Antibiotic and Cu²⁺ Co-Selection in MFCs Treating Swine Wastewater: Antibiotic Resistance Genes Dynamics and Removal Performance

Intensive livestock production relies heavily on sulfonamides, quinolones and Cu/Zn additives, creating potent co-selective pressures that accelerate the environmental spread of antibiotic-resistance genes (ARGs). This study examined the effects of combined sulfamethoxazole (SMX), ciprofloxacin (CIP), and copper (Cu²⁺) in swine wastewater on microbial fuel cell (MFC) performance and ARGs accumulation. Closed-circuit MFCs achieved 95.9% COD removal in pristine influent, 88-90% under antibiotic stress and 81% when Cu²⁺ was present, while maintaining >99% removal of both SMX and CIP via adsorption and biodegradation. Although Cu²⁺ reduced coulombic efficiency by ~30%, stable electricity generation persisted. Cu²⁺ co-stress doubled class 1 integron abundance and tripled sul, qnr, and cus gene copies relative to antibiotic-only conditions. Anodic polarization enriched electroactive and Cu-tolerant taxa (e.g., Trichococcus and Rhodococcus) associated with ARGs, mobile genetic elements, and metal-resistance genes. PICRUSt2 analysis indicated upregulation of DNA repair, metal efflux, and electron-transfer-related pathways, suggesting functional coupling between bioelectrochemical activity and resistance propagation. These findings demonstrate that while MFCs efficiently remove organic matter and antibiotics, they can simultaneously intensify resistance-gene evolution through quantitative, host-associated and functional mechanisms, highlighting the urgent need for post-treatment barriers or operational optimization to curb ARG dissemination from livestock-wastewater treatment systems.