From environmental gradients to putative intrinsic resistance: Deciphering the soil resistome across Mongolian Plateau grasslands

Soils are important reservoirs of antibiotic resistance genes (ARGs), yet the ecological differentiation and host associations of grassland resistomes remain poorly understood. We investigated topsoil resistomes across desert steppe, typical steppe, and meadow steppe of the Mongolian Plateau using short- and long-read metagenomics. The resistome comprised 28 ARG types and 819 subtypes, dominated by rifamycin, vancomycin, and multidrug resistance genes. ARG abundance increased from desert steppe to meadow steppe. Soil pH, total organic carbon, total nitrogen, and climatic variables were strongly associated with resistome turnover, whereas grazing showed weaker associations. Mobile genetic element (MGE) abundance correlated positively with ARG abundance, but only 3.37% of ARG-carrying contigs contained detectable MGEs. An operational multi-indicator framework distinguished putative intrinsic and putative acquired ARGs with contrasting host and environmental associations. Both fractions were dominated by Actinomycetota, although Pseudomonadota contributed relatively more to putative acquired ARGs. Putative intrinsic ARGs were associated mainly with nutrient-related soil variables, whereas putative acquired ARGs showed stronger climatic and edaphic associations. Long-read assemblies markedly reduced the unclassified fraction. Among 195 ARG-carrying metagenome-assembled genomes, 65.6% represented reference-poor or previously uncultured lineages. These findings reveal spatially structured and heterogeneous grassland resistomes linked to ARG composition, host affiliation, and genomic context.