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 plants remains limited and the relative importance of operational and physicochemical drivers is poorly resolved. Here, we profiled stage-resolved resistome and mobilome dynamics across substrates, digestates, and post-digestion storages from ten Swedish farm-scale AD plants (42 samples). High-throughput qPCR (ResistoMap) was combined with absolute 16S rRNA gene quantification and 16S rRNA gene amplicon sequencing to quantify ARG/MGE abundances and characterise bacterial community structure. ARGs and MGEs showed heterogeneous changes during digestion and subsequent storage, with both increases and decreases across plants. Non-metric multidimensional scaling (NMDS) suggested that higher pH and free ammonia were associated with decreasing trends in ARG and MGE relative abundance. Stage-resolved correlation networks revealed a dominant ARG-MGE association backbone that remained largely conserved from substrates to digestates and storage, whereas host-ARG and host-MGE associations were extensively restructured across processing stages. Collectively, these results show that stage-resolved, network-informed analyses offer a more informative framework than pooled, static approaches for evaluating resistance persistence and potential environmental risks associated with digestate application.