Thermophilic Parageobacillus toebii G12 suppresses ARG enrichment during chicken manure composting even with multidrug-resistant plasmid-harboring bacteria as a stressor.

The enrichment of antibiotic resistance genes (ARGs) during composting poses a substantial risk to the safe utilization of chicken manure. However, the role of multidrug-resistant plasmid (MRP)-harboring bacteria in ARG dissemination and microbial succession during composting remains poorly understood. In this study, we investigated the regulatory effects of thermophilic Parageobacillus toebii G12 (G12) on ARG dynamics, as well as its performance under interference from MRP-harboring bacteria. Our results showed that G12 inoculation elevated compost temperature, extended the thermophilic phase, and accelerated maturation. It increased overall ARG removal to 60.9 % and 55.6 % in the conventional and MRP-contaminated systems, respectively, and reduced persistent ARG enrichment by 71.4 % and 66.3 %, respectively. Although MRP-associated stress shifted the suppression preference of G12 from protection-type to alteration/replacement-type persistent ARGs, the strain consistently suppressed efflux- and inactivation-type persistent ARGs. Moreover, G12 markedly reduced the coverage depth of MRPs after composting, thereby alleviating MRP-associated stress. Integrated analyses, including co-occurrence network, Mantel test, partial least squares path modeling, and metagenome-assembled genome approaches, revealed that G12 reshaped the microbial community by suppressing the dominant host Pseudomonadota, thereby reducing its efflux-type persistent ARGs, while directly diminishing integration/excision-type mobile genetic elements to curtail horizontal gene transfer potential. This study elucidates the core mechanisms by which G12 counteracts ARG enrichment and demonstrates its efficacy even under MRP-associated stress, providing a theoretical foundation for mitigating antimicrobial resistance risks in manure composting.