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

Introduction: Poultry farms have been recognized as environments prone to fungal contamination. However, the occurrence of azole-resistant Aspergillus fumigatus and its cytotoxic potential remain insufficiently characterized. This study aimed to characterize the occurrence, cytotoxic potential, and azole-resistance profile ofAspergillus section Fumigati in poultry farms. Methods: A total of 420 samples, including air (n = 47), electrostatic dust cloths (n = 87), bedding (n = 87), feed (n = 95), swabs (n = 87), workers' masks (n = 2), and broiler breast (n = 15), were obtained. Fungal characterization was performed through culture-based methods (27 °C and 37 °C), followed by azole resistance screening according to EUCAST guidelines. Resistant isolates were subjected to whole-genome sequencing and a targeted analysis of cyp51A mutations. The cytotoxicity potential of fungal isolates and environmental samples was evaluated using selected cell lines representing respiratory organs (human alveolar [A549]) and detoxification organs (swinekidney [SK]/hepatocellular carcinoma [HepG2]). Results: Seven putative Aspergillus fumigatus isolates exhibited pan-azole resistance (MICs: ≥ 2 mg/L ITR/VOR; ≥ 1mg/L for POS). Four isolates carried the TR34/L98H mutation and were recovered from bedding (n = 3) and air (n = 1), suggesting the presence of resistant A. fumigatus in poultry farm matrices. Although 12% of isolates induced toxicity on both cell lines (17/145), no significant association was observed between isolate cytotoxicity and environmental sample toxicity, suggesting that additional biological and chemical components may contribute to the overall toxicological profile of farm environments. Discusion: The study highlights the occurrence of azole-resistant A. fumigatus in poultry farms and support integrated surveillance approaches addressing antifungal resistance and environmental exposure risks in poultry production.