Stage-Resolved Dynamics of Antibiotic Resistance Genes and Mobile Genetic Elements in Full-Scale Anaerobic Digestion Systems

Abstract Anaerobic digestion (AD) has the potential to reduce antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) during waste treatment, yet evidence from full-scale systems remains limited. In particular, process-stage dynamics and host-associated resistance mechanisms are poorly understood, and the relative importance of operational and physicochemical drivers remains unresolved. Here, we profiled process-oriented, stage-resolved resistome and mobilome dynamics across substrates, digestates, and storages from ten farm-scale AD plants, resolving putative host-ARG-MGE coabundance associations across sequential process stages. An integrated framework combining high-throughput qPCR and 16S rRNA gene sequencing was used to quantify ARG and MGE dynamics and characterize microbial community structure. Across different AD systems, ARGs and MGEs exhibited heterogeneous abundance changes, with no consistent patterns. However, elevated pH and NH3 were associated with decreasing trends but were not solely determinative. Stage-resolved correlation networks revealed that ARG-MGE associations formed a conserved structural backbone, whereas putative host associations underwent continuous reshuffling across process stages. This indicates that resistance persistence is driven less by stable taxon associations and more by the redistribution of ARG-MGE units among microbial taxa. Collectively, these results demonstrate that stage-resolved, network-informed analyses provide an informative framework for evaluating resistance persistence and potential environmental risks associated with digestate application.