Pan-Azole-Resistant Aspergillus fumigatus Detected in Poultry Farm Bedding and Air

On poultry farms across Madeira Island, Portugal, researchers have recovered strains of the fungus Aspergillus fumigatus that resist every first-line azole antifungal drug used in human medicine. Four of these isolates carried the TR34/L98H mutation, the genetic signature most closely associated with the environmental emergence of azole resistance worldwide. The finding, published in Frontiers in Fungal Biology, marks the first report of putative pan-azole-resistant A. fumigatus harboring this mutation in environmental samples from poultry farms, and it places animal bedding material and indoor air at the center of a growing One Health concern.

A. fumigatus is the principal cause of aspergillosis, a spectrum of infections ranging from allergic airway disease to life-threatening invasive illness. The fungus produces extremely small conidia that are easily inhaled deep into the respiratory tract, and it can survive and grow across a wide range of environmental conditions, including at and above human body temperature. It also manufactures toxic metabolites known as mycotoxins. In 2022, the World Health Organization designated A. fumigatus a priority fungal pathogen, underscoring the urgent need for global surveillance as resistance to azoles, the first-line class of antifungal agents, continues to rise.

The research team, led by Bianca Gomes of the Center for Ecology, Evolution and Environmental Change at the University of Lisbon and colleagues from several Portuguese institutions, collected 420 samples from five poultry farms comprising 14 poultry pavilions. The matrices included indoor air, electrostatic dust cloths, wood shavings bedding, feed, surface swabs, workers’ filtering respiratory protection devices, and broiler breast tissue. Sampling spanned four stages of the production cycle, from cleaned and disinfected empty pavilions through the first, second, and third weeks of bird growth, across two consecutive production cycles in summer and winter.

Fungal characterization relied on culture-based methods at two temperatures. Plates incubated at 27 degrees Celsius captured the general environmental mycobiota, while incubation at 37 degrees Celsius selected thermotolerant species capable of growing at human body temperature, a critical indicator of pathogenic potential. Detection rates at 37 degrees Celsius reached 95.78 percent across all matrices and production stages, compared with only 14.64 percent at 27 degrees Celsius. Section Fumigati was particularly predominant in air and bedding at the higher temperature, representing more than 79 percent of recovered fungi in both matrices. The team notes that exposure assessments targeting this section should include culture at 37 degrees Celsius, since lower temperatures may promote faster-growing fungi and underestimate the presence of potentially pathogenic Aspergillus species.

From 253 recovered isolates, 173 grew at 37 degrees Celsius and were selected for further analysis. Sub-culturing on Potato Dextrose Agar at 50 degrees Celsius identified 164 putative A. fumigatus isolates, since growth at such elevated temperatures is indicative of A. fumigatus sensu stricto rather than morphologically similar cryptic species within the section. Azole resistance screening followed EUCAST guidelines using the azole-containing agar plate method, with conidial suspensions inoculated onto four-well RPMI-1640 agar plates containing 4 milligrams per liter itraconazole, 2 milligrams per liter voriconazole, and 0.5 milligrams per liter posaconazole. Seven isolates, six from bedding and one from air, showed visual growth on the azole-supplemented wells and were selected for confirmatory testing.

Broth microdilution susceptibility testing according to the EUCAST reference method E.Def 9.4 confirmed pan-azole resistance in all seven isolates. Minimum inhibitory concentrations ranged from 2 to 8 milligrams per liter for itraconazole, 2 to 4 for voriconazole, and 1 to 2 for posaconazole, all exceeding the EUCAST clinical breakpoints. Whole-genome sequencing using Oxford Nanopore technology generated 13.02 million reads and 48.76 gigabases of sequence data, with sequencing depth between 168.7-fold and 313.7-fold and 96 to 98 percent of reads successfully mapped against the A. fumigatus Af293 reference genome. Taxonomic classification using Kraken2 confirmed the predominance of A. fumigatus reads across all datasets, with less than 1 percent of reads remaining unclassified.

Targeted analysis of the cyp51A gene, which encodes the azole target enzyme, identified the TR34/L98H mutation in four isolates. This mutation combines a 34-base-pair tandem repeat insertion in the promoter region with a leucine-to-histidine substitution at position 98 in the coding region, altering the azole target enzyme and reducing antifungal binding affinity. The four TR34/L98H isolates were recovered from bedding and air, with three from bedding and one from air. The remaining three isolates lacked tandem repeats in the promoter but carried the mutations F46Y, M172V, N284T, D255E, and E427K, along with four indels upstream of the promoter. While the functional impact of these distal promoter alterations remains unclear, changes in the cyp51A promoter have been associated with altered gene expression and reduced azole susceptibility, making them candidate genetic features that may contribute to the resistant phenotype. Further studies, including gene expression analysis and functional validation, are required to determine their role.

The environmental use of azole fungicides has been linked to the emergence and dissemination of TR-based resistance mechanisms in A. fumigatus, contributing to the global spread of resistant lineages characterized by multi-azole resistance and increased recombination rates. The TR34/L98H clade frequently harbors the hypermutator variant msh6-G233A, which has been associated with elevated mutation rates and adaptive capacity, potentially facilitating the evolution of antifungal resistance. The presence of this variant was not assessed in the recovered isolates, so its occurrence cannot be established. Nonetheless, the detection of TR34/L98H isolates in poultry farm environments highlights the importance of continued One Health surveillance to monitor the occurrence and potential dissemination of azole-resistant A. fumigatus across environmental, animal, and human interfaces. The common use of wood shavings, possibly treated with azoles, as animal bedding material may promote the emergence of resistant mycobiota within poultry farms, although azole residues in bedding were not assessed in this study.

Cytotoxicity assays were performed on 145 successfully re-isolated section Fumigati isolates using the MTT assay with two cell lines: human alveolar lung carcinoma cells (A549), representing the primary site of exposure to airborne fungal spores, and human hepatocellular carcinoma cells (HepG2), representing potential systemic effects of mycotoxin exposure. A reduction in cell viability below 70 percent was considered indicative of a cytotoxic effect. Overall, 46.20 percent of isolates induced toxicity on HepG2 cells, reducing viability to as low as 34 percent, while 16.55 percent reduced A549 viability to as low as 57 percent. Twelve percent of isolates induced toxicity on both cell lines. Isolates recovered from air samples showed the highest frequency of cytotoxicity in HepG2 cells at 65.8 percent, whereas cytotoxicity in A549 cells was generally less frequent across all matrices.

When the cytotoxicity of fungal isolates was compared with previously obtained cytotoxicity data from their corresponding environmental samples, no significant association was detected. Overall, 76.90 percent of environmental samples induced cytotoxicity, whereas only 10.77 percent of the corresponding isolates exhibited cytotoxic effects, with very low concordance between the two types of samples. This pattern of discordance, dominated by cytotoxic environmental samples with noncytotoxic isolates, suggests that cultivable fungi alone are not the main contributors to the observed toxic profile. The team also screened 38 mycotoxins in environmental samples, detecting deoxynivalenol, fumonisins, and zearalenone across all three matrices. For swine kidney cells, a significant association was detected between cytotoxicity and mycotoxin detection, with cytotoxic samples approximately twice as likely to contain detectable mycotoxins. For A549 cells, no significant association was observed, suggesting that matrix type and production stage may have a greater influence on cytotoxicity than mycotoxin detection alone. Collectively, these findings reinforce the need for integrated surveillance strategies targeting both Aspergillus species and mycotoxins in poultry production, extending to poultry residues and their safe land application, while research into sustainable antifungal alternatives, including plant essential oils, may help reduce reliance on azoles and mitigate the selective pressure driving resistance.

Subject of Research: Microbiology

Article Title: Detection of TR34/L98H pan-azole-resistant Aspergillus fumigatus in poultry farm environments

Article References: Gomes, B., Dias, M., Cervantes, R., Gonçalves, L., Carolino, E., Ramos, M. J. N., Nunes, M., Dias, R., & Viegas, C. (2026). Detection of TR34/L98H pan-azole-resistant Aspergillus fumigatus in poultry farm environments. Frontiers in Fungal Biology, 7, Article 1858050. https://doi.org/10.3389/ffunb.2026.1858050

Image Credits: AI Generated

DOI: 10.3389/ffunb.2026.1858050

Keywords: Aspergillus fumigatus, azole resistance, TR34/L98H, poultry farms, One Health, antifungal resistance, mycotoxins, Detection, TR34, L98H, pan-azole-resistant, Aspergillus