Particle-scale distribution of antibiotic resistance genes in municipal wastewater across short-term and variable...

Antibiotic resistance genes (ARGs) are increasingly recognized as persistent contaminants in aquatic environments, with wastewater treatment plants (WWTPs) serving as critical control points for their removal and downstream dissemination. This study investigated the fate, particle size distribution, and hydrodynamic sensitivity of four clinically relevant ARGs, blaTEM, mefA, tetW, and vanA, across primary influent, primary effluent, and final effluent of a municipal WWTP in Bloomington, Indiana. ARGs were quantified using digital PCR and partitioned across particle size classes (60 µm, 0.45 µm, and 0.025 µm) via cascade filtration. All targets exhibited significant reductions between primary influent and final effluent (p < 0.0001), with log₁₀ reductions ranging from 1.5 (blaTEM) to 2.3 (vanA). Despite treatment, ARGs persisted in final effluent at concentrations up to 4.0-6.2 log₁₀ gene copies L^-1. Particle association analyses revealed a shift from dominance in the >60 µm fraction in influent to increased association with the 0.45 µm fraction in effluent, particularly for blaTEM, which exhibited up to 76.5% association with this size class in final effluent. Controlled hydrodynamic experiments demonstrated that high-speed mixing (640 rpm) promoted transient redistribution toward smaller particles (<0.025 µm), consistent with particle disintegration and extracellular DNA release, whereas low-speed conditions (50 rpm) favored stable aggregation. These findings demonstrate that ARG-particle associations are dynamic, gene-specific, and sensitive to flow conditions, highlighting the need to consider physical partitioning and hydrodynamics when modeling ARG transport, exposure risk, and treatment efficacy.