Stage-Dependent Dynamics of Soil Antibiotic Resistance Genes under Long-Term Pollutant Exposure

Abstract Agricultural soils are key environmental reservoirs of antibiotic resistance genes (ARGs), yet the long-term dynamics of ARGs and their ecological context under sustained pollutant exposure remain poorly understood. Here, we conducted a 366-day soil microcosm experiment in which agricultural soils were exposed to single and combined heavy metal and polycyclic aromatic hydrocarbon (PAH) contamination, with metagenomic profiling across 12 time points. Relative to initial levels, ARG relative abundance across pollution treatments increased by an average of 23% by day 114 and reached 62% above baseline by day 254, after which it stabilized through day 366. These temporal patterns were associated with shifts in the correlates of ARG variation across biological scales: early-stage ARG variation was primarily associated with bacterial community composition, middle-stage variation was most strongly associated with mobile genetic element (MGE) abundance and mobility-related signals, and late-stage variation was more consistently associated with tolerance-related functional traits. Genome-resolved analyses further revealed stronger microdiversity signals from the middle stage onward, and these stage-associated ecological and molecular patterns were generally more pronounced under combined pollution than under single-pollutant exposure. Taken together, these findings indicate a stage-dependent pattern of ARG dynamics under long-term pollutant exposure and provide a temporal framework for understanding long-term resistome reorganization in polluted agricultural soils.