Real-world aged microplastics exacerbate antibiotic resistance genes dissemination in anaerobic sludge digestion via enhancing microbial metabolite communication-driven pilus conjugative transfer

The dissemination of antibiotic resistance genes (ARGs) facilitated by coexisting microplastics (MPs) in the “source-sink” hotspots of waste activated sludge (WAS) raises great concern. Despite real-world MPs undergoing aging, whether and how naturally aged microplastics (AMPs) affect ARG dissemination during sludge treatment remains largely unknown. Herein, we systematically explored the evolved effects and underlying mechanisms of environmentally relevant MPs (0, 3, and 30 mg/kg TS) aging on ARG propagation in anaerobic sludge digestion via multi-omics analyses. Specifically, microplastic exposure increased total ARG abundance by 2.59–15.31 % with enriched mobile genetic elements (MGEs, 0.22–16.71 %). These effects were escalated at higher microplastic dosages and aging degrees. Mechanistically, metagenomic and metaproteomic analyses revealed the drivers for ARG amplification in the sludge digester evolved from the pristine microplastics (PMPs)-induced higher oxidative stress and membrane permeability to AMPs-induced higher multidrug efflux coupled with pilus-mediated conjugation. Subsequently, metagenomic binning identified key multidrug-resistant hosts of Sedimentibacter, Alicycliphilus, and Sulfuricurvum genera. Moreover, high-resolution metabolomics and reactomics network analysis uncovered that AMPs stimulated microbial metabolite turnover, particularly of nitrogenous and sulfurous compounds, and enhanced the complexity and communication frequency of molecular transformation networks centered on lignin and protein nodes, thereby promoting ARG exchange. Finally, Mantel tests reconfirmed that reactive oxygen species level (Mantel's r = 0.93, p = 0.04) and metabolite network connectivity (Mantel's r = 0.82, p = 0.04) are paramount drivers of ARG spread. These findings offer novel insights into the ARG amplification risk driven by MPs aging, guiding targeted strategies to mitigate ARG spread and improve resource recovery in sludge bioengineering systems.