Field-scale isotope tracing reveals combined organic fertilizer–antibiotic effects in amplifying antimicrobial resistance in the soil–lettuce continuum

Antimicrobial resistance (AMR) in agricultural systems poses a critical “One Health” challenge, impacting animal, environmental, and human health. However, the field-scale dynamics of antibiotics within the soil–plant continuum, and their combined effects with organic fertilizers in driving antibiotic resistance genes (ARGs), remain poorly resolved. Here, we applied a stable isotope tracing approach using fully 13C-labeled sulfamethoxazole (13C₁₀-SMX) in a lettuce field to track antibiotic dynamics across the soil–plant continuum. The half-lives of newly introduced 13C₁₀-SMX in the labeled antibiotic (LA) and combined organic fertilizer and labeled antibiotic (OF&LA) treatments were 34.66 and 24.76 days, respectively. Rhizosphere soils showed early accumulation, with root-to-leaf translocation factors ranging from 0.16 to 0.34. OF&LA treatment amplified the relative abundance of ARGs by up to 5.35-fold in soils and 2.38-fold in roots, sustaining broad ARG enrichment. Mobile genetic elements (MGEs) emerged as the strongest direct driver of ARGs (β = 0.88), while 13C₁₀-SMX exerted stronger indirect effects on ARGs than fertilizer inputs. Notably, nine high-risk ARGs were detected in lettuce leaves despite negligible antibiotic residue levels. These findings provide direct field-scale evidence that antibiotics and organic fertilizers jointly contribute to AMR propagation, highlighting the urgent need for integrated management strategies to mitigate agricultural AMR risks at the human–animal–environment interface.