Swine waste stabilization ponds as hotspots for antimicrobial resistance gene accumulation: a longitudinal metagenomic study

Using next-generation sequencing, this study provides a comprehensive longitudinal assessment of bacterial communities, antimicrobial resistance genes (ARGs), mobile genetic elements (MGEs), and metabolic pathways in a full-scale swine waste treatment system in Brazil. Samples were collected from the first (WSP1) and final (WSP4) waste stabilization ponds of a farrow-to-finish farm during four sampling events between October 2022 and January 2023. Antibiotic molecules were additionally identified and quantified using solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. Bacterial community composition remained remarkably stable over time. Similarly, the resistome and mobilome showed pronounced temporal stability, although a consistently higher relative abundance of ARGs and MGEs was observed in the final treatment process (WSP4). Genes encoding resistance markers of human-health relevance were detected in WSP4, including Paer_PhoP_CST, associated with polymyxin (colistin) resistance; PRC-1, linked to resistance to third-generation cephalosporins; and quinolone resistance determinants such as adeF, Paer_parE_FLO, and Mtub_gyrB_FLO. Genes encoding efflux pump complexes associated with multidrug resistance were also identified, including Paer_CpxR, PmpM, YajC, MuxB, and MexW. Supporting these findings, fluoroquinolones (ciprofloxacin and norfloxacin), lincomycin, and tetracycline molecules were detected in the waste ponds, indicating sustained selective pressure within the system. The accumulation of clinically relevant resistance determinants in the final of the waste treatment process, whose effluent is reused for agricultural irrigation, highlights waste stabilization ponds as potential hotspots for the persistence and environmental dissemination of antimicrobial resistance. These findings underscore the urgent need for improved monitoring and management of livestock waste treatment systems to mitigate antimicrobial resistance dissemination across agroecosystems.