Hydrological Regime Governs the Fate of Manure-Derived Antibiotic Resistance Genes in Rice–Faba Bean Rotation

Background/Objectives: The proliferation of antibiotic resistance genes (ARGs) in agricultural soils poses a growing One Health threat. However, how hydrological fluctuations in paddy–upland rotations shape the soil resistome remains unclear. This study examined the spatiotemporal dynamics and drivers of soil ARGs in a manure-amended rice–faba bean rotation system. Methods: A one-year field experiment was conducted using quantitative PCR and 16S rRNA gene sequencing to characterize ARGs, mobile genetic elements (MGEs), and bacterial communities. Results: The hydrological regime and manure application interactively drove resistome succession. Manure introduced diverse ARGs and MGEs, whereas redox fluctuations restructured bacterial host communities and ARG–MGE networks. Compared with the wet phase, the dry phase enriched intI1 by approximately two orders of magnitude. Specifically, the log10-transformed intI1/16S rRNA gene copy-number ratio increased from −7.11 to −5.14, indicating greater potential for horizontal gene transfer. The fallow transition period represented a critical resistome-restructuring stage with lasting legacy effects. Structural equation modeling identified a dominant carbon–microbe–MGE cascade. Active organic carbon fractions, including dissolved organic carbon (DOC) and readily oxidizable carbon (ROC), shaped microbial community structure, which regulated MGE abundance and ultimately ARG dynamics. MGEs were the strongest direct driver of ARGs (path coefficient = 0.575, p < 0.001). Conclusions: Wet–dry alternation modulates soil resistance risks through the carbon–microbe–MGE cascade. The fallow transition period therefore provides a key target for managing hydrology and carbon inputs to mitigate ARG dissemination in sustainable agroecosystems.