Abstract
The environmental dissemination of antibiotic resistance genes (ARGs), particularly in manure-amended soils, poses a growing threat to public health due to the potential transfer of ARGs to humans and animals. Effective strategies are urgently needed to mitigate ARG spread in agricultural settings. Biochar, an eco-friendly soil amendment, shows promise for pollution control, yet its role in suppressing ARG horizontal gene transfer remains unclear. Here, metagenomic analysis showed that manure application significantly increased the relative abundance of ARGs in soil microbiota, whereas biochar amendment reduced it. To determine whether biochar suppresses ARG dissemination by inhibiting horizontal transfer, we established a soil microcosm. Manure application increased the conjugative transfer ratio by 3-fold, whereas biochar effectively suppressed this transfer-reducing it to levels observed in unamended soils. Cell sorting and 16S rRNA gene amplicon sequencing demonstrated that biochar treatment reduced the diversity of transconjugant pools at both phylum and genus levels. Transconjugants were primarily affiliated with Pseudomonadota, Bacillota, and Actinomycetota, with Massilia, Delftia, and Ammoniphilus being the most abundant genera in biochar treatment soil. Mechanistic investigations revealed that biochar-mediated inhibition of ARG transfer was linked to reduced ATP energy supply, decreased reactive oxygen species production, and lower cell membrane permeability, and diminished bioavailability of heavy metals and antibiotics. Additionally, biochar altered soil enzyme activity and microbial community structure, further limiting ARG dissemination. The findings provide insights into biochar-induced mitigation of ARG spread in manure-amended soils and highlight its potential as an effective strategy for controlling environmental ARG transmission.