Breaking the cycle of resistance: Hyperthermophilic composting establishes a multi-layered defense against ARG...

The environmental dissemination of antibiotic residues and antibiotic resistance genes (ARGs) from livestock manure poses major risks to public health. Here, we established a hyperthermophilic composting (HC, >80℃) system alongside conventional composting (CK) to elucidate the mechanisms driving antibiotic and ARG removal. By integrating metagenomics, Quantitative PCR (qPCR), and machine learning, we systematically assessed antibiotic degradation, ARG/MGE dynamics, and microbial functional shifts. HC rapidly reached 87.3 ℃ within 24 h and maintained extreme thermophilic conditions, achieving higher TOC degradation (36.58 % vs. 22.59 % in CK), accelerated humification (HA/FA=2.19 by day 15), and efficient removal of oxytetracycline, enrofloxacin, and sulfamethoxazole (98.44 %, 92.34 %, and 99.63 %, respectively), thereby shortening the selective pressure window. Metagenomics identified 796 ARGs and 128 MGEs; key ARGs (macB, bcrA, tetA(58)) declined markedly in HC, with ARG and MGE abundances strongly correlated and virulence factor genes synchronously reduced. qPCR confirmed rapid ARG/MGE attenuation in HC without the rebound observed in CK. Partial least squares path modeling (GOF=0.858) showed MGEs were the primary driver of ARG dissemination (path coefficient=0.790), while temperature exerted indirect effects by reshaping thermophilic communities, enhancing metabolism, and suppressing antibiotics and MGEs. Random forest further highlighted MGEs (integrase, transposase, IS91/ISCR) and humification indices (HA/FA/HS) as key factors, with Bacillus and Truepera as major contributors under HC. Overall, HC established a multilayered "high temperature-community restructuring-functional metabolism-MGE suppression" barrier, enabling faster, more stable and safer resistance risk control than CK. This provides a scalable pathway for safe manure valorization and ARG mitigation under the One Health framework.