Pressure-induced occurrence and distribution of antibiotic resistance genes in extracellular and intracellular...

Extracellular polymeric substances (EPS) form a multilayered matrix that governs the retention, transformation, and propagation of antibiotic resistance genes (ARGs) in activated sludge. This study systematically examined the stratified distribution of tetracycline resistance genes (tet) across slime EPS (SEPS), loosely bound EPS (LB), tightly bound EPS (TB), and intracellular polymeric substances (IPS) in sequencing batch reactors (SBR) and membrane bioreactors (MBR). Tetracycline accumulated predominantly in SEPS and LB, where elevated selective pressure promoted tet gene enrichment. ARG profiles shifted over time from efflux-pump genes [tet A, tet C, tet G] to ribosomal-protection [tet M] and enzymatic-deactivation [tet X] mechanisms. Protein secondary structure analysis revealed more compact EPS in MBR, which enhanced DNA retention and reduced ARG release into effluent. Correlation analysis showed that tetracycline concentration, rather than biomass abundance, was the dominant driver of ARG proliferation, highlighting the likely role of eDNA and horizontal gene transfer. These findings underscore the mechanistic role of EPS microenvironments in ARG dissemination and provide actionable strategies for mitigating antibiotic resistance in wastewater treatment systems.