Soil charosphere as a hotspot for antibiotic resistance genes and potential cross-trophic interactions

Extensive biochar application has created a distinct soil microenvironment-the charosphere-that supports intense microbial activity and may serve as a hotspot for antibiotic resistance genes (ARGs), yet the underlying biological drivers of ARG enrichment in this niche remain poorly understood. Here we investigated whether the ARGs are enriched in charosphere compared to the surrounding bulk soil and whether protist consumers influence ARG profiles through trophic interactions. To address this gap, we collected acidic and alkaline field soils treated with biochar to compare the differences in resistome profiles, bacterial, and protist communities between the charosphere and bulk soils. Our results show that ARG richness and abundance were significantly elevated in the charosphere (2.18-2.26-fold and 3.53-3.65-fold higher, respectively), in particular for aminoglycoside and multidrug resistance genes. These enrichments coincided with significantly higher bacterial (14.2%-21.7%) and protist (4.9%-39.0%) richness in the charosphere, and procrustes analysis revealed strong correlations among ARG, bacterial, and protist community compositions. Cross-trophic networks analysis further demonstrated that the interactions among protists, bacteria, and ARGs were notably more complex in the charosphere than in bulk soil, evidenced by a greater number of nodes and links, particularly within the protist-bacteria-ARG subnetwork. Overall, our findings suggest that protist may contribute to the enrichment of ARGs in charosphere, emphasizing the need to reconsider biochar application strategies from a One Health perspective.