Community science uncovers environmental azole resistance in Aspergillus fumigatus across Norway, including a novel tandem repeat variant (TR 62 )

Azole-resistant Aspergillus fumigatus (AR Af ) is an emerging global health concern, mainly driven by environmental selection caused by azole fungicide use. Despite reports of environmental resistance across Europe, data from northern regions remain scarce. Here, we conducted the first nationwide investigation of environmental AR Af in Norway using a community science-based air sampling approach. Volunteers across the country collected airborne fungal spores using passive sticky-seal traps during three sampling periods between 2022 and 2023. In total, 2,332 air samples from 398 locations were analyzed, yielding 1,583 A. fumigatus isolates. Azole resistance screening identified 15 resistant isolates (0.95%) from 14 locations across four out of five Norwegian regions. Minimum inhibitory concentrations (MICs) confirmed resistance to medical azoles and elevated MICs toward azole fungicides. Whole-genome sequencing revealed known environmentally associated resistance mechanisms, including TR 34 /L98H ( n = 11) and TR 46 /Y121F/T289A ( n = 2), as well as a TR 46 /Y121F/T289A/S363P/I364V/G448S variant ( n = 1). In addition, a previously undescribed promoter tandem repeat variant (TR 62 ) was identified in one isolate that exhibited multi-azole resistance. Population genomic analyses indicated that resistant isolates were genetically diverse and not restricted to a single clonal lineage, suggesting both local expansion and recombination within a structured population. Although the observed resistance frequency was low, the presence of globally disseminated resistance mechanisms in airborne spores across Norway demonstrates that environmental azole resistance is established in the region. This study provides baseline data on the frequency and distribution of environmental AR Af in Norway and contributes to international efforts to strengthen monitoring of antifungal resistance. IMPORTANCE Azole-resistant Aspergillus fumigatus (ARAf) is an emerging public health concern, yet data from northern Europe remain limited. This study provides the first nationwide investigation of the frequency, distribution, and genetic diversity of environmental ARAf in Norway. By combining community-based environmental sampling with whole-genome sequencing, we demonstrate that resistance-associated genotypes of international concern are established in the Norwegian environment and occur across multiple regions. These findings provide important baseline data for future surveillance and contribute to global efforts to monitor and understand the emergence and spread of antifungal resistance.