Longitudinal wastewater metagenomics reveals distinct environmental and anthropogenic associations with resistance, virulence, and viral communities.

Urban wastewater systems are reservoirs of antimicrobial resistance genes (ARGs) and virulence factor genes (VFGs), increasingly recognized as emerging environmental pollutants. However, longitudinal evidence linking tourism-related human mobility to its dynamics remains limited in the Southern Hemisphere. We evaluated whether tourism seasonality, used as a proxy for transient population load, is associated with changes in the wastewater resistome, virulome, and virome in Natal (Northeast Brazil). Using year-long shotgun metagenomics (June 2021-May 2022) of 24 monthly pooled metagenomes (12 composites × 2 replicates) from three wastewater treatment plants, we observed differential enrichment patterns despite stable bacterial community composition dominated by Aliarcobacter. Redundancy analysis (RDA) demonstrated that the model for bacterial community explained 40.1% of the total variance (F = 1.79, p = 0.039), with tourism showing marginal effects. In contrast, precipitation was not significant (p = 0.262). RDA also revealed that precipitation was associated with ARG distribution (p = 0.027) and that VFG composition was associated with international tourism (p = 0.002). ARGs were more abundant during high-precipitation periods, whereas VFGs showed higher relative abundance during international tourism peaks. Metagenome-assembled genomes (n = 95) revealed 33 multidrug-resistant hosts, including understudied taxa such as Tolumonas and the family Aquaspirillaceae, harboring plasmids (e.g., IncFIB(K)). Co-occurrence networks showed that viruses were positively correlated with ARGs and negatively correlated with VFGs, except for crAssphage, which was associated with virulence traits. These findings reveal distinct environmental and human mobility factors underlying wastewater microbial dynamics. We underscore the importance of integrating longitudinal metagenomics into seasonally adjusted surveillance frameworks to mitigate antimicrobial resistance as an emerging form of environmental pollution.