Comparative air fungal microbiomes in a metropolis and a provincial hospitals in Thailand

Hospital environments harbor diverse airborne fungal communities, which may pose health risks to patients and healthcare workers. This study systematically characterized and compared the airborne fungal microbiomes (mycobiomes) of two Thai hospitals representing distinct geographic contexts: a metropolitan hospital (King Chulalongkorn Memorial Hospital, Bangkok) and a provincial hospital (Naresuan University Hospital, Phitsanulok). High-throughput internal transcribed spacer 1 (ITS1) amplicon sequencing, combined with multivariate, functional, and source-tracking analyses, revealed pronounced spatial heterogeneity in hospital air mycobiomes. The provincial hospital exhibited significantly higher fungal richness (operational taxonomic units and Chao1 index), whereas community evenness (Shannon index) remained comparable between hospitals. Beta-diversity analyses demonstrated strong clustering by geographic setting, identifying hospital location as the primary driver of fungal community variation, followed by area type and high-efficiency particulate air (HEPA) filtration. Distinct taxonomic patterns were observed, with Cladosporium , Schizophyllum , and members of Cordycipitaceae enriched in the metropolitan hospital, while Aspergillus and Wallemia predominated in the provincial hospital. Operating rooms showed additional structuring associated with HEPA filtration and surgical activities. Functional annotation revealed a higher relative abundance of fungi assigned to pathotrophic ecological guilds in the provincial hospital, particularly in restaurant and examination areas. Several taxa with reported associations with opportunistic infections, including Eupenidiella , Hortaea werneckii , Sterigmatomyces halophilus and Toxicocladosporium irritans , were identified as area-specific biomarkers based on FUNGuild ecological annotations. SourceTracker2 analysis indicated soil the largest identified fungal source in both hospitals, followed by human-associated and urban environmental inputs. Together, these findings highlighted the combined effects of geographic, area type (human activities), and ventilation design on hospital air DNA mycobiomes and support targeted airborne fungal monitoring strategies to enhance infection prevention and patient safety.