Composted sewage sludge reshapes soil resistome connectivity and enhances antibiotic resistance gene dissemination in paddy fields.

The reuse of organic waste streams, such as composted sewage sludge (CSS), provides agronomic benefits, but also represents a critical pathway for the environmental dissemination of antibiotic resistance genes (ARGs). The consequences of CSS application for soil resistome dynamics and microbial ecology remain insufficiently understood. Here, we investigated paddy soils in Tsuruoka, Japan, under three fertilization treatments: CSS, chemical fertilizer (CF), and their combination (CSS + CF). Quantitative PCR targeted total bacterial (16S rRNA), fecal indicators (E. coli, Enterococcus spp.), mobile genetic element (MGE) (inlt1), and five ARGs (sul1, blaTEM, blaCTX-M Groups 1, 2, and 9). CSS amended soils showed elevated levels of intI1, sul1, and blaTEM in 2023, with blaTEM persisting into 2024. Although both E. coli and Enterococcus spp. showed weak correlations with ARGs, only Enterococcus spp. (ρ = 0.37, p < 0.05) showed statistically significant associations with intI1. Network analysis demonstrated that CSS fostered a highly interconnected resistome with sul1 emerging as a central hub linking multiple bacterial families. In contrast, CF maintained a sparse modular structure while CSS + CF generated an intermediate network. Collectively, these results demonstrate that CSS amplifies the potential for ARG dissemination by fostering a highly connected resistome, whereas co-application with chemical fertilizers partially disrupts this connectivity, thereby reducing dissemination risk in the soil environment. Our findings provide field-based evidence of the environmental impacts of waste-derived fertilization and underscore the need for integrated fertilization strategies and ARG surveillance to promote sustainable soil management and safeguard environmental health.