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Abstract Activated sludge is the centerpiece of biological wastewater treatment, as it facilitates removal of sewage-associated pollutants, fecal bacteria, and pathogens from wastewater through semi-controlled microbial ecology. It has been hypothesized that horizontal gene transfer facilitates the spread of antibiotic resistance genes within the wastewater treatment plant, in part because of the presence of residual antibiotics in sewage. However, there has been surprisingly little evidence to suggest that sewage-associated antibiotics select for resistance at wastewater treatment plants via horizontal gene transfer or otherwise. We addressed the role of sewage-associated antibiotics in promoting antibiotic resistance using lab-scale sequencing batch reactors fed field-collected wastewater, metagenomic sequencing, and our recently developed bioinformatic tool Kairos. Here, we found confirmatory evidence that fluctuating levels of antibiotics in sewage are associated with horizontal gene transfer of antibiotic resistance genes, microbial ecology, and microdiversity-level differences in resistance gene fate in activated sludge.