Barrier or reservoir? Tertiary media filters reshape antibiotic resistance gene profiles in full-scale wastewater treatment plants

Media filtration units are widely used to polish secondary effluent in wastewater treatment plants (WWTPs), yet their influence on antibiotic resistance gene (ARG) profiles remains poorly characterised under full-scale conditions. To test whether these filters act as barriers that attenuate ARGs or as compartments in which selected ARGs persist, shotgun metagenomic sequencing was applied to six sampling compartments at three municipal WWTPs, including filter-media biomass and backwash effluent. At the whole-plant scale, 122 ARG subtypes consistently decreased and 54 increased in relative abundance across all three facilities. Within the filtration units, this pattern reversed: 89 subtypes increased and 21 decreased, including increases in 30 of 37 high-confidence subtypes. Filtration-associated increases were dominated by efflux pump-related and polymyxin-resistance-related annotations, whereas target alteration and ribosomal protection genes decreased mainly during pre-filter treatment. Across the water-backwash step, 78.0 ± 9.0% of ARG subtypes detected in filter media remained detectable, with subtype-specific turnover rather than uniform removal. Taxonomic labels co-occurring with ARGs shifted toward environment-associated genera in filter effluent, most consistently Stenotrophomonas . ARG-bearing contigs detected after filtration were more frequently co-localised with pathogen–host interaction, heavy metal resistance, and virulence factor annotations than with mobile genetic element annotations. Overall, more ARG subtypes showed relative decreases across the whole treatment train, whereas tertiary filtration was associated with stage-specific relative increases and taxonomic-context turnover. Being DNA-based and relative, these data do not establish absolute ARG amplification, host identity, or clinical risk. These findings support ARG-aware evaluation of tertiary filtration performance and backwash management in full-scale wastewater treatment.