Carbon Source Diversity Shapes DOM Chemodiversity and Antibiotic Resistome in Earthworm Casts

Abstract The earthworm gut acts as a hotspot for reprocessing ingested organic matter, thereby altering carbon availability. These changes reshape microbial communities and competitive interactions, potentially affecting the dissemination of antibiotic resistance genes (ARGs) in the gut. Beyond substrate amount, substrate diversity shapes these processes, yet its role remains unknown. We established microcosm experiments with a substrate-diversity gradient, combining FT-ICR MS and metagenomics to investigate dissolved organic matter (DOM) chemistry, microbial traits and ARGs in earthworm guts. Higher substrate diversity enriched carbon-centered metabolic genes and increased gut DOM availability, possibly through the proliferation of polysaccharide-degrading microorganisms with larger genomes. However, higher carbon availability may have increased microbial nutrient demand, accompanied by reductions in CHON-containing compounds and stronger microbial competition. ARG enrichment was associated with greater potential for microbial competition, as indicated by stronger negative microbial interactions and higher competition indices derived from genome scale metabolic models. In vitro monoculture of gut-derived pathogenic Klebsiella pneumoniae showed that substrate diversity enriched multidrug- and β-lactam-resistance genes and reshaped the resistome. Collectively, these findings suggest that increased substrate diversity modifies earthworm-driven carbon reprocessing, may reshape microbial resource competition and promote ARG enrichment, providing insights into the linkage between organic matter processing and resistome in soil fauna.