Antimicrobial resistance (AMR) and air pollution are critical global health challenges, but their interplay remains poorly understood, particularly in Europe. Serbia, characterized by extensive antibiotic use, high prevalence of multidrug-resistant isolates and severe air pollution, provides a relevant model to study airborne AMR dissemination. During the heating season, air samples were collected at eight locations in Belgrade, representing industrial, traffic loaded and background environments. Shotgun metagenomics, co-occurrence networks and NMDS ordinations were applied to investigate the relationships between atmospheric pollutants, antibiotic resistance genes (ARGs), biocide resistance genes (BRGs), metal resistance genes (MRGs) and mobile genetic elements (MGEs). Autumn microbiomes were dominated by Lactococcus spp., whereas winter lacked such dominance. ARGs associated with antibiotic inactivation accounted for > 50% in autumn and > 75% in winter, with β-lactam resistance (bla_TEM) predominating in both seasons. Winter resistomes also showed more consistent patterns of BRGs and MRGs, with multibiocide/acid and multimetal resistance prevailing. Integron analysis revealed predominance of class 1 integrons (intI1) commonly associated with Escherichia coli. Plasmid-related contigs were most similar to sequences reported in Acinetobacter baumannii and E. coli, while plasmid signatures related to Lactococcus lactis were also detected in autumn. Crucially, the network analysis revealed a seasonal restructuring of the airborne resistome. Autumn networks displayed fragmented structure, showing antagonism between Lactococcus and Escherichia, whereas winter networks coalesced into a densely interconnected superhub that could facilitate horizontal gene transfer and co-selection of resistance determinants. These findings suggest that prolonged air pollution and seasonality jointly shape airborne resistomes, reinforcing the need for integrated environmental and AMR surveillance in highly polluted urban areas.

The emergence of multidrug-resistant pathogens has driven the development of novel antimicrobial peptides (AMPs) as therapeutic alternatives. Lactolisterin LBU (LBU) is a bacteriocin with promising activity against Gram-positive bacteria, including Staphylococcus aureus. In this study, we designed and evaluated a panel of amino acid variants of LBU to investigate domain–activity relationships and improve activity. Peptides were commercially synthesized, and their effect was evaluated for minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), hemolytic activity, cytotoxicity, in vivo toxicity, and virulence modulation. AlphaFold3 structural prediction of LBU revealed a four-helix topology with amphipathic and hydrophobic segments. Helical wheel projections identified helices I and IV as amphipathic, suggesting their potential involvement in membrane interaction and activity. Glycine-to-alanine substitutions at helix I markedly increased antimicrobial activity but altered toxicity profiles. In contrast, changes at helix junctions and kinks reduced antimicrobial activity. We also showed differential regulation of virulence genes upon sub-MIC treatment. Overall, rational substitution enabled identification of residues critical for activity and toxicity, providing insights into therapeutic tuning of lactolisterin-based peptides.

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.