Airborne Pathogens and Virulence Factors in Belgrade Metropolitan Area Metagenomic Insight

Introduction: Air pollution has been recognized as one of the greatest threats to human health. The particulate matter-PM2.5, main air pollution indicator, is found to contain numerous bacterial pathogens. Considering that Serbia is struggling with excessive antibiotics use and misuse, a high percentage of multidrug-resistant (MDR) bacterial isolates, and poor air quality, made Serbian capital Belgrade interesting research model for the effects of air pollution on the abundance and diversity of airborne pathogens. Objective: The presence of MDR pathogens, virulence factors as well as pathogen-host interactions are studied in the airborne metagenome samples from seven locations of Belgrade metropolitan area collected during the summer season of 2024. Method: Air samples were collected from seven Belgrade locations using hydrophobic polypropylene membrane filters (air volume of 30 m³). Total DNA was analysed by shotgun metagenomic sequencing, followed by bioinformatic processing (Novogene, UK). Taxonomic diversity/abundance, virulence factors and pathogen-host interactions were analysed using MEGAN, VFDB, and PHI databases, respectively. Findings: Important human and animal pathogens were found in analysed metagenomes: Acinetobacter schindleri, Streptococcus pneumonie, Streptococcus oralis, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Ewingella americana, Burkholderia multivorans, Legionella pneumophila, Salmonella enterica and Brachybacterium muris. Genes encoding for motility, adherence, immune modulation, exotoxins and biofilm formation were also found. Additionally, PHI analysis revealed the presence of different type of infections caused by pathogenic bacteria – nosocomial and urinary tract infections, pneumonia, salmonellosis, soft rot. Conclusion: This study indicated that Belgrade air could serve as a reservoir for pathogenic bacteria, virulome and numerous types of infections.