When resolved in a high-percentage (e.g., 2.5%) agarose gel, the size shift is visualized as specific RFLP patterns for TR₃₄/L98H- and TR₄₆-carrying isolates. Other substitutions cannot be found using this method, as their RFLP patterns correspond to the wild-type patterns. The method may, however, be suitable to detect other promoter variants.