Introduction: The frequency in detection of azole-resistant Aspergillus fumigatus isolates has increased since 2010. In Portugal, the section Fumigati is one of the most frequent, and resistant strains to have been found in clinical and environmental contexts. Although several cryptic species within the Fumigati section show intrinsic resistance to azoles, one factor driving (acquired) resistance is selective pressure deriving from the extensive use of azoles. This is particularly problematic in occupational environments where high fungal loads are expected, and where there is an increased risk of human exposure and infection, with impact on treatment success and disease outcome. The mechanisms of resistance are diverse, but mainly associated with mutations in the cyp51A gene. Despite TR₃₄/L98H being the most frequent mutation described, it has only been detected in clinical specimens in Portugal. Methods: We analyzed 99 A. fumigatus isolates from indoor environments (healthcare facilities, spas, one dairy and one waste sorting unit) collected from January 2018 to February 2019 in different regions of Portugal. Isolates were screened for resistance to itraconazole, voriconazole and posaconazole by culture, and resistance was confirmed by broth microdilution. Sequencing of the cyp51A gene and its promoter was performed to detect mutations associated with resistance. Results: Overall, 8.1% of isolates were able to grow in the presence of at least one azole, and 3% (isolated from the air in a dairy and from filtering respiratory protective devices in a waste sorting industry) were pan-azole-resistant, bearing the TR₃₄/L98H mutation. Conclusion: For the first time in Portugal, we report environmental isolates bearing the TR₃₄/L98H mutation, isolated from occupational environments. Environmental surveillance of the emergence of azole-resistant A. fumigatus sensu stricto strains is needed, to ensure proper and timely implementation of control policies that may have a positive impact on public and occupational health.

For decades, occupational exposure to flour dust has been linked to a range of respiratory diseases, including occupational asthma, thought to result from exposure to fungi present in the flour. Antifungal resistance is of increasing prevalence in clinical settings, and the role of occupational and environmental exposures, particularly for specific fungal species, is of concern. Occupational exposure to flour dust can occur in a range of occupational settings, however, few studies have focused on restaurant workers. The objective of this study was to measure occupational exposure to flour and microbial contamination, including azole resistance screening, in two small commercial bakeries and in a pizzeria. Personal full shift inhalable dust measurements were collected from workers, and were analyzed for inhalable dust and fungi, bacteria, azole resistance, and mycotoxins. Samples of settled dust were collected, and electrostatic dust cloths (EDC) were deployed and analyzed for microbial contamination, including azole resistance screening, and mycotoxins. Geometric mean exposures of 6.5 mg m⁻³ were calculated for inhalable dust, however, exposures of up to 18.30 mg m⁻³ were measured—70% of personal exposure measurements exceeded the occupational exposure limit for flour dust of 1.0 mg m⁻³. The air and EDC fungal counts were similar to those reported in previous studies for similar occupational environments. The fungi were dominated by Penicillium genera, however Aspergillus genera, including Fumigati and Flavi sections, were observed using culture-based methods, and the Fumigati section was also observed by molecular tools. Both Aspergillus sections were identified on the azole resistance screening. Mycotoxins were also detected in the settled dust samples, dominated by deoxynivalenol (DON). The role of environmental exposure in both the development of antimicrobial resistance and the total mycotoxin body burden is a growing concern; therefore, the presence of azole-resistant fungi and mycotoxin contamination, although low in magnitude, is of concern and warrants further investigation.

Association between selection pressure caused by the use of azole fungicides in sawmills and the development of fungal resistance has been described. The aim of this study was to implement an algorithm to assess the presence of Aspergillus section Fumigati resistant strains in sawmills. Eighty-six full-shift inhalable dust samples were collected from eleven industrial sawmills in Norway. Different culture media were used and molecular identification to species level in Aspergillus section Fumigati was done by calmodulin sequencing and TR₃₄/L98H and TR₄₆/Y121F/T289A mutations were screened by real-time PCR assay and confirmed by cyp51A sequencing. Six Fumigati isolates were identified as A. fumigatus sensu stricto and two of these grew on azole-supplemented media and were further analyzed by real-time PCR. One was confirmed to be a TR₃₄/L98H mutant. The obtained results reinforce the need to assess the presence of A. fumigatus sensu stricto resistant isolates at other workplaces with fungicide pressure.