Identification of key species and molecular mechanisms driving conjugative transfer of antibiotic resistance genes in swine manure-derived bacterial communities.

The spread of antimicrobial resistance in livestock environments poses a major public health risk. Conjugative transfer plays a key role in antimicrobial resistance transmission, but the diversity of bacterial hosts involved and the molecular mechanisms driving conjugative transfer within complex microbial communities remain poorly understood. To address this, we investigated plasmid-mediated conjugation in both a swine manure-derived bacterial community and isolated strains from manure. Our study identified 53 OTUs as plasmid recipients, with 66 % belonging to Proteobacteria. Exposure to subinhibitory doxycycline levels decreased the diversity of transconjugants, but conjugation-related gene expression was significantly upregulated, which also became apparent in a marked increase in conjugation frequency. Increased conjugation frequency correlated with increased ATP, ROS and eLDH levels both in the complex bacterial community and in pairwise strains, pointing to the physiological shifts occurring in species that engage in conjugation. Among the identified recipients, Bacillus velezensis exhibited the highest conjugation frequency, likely due to the upregulation of its two-component system, quorum sensing pathways, and strong biofilm-forming ability. Our findings provide new insights into conjugative transfer in livestock manure, identifying potential key spreaders and highlighting opportunities for targeted intervention strategies to mitigate antimicrobial resistance transmission, thereby enhancing its sustainability as a fertilizer.