Seasonal shifts in the Belgrade airborne resistome and virulome: A metagenomic perspective

The atmosphere is a dynamic reservoir for microorganisms and antimicrobial resistance genes (ARGs), yet the seasonal interplay of microbial communities, resistance and virulence determinants with environmental conditions remains poorly characterized, particularly in polluted urban areas. This study presents year-round (summer 2024-spring 2025) shotgun metagenomic monitoring of airborne microbiomes across the Belgrade metropolitan area, a European air pollution hotspot. While community composition shifted seasonally, with an enrichment of Bacillota in autumn and stress-tolerant genera in winter, opportunistic pathogens including Pseudomonas and Acinetobacter were detected year-round. The airborne resistome and mobilome exhibited pronounced seasonal restructuring, with winter showing the highest diversity of resistance genes and plasmid-associated sequences. Mobility-associated genes, including unique toxins and plasmid maintenance systems, were also most prominent in winter. Pathogen-host interaction profiling revealed a functional shift from respiratory and colonization-associated Gram-positive taxa such as Streptococcus pneumoniae and Staphylococcus aureus in autumn to enteric pathogens like Escherichia coli and Salmonella enterica in winter. Network analysis showed that winter formed the densest co-occurrence network, suggesting enhanced potential for co-selection of resistance and virulence traits. Specific plasmid-associated ARGs displayed seasonal patterns, with bla CTX-M linked to multiple plasmids in summer, while bla TEM and aph genes were more prominent in winter. Our findings illustrate that seasonal variations in the airborne genetic landscape are linked to environmental factors and fluctuating reservoirs of clinically relevant resistance and virulence determinants. This highlights the need for integrated longitudinal aerobiome surveillance to understand its implications for public health within the One Health framework.