Genomic Context of Azole Resistance Mutations in Aspergillus fumigatus Determined Using Whole-Genome Sequencing

Abstract

A rapid and global emergence of azole resistance has been observed in the pathogenic fungus Aspergillus fumigatus over the past decade. The dominant resistance mechanism appears to be of environmental origin and involves mutations in the cyp51A gene, which encodes a protein targeted by triazole antifungal drugs. Whole-genome sequencing (WGS) was performed for high-resolution single-nucleotide polymorphism (SNP) analysis of 24 A. fumigatus isolates, including azole-resistant and susceptible clinical and environmental strains obtained from India, the Netherlands, and the United Kingdom, in order to assess the utility of WGS for characterizing the alleles causing resistance. WGS analysis confirmed that TR34/L98H (a mutation comprising a tandem repeat [TR] of 34 bases in the promoter of the cyp51A gene and a leucine-to-histidine change at codon 98) is the sole mechanism of azole resistance among the isolates tested in this panel of isolates. We used population genomic analysis and showed that A. fumigatus was panmictic, with as much genetic diversity found within a country as is found between continents. A striking exception to this was shown in India, where isolates are highly related despite being isolated from both clinical and environmental sources across >1,000 km; this broad occurrence suggests a recent selective sweep of a highly fit genotype that is associated with the TR34/L98H allele. We found that these sequenced isolates are all recombining, showing that azole-resistant alleles are segregating into diverse genetic backgrounds. Our analysis delineates the fundamental population genetic parameters that are needed to enable the use of genome-wide association studies to identify the contribution of SNP diversity to the generation and spread of azole resistance in this medically important fungus.

Importance: Resistance to azoles in the ubiquitous ascomycete fungus A. fumigatus was first reported from clinical isolates collected in the United States during the late 1980s. Over the last decade, an increasing number of A. fumigatus isolates from the clinic and from nature have been found to show resistance to azoles, suggesting that resistance is emerging through selection by the widespread usage of agricultural azole antifungal compounds. Aspergillosis is an emerging clinical problem, with high rates of treatment failures necessitating the development of new techniques for surveillance and for determining the genome-wide basis of azole resistance in A. fumigatus.

FIG 1

Circos (72) image of normalized whole-genome depth of coverage of all 24 A. fumigatus isolates (plotted as listed in the key), averaged over 10,000-bp bins. Black circles mark the presence of the TR₃₄/L98H mutation. Chromosomes 1 and 6 show large deletions spanning >300 kbp in most isolates, except AF65 and AF293, while chromosome 8 displays a 60-kbp deletion in all isolates except AF65, 09-7500806, 12-7504652, and 12-7504462.

FIG 2

Phylogenetic analysis of A. fumigatus isolates representing azole-resistant and -susceptible genotypes from India, the Netherlands, and the United Kingdom. Bootstrap analysis was performed on WGST SNP data from 24 A. fumigatus genomes to generate an unrooted maximum-likelihood phylogeny, with all branches supported to 87% or higher. Isolates are color coded according to country of origin (red, India; green, the Netherlands; blue, United Kingdom), and environmental isolates are indicated by a tree symbol. Isolates marked with a black circle contain the TR₃₄/L98H mutation in the cyp51A gene. Branch lengths represent the numbers of SNPs between taxa.

FIG 3

Recombination analysis, using LDhat interval (42), of A. fumigatus isolates from the United Kingdom, the Netherlands, and India. The black peaks represent the recombination rate across the whole A. fumigatus genome, and the vertical red lines mark the chromosome boundaries.