Bioaerosols in the Athens Metro: Metagenetic insights into the PM₁₀ microbiome in a naturally ventilated subway station

To date, few studies have examined the aerosol microbial content in Metro transportation systems. Here we characterised the aerosol microbial abundance, diversity and composition in the Athens underground railway system. PM₁₀ filter samples were collected from the naturally ventilated Athens Metro Line 3 station "Nomismatokopio". Quantitative PCR of the 16S rRNA gene and high throughput amplicon sequencing of the 16S rRNA gene and internal transcribed spacer (ITS) region was performed on DNA extracted from PM₁₀ samples. Results showed that, despite the bacterial abundance (mean = 2.82 × 10⁵ 16S rRNA genes/m³ of air) being, on average, higher during day-time and weekdays, compared to night-time and weekends, respectively, the differences were not statistically significant. The average PM₁₀ mass concentration on the platform was 107 μg/m³. However, there was no significant correlation between 16S rRNA gene abundance and overall PM₁₀ levels. The Athens Metro air microbiome was mostly dominated by bacterial and fungal taxa of environmental origin (e.g. Paracoccus, Sphingomonas, Cladosporium, Mycosphaerella, Antrodia) with a lower contribution of human commensal bacteria (e.g. Corynebacterium, Staphylococcus). This study highlights the importance of both outdoor air and commuters as sources in shaping aerosol microbial communities. To our knowledge, this is the first study to characterise the mycobiome diversity in the air of a Metro environment based on amplicon sequencing of the ITS region. In conclusion, this study presents the first microbial characterisation of PM₁₀ in the Athens Metro, contributing to the growing body of microbiome exploration within urban transit networks. Moreover, this study shows the vulnerability of public transport to airborne disease transmission.