Aspergillus fumigatus isolated from diverse wildfowl exhibit distinct antifungal susceptibility profiles driven by genetic and non-genetic determinants

Invasive aspergillosis (IA) is a fungal infection caused by Aspergillus species affecting humans and animals, including birds. Such infections have severe impacts on host health, with the efficacy of current treatment options dwindling against rising rates of antifungal resistance. This scenario represents a critical One Health challenge influenced by climate change at the intersection of animal, human, and environmental health. In this study, we isolated and identified four fungal isolates from infected wildfowl in Southern Ontario, Canada, as Aspergillus fumigatus. Antifungal susceptibility assays against amphotericin B, itraconazole, voriconazole and terbinafine were performed following the Clinical and Laboratory Standards Institute guidelines for filamentous fungi. All strains displayed similar sensitivity to amphotericin B and itraconazole, whereas differences were observed in the response to voriconazole and terbinafine. Next, we performed whole genome sequencing integrated with a comparative genomic analysis to define differences across isolates potentially influencing antifungal susceptibility. As expected, the isolates were phylogenetically similar but demonstrated distinct clustering with A. fumigatus isolate AfB6 mapping closely with the ATCC reference strain compared to the other isolates (i.e., AfB2, AfB8, and AfB7). Notably, single nucleotide polymorphisms (SNPs) were detected across the strains with some correlation between SNPs in antifungal resistance-associated genes and susceptibility profiles; however, antifungal tolerance towards terbinafine was not directly correlated with genetic factors. These data suggest that A. fumigatus isolated from wildfowl with lethal infections from Southern Ontario, Canada, have varying levels of susceptibility to known antifungals and that drivers beyond the anticipated genetic factors influence antifungal response.