Aging attenuates threat: how moderate aging of microplastics suppresses antibiotic resistance gene proliferation during sludge anaerobic digestion.

Microplastics (MPs) are known to promote antibiotic resistance gene (ARG) dissemination in waste activated sludge; however, most existing evidence is based on unaged MPs, and the influence of aging degree remains poorly understood. This study systematically investigated how varying aging degrees of polyethylene (PE) and polypropylene (PP) MPs modulate ARG profiles and transfer mechanisms during anaerobic digestion. The results demonstrated a non-monotonic effect of aging degree on ARG proliferation, with moderate aging of MPs showing the strongest attenuation of ARG promotion. Under moderate carbonyl indices (CI) of 0.104 for PE-MPs and 0.219 for PP-MPs, the average reduction of the most affected ARGs reached 40% and 50%, respectively, compared with the unaged MPs. Metagenomic analysis further revealed that moderate aging of MPs reduced both the abundance and diversity of ARGs stimulated by unaged MPs. Mechanistically, unaged MPs induced multiple biological responses. These included enrichment of dominant ARG-hosting genera within Proteobacteria and Chloroflexi, elevated oxidative stress, increased membrane permeability, and activation of horizontal gene transfer (HGT) pathways, including the type IV secretion system (T4SS), quorum sensing (QS), and two-component systems (TCS). Conversely, aging weakened these microbial signaling and stress responses at moderate aging degrees but led to a rebound at higher aging degrees, thereby modulating HGT potential in a non-monotonic manner. These findings indicate that aging of sludge-relevant MPs (PE and PP) fundamentally alters their ecological impact on the sludge resistome, highlighting the necessity of incorporating aging dynamics into the risk assessment of MPs in engineered ecosystems.