Antibiotic stress alters lysogeny-lysis dynamics and drives phage-mediated transfer of antibiotic resistance genes in the activated sludge process.

The spread of antibiotic resistance genes (ARGs) in wastewater treatment systems poses a significant public health concern, yet the role of bacteriophages (phages), particularly temperate phages, in mediating horizontal gene transfer (HGT) of ARGs under antibiotic stress remains poorly understood. This study investigated the effects of escalating ciprofloxacin (CIP; 0-200 μg/L)-selected as a representative antibiotic due to its frequent occurrence and persistence in wastewater-on phage lysogeny-lysis dynamics and phage-mediated ARG transfer in a laboratory-scale activated sludge reactor. Integrating metaviromic and metagenomic analysis revealed that the phage-mediated ARG-HGT events mainly occurred at the highest CIP concentration stage (200 μg/L), indicating that high-level antibiotic stress is essential for triggering significant ARG transfer. Notably, all these HGT events were associated with temperate phages. The HGT-associated ARGs may confer host resistance to antibiotics, as supported by the ARG expression and antibiotic resistance activity experiment. Although temperate dynamics generally shifted toward lysogeny under escalating stress, most of the temperate phages involved in ARG-HGT became more active at higher CIP concentration stages, which may facilitate host survival under stress conditions.