Aspergillus fumigatus is a ubiquitous fungus and the main agent of aspergillosis, a common fungal infection in the immunocompromised population. Triazoles such as itraconazole and voriconazole are the common first-line drugs for treating aspergillosis. However, triazole resistance in A. fumigatus has been reported in an increasing number of countries. While most studies of triazole resistance have focused on mutations in the triazole target gene cyp51A, >70% of triazole-resistant strains in certain populations showed no mutations in cyp51A. To identify potential non-cyp51A mutations associated with triazole resistance in A. fumigatus, we analyzed the whole genome sequences and triazole susceptibilities of 195 strains from 12 countries. These strains belonged to three distinct clades. Our genome-wide association study (GWAS) identified a total of six missense mutations significantly associated with itraconazole resistance and 18 missense mutations with voriconazole resistance. In addition, to investigate itraconazole and pan-azole resistance, Fisher’s exact tests revealed 26 additional missense variants tightly linked to the top 20 SNPs obtained by GWAS, of which two were consistently associated with triazole resistance. The large number of novel mutations related to triazole resistance should help further investigations into their molecular mechanisms, their clinical importance, and the development of a comprehensive molecular diagnosis toolbox for triazole resistance in A. fumigatus.

Aspergillus fumigatus is a ubiquitous saprophytic mold that can cause a range of clinical syndromes, from allergic reactions to invasive infections. Amphotericin B (AMB) is a polyene antifungal drug that has been used to treat a broad range of systemic mycoses since 1958, including as a primary treatment option against invasive aspergillosis in regions with high rates (≥10%) of environmental triazole resistance. However, cases of AMB-resistant A. fumigatus strains have been increasingly documented over the years, and high resistance rates were recently reported in Brazil and Canada. The objective of this study is to identify candidate mutations associated with AMB susceptibility using a genome-wide association analysis of natural strains, and to further investigate a subset of the mutations in their putative associations with differences in AMB minimum inhibitory concentration (MIC) and in growths at different AMB concentrations through the analysis of progeny from a laboratory genetic cross. Together, our results identified a total of 34 candidate single-nucleotide polymorphisms (SNPs) associated with AMB MIC differences—comprising 18 intergenic variants, 14 missense variants, one synonymous variant, and one non-coding transcript variant. Importantly, progeny from the genetic cross allowed us to identify putative SNP–SNP interactions impacting progeny growth at different AMB concentrations.

Aspergillus fumigatus is a saprophytic mold and an opportunistic pathogen with a broad geographic and ecological distribution. A. fumigatus is the most common etiological agent of aspergillosis, affecting over 8,000,000 individuals worldwide. Due to the rising number of infections and increasing reports of resistance to antifungal therapy, there is an urgent need to understand A. fumigatus populations from local to global levels. However, many geographic locations and ecological niches remain understudied, including soil environments from arctic regions. In this study, we isolated 32 and 52 A. fumigatus strains from soils in Iceland and the Northwest Territories of Canada (NWT), respectively. These isolates were genotyped at nine microsatellite loci and the genotypes were compared with each other and with those in other parts of the world. Though significantly differentiated from each other, our analyses revealed that A. fumigatus populations from Iceland and NWT contained evidence for both clonal and sexual reproductions, and shared many alleles with each other and with those collected from across Europe, Asia, and the Americas. Interestingly, we found one triazole-resistant strain containing the TR₃₄ /L98H mutation in the cyp51A gene from NWT. This strain is closely related to a triazole-resistant genotype broadly distributed in India. Together, our results suggest that the northern soil populations of A. fumigatus are significantly influenced by those from other geographic regions.

Aspergillus fumigatus is a globally distributed mold and a major cause of opportunistic infections in humans. Because most infections are from environmental exposure, it is critical to understand environmental populations of A. fumigatus. Soil is a major ecological niche for A. fumigatus. Here, we analyzed 748 soil isolates from 21 locations in six provinces and one territory in Canada. All isolates were genotyped using nine microsatellite markers. Due to small sample size and/or close proximities for some local samples, these isolates were grouped into 16 local geographic and ecological populations. Our results indicated high allelic and genotypic diversities within most local and provincial populations. Interestingly, low but statistically significant genetic differentiations were found among geographic populations within Canada, with relatively similar proportions of strains and genotypes belonging to two large genetic clusters. In Hamilton, Ontario, and Vancouver, BC, where two and three ecological populations were analyzed, respectively, we found limited genetic difference among them. Most local and provincial populations showed evidence of both clonality and recombination, with no population showing random recombination. Of the 748 soil isolates analyzed here, two were resistant to triazole antifungals. We discuss the implications of our results to the evolution and epidemiology of A. fumigatus.

The wild edible mushroom Cantharellus enelensis is a recently described species of the golden chanterelles found in eastern North America. At present, the genetic diversity and population structure of C. enelensis are not known. In this study, we analyzed a total of 230 fruiting bodies of C. enelensis that were collected from three regions of Canada: near the east and west coasts of Newfoundland (NFLD), with 110 fruiting bodies each, and around Hamilton, Ontario (10 fruiting bodies). Among the 110 fruiting bodies from each coast in NFLD, 10 from 2009 were without specific site information, while 100 sampled in 2010 were from each of five patches separated by at least 100 m from each other. Each fruiting body was genotyped at three microsatellite loci. Among the total 28 multilocus genotypes (MLGs) identified, 2 were shared among all three regions, 4 were shared between 2 of the 3 regions, and the remaining 22 were each found in only 1 region. Minimal spanning network analyses revealed several region-specific MLG clusters, consistent with geographic specific mutation and expansion. Though the most frequently observed MLGs were shared among local (patch) and regional populations, population genetic analyses revealed that both local and regional geographic separations contributed significantly to the observed genetic variation in the total sample. All three regional populations showed excess heterozygosity; for the eastern NFLD population, we reject the null hypothesis of Hardy–Weinberg equilibrium (HWE) at all three loci. However, the analyses of clone-corrected samples revealed that most loci were in HWE. Together, our results suggest that the three discrete regional populations of C. enelensis were likely colonized from a common refugium since the last ice age. However, the local and regional populations are diverging from each other through mutation, drift, and selection at least partly due to heterozygous advantage.