Temperature-driven dynamics of intracellular and extracellular antibiotic resistance genes during aerobic composting: Insights from qPCR and metagenomic analysis

The widespread use of antibiotics in animal farming has accelerated the dissemination of antibiotic resistance genes (ARGs). Aerobic composting is an effective method for managing animal manure, yet its effects on intracellular (iARGs) and extracellular ARGs (eARGs) under different temperature regimes including control (LT), gradual increase temperature (GT), and initially enriched temperature (HT) remain unclear. This study investigated the dynamics of iARGs and eARGs across these temperature gradients during composting. Initial composting substrate harbored higher levels of iARGs than eARGs. After composting, ARG rebound was primarily driven by eARG enrichment. On day 40, iARGs decreased by 3.1 logs in HT, 1.2 logs in LT, and 1 log in GT, while eARGs decreased by 0.9 log only in HT but increased in LT and GT. Initially phyla Firmicutes and Proteobacteria were dominated, the microbial community shifted to Bacteroidetes in LT, Actinobacteria in GT, and retained Firmicutes dominance in HT on day 40. Metagenomic analysis revealed that 68 % ARG reduction was associated with plasmid-borne ARGs, with reductions of 83.4 % in HT, 68.2 % in GT, and 51.6 % in LT, whereas non-plasmid ARGs were reduced by up to 75 % across all treatments. Notably, plasmid conjugation was significantly inhibited under HT and GT conditions.