Limited ARG removal but stable resistome dynamics in a surface flow constructed wetland

Improperly treated wastewater and surface runoff can degrade water quality by introducing microbial contaminants, including antibiotic-resistant bacteria (ARB) and their genes (ARGs). Constructed treatment wetlands (CTWs) offer a low-resource solution for managing impaired watersheds. However, their ability to mitigate microbial contaminants, particularly ARGs, requires further study. In this study, 62 water samples from Banklick Creek CTW (BCTW) were shotgun sequenced to assess ARG dynamics and removal characteristics. Results showed minimal resistome attenuation, likely due to the wetland's horizontal surface flow design with short, variable hydraulic residence times (0.48-3.1 days). Despite this, 198 low-abundance ARGs were removed, accounting for a median of 0.52 % (0-3.1 %) of total ARG abundance upstream. The core resistome, comprising 95.6 ± 1.9 % of total ARG abundance, was stable and mainly consisted of multidrug efflux systems carried by bacterioplankton and macrophyte symbionts, indicating a native resistome reflective of regional pollution history. Resistome and microbiome structures were highly correlated (R² = 0.808), with ARGs rarely co-occurring with mobile genetic elements, indicating limited intercellular transfer potential. No significant correlations were found between resistome dynamics and human fecal (HF183, crAssphage) or avian (GFD) biomarkers. Although several class-one integron-integrase (intI1) contigs were enriched in treatment channels, gene cassette cargo was void of ARGs. As detection of intI1 via qPCR is generally considered indicative of resistome mobility potential, this finding carries important implication for intI1 qPCR assay selection (i.e., targeting clinical intI1 mosaics) and over-interpretation of ARG spread in the environment.