Antimicrobial resistance as a signature of soil restoration across a 143-year chronosequence.

Restoring agriculturally degraded habitats to species-rich grasslands is a vital conservation objective. During restoration, how the soil resistome matures alongside microbial community composition and function remains unclear. Here, we tested two competing hypotheses: whether the soil resistome co-occurs through a microbial structural maturation, in which soil restoration is associated with higher-order biotic interactions, or whether antimicrobial resistance (AMR) is instead associated with the competitive pressures and high bacterial taxonomic richness found in disturbed, eutrophic arable land. Using a unique land-use chronosequence on Salisbury Plain, UK, we investigated the trajectory of ecosystem reassembly following the cessation of agricultural activity. Our results demonstrate that AMR abundance increases significantly with restoration age, reaching a maximum in >143-year-old soils. The strongest predictor of this rise in AMR abundance was an increasing microbial eukaryotic signature rather than increasing microbial density, suggesting that resistome expansion is not associated with generalized spatial competition, but rather, co-occurs with structural maturation of the microbiome. We observed an order of magnitude increase in antibiotic biosynthetic potential, dominated by the emergence of streptomycin clusters. Microbial reorientation during soil maturation mirrors the expansion of a core resistome comprised of ancient, intrinsic mechanisms, such as Major Facilitator Superfamily (MFS) efflux pumps and RNA Polymerase-Binding Protein A (RbpA) target protection, in older soils. We demonstrate that endogenous AMR is a hallmark of healthy, restored soil ecosystems rather than a marker of anthropogenic soil degradation.