Plant spatial compartmentalization buffers bacteriome structure and function under antibiotic stress.

Agricultural antibiotic contamination poses increasing threats to crop productivity and ecosystem stability through disruption of the plant-associated microbiome. While antibiotic impacts on bulk soil and rhizosphere communities are documented, the extent to which spatial compartmentalization across the plant-soil continuum buffers these effects remains poorly understood. Here, we investigated how compartment-specific selective pressures influence bacterial community assembly, functional resilience, and interaction networks under antibiotic stress. Lettuce (Lactuca sativa) was grown under five treatments in a completely randomized greenhouse design: T1 (sulfamethoxazole [SMX], 3 mg kg⁻¹ + manure + plant), T2 (trimethoprim [TMP], 3 mg kg⁻¹ + manure + plant), T3 (manure + plant, antibiotic-free control), T4 (manure only, plant-free control), and T5 (soil only, negative control). Bacterial communities were profiled across bulk soil, rhizosphere, and endosphere compartments using full-length 16 S rRNA gene sequencing. Spatial compartmentalization emerged as the primary driver of bacteriome structure and functional potential, surpassing antibiotic treatment effects across all analytical approaches. PERMANOVA revealed significant compartment-driven community structuring (R² = 0.189, P = 0.001), while treatment effects were non-significant (R² = 0.145, P = 0.116). Endosphere communities exhibited substantially lower alpha diversity than bulk soil and rhizosphere (P = 0.0001), with significant treatment × compartment interactions (P = 0.007). Antibiotic treatments selectively enriched xenobiotic degradation (P = 0.042) and secondary metabolism functions, particularly in bulk soil, without systematically increasing pathogen-associated or resistance-related functions. Network analysis revealed reduced bacterial connectivity under antibiotic pressure, yet cooperative interactions dominated across all treatments. Compositional differential abundance testing (ALDEx2) detected no significantly altered taxa for primary antibiotic contrasts (T1 vs. T3, T2 vs. T3), indicating context-driven rather than antibiotic-driven compositional changes. Functional diversity was significantly structured by compartment (Shannon P = 0.0017; richness P = 0.0039), while core plant-beneficial functions remained stable across treatments, with large effect sizes (Cohen's d ≥ 0.8) restricted to antibiotic degradation and secondary metabolism pathways. Our findings demonstrate that plant-microbe spatial structuring provides an ecological buffer that maintains core bacteriome functions against pharmaceutical disturbance, preserving plant-beneficial capabilities despite compositional shifts. The selective enrichment of antibiotic degradation pathways suggests potential for microbiome-assisted mitigation of pharmaceutical residues in agricultural systems. These results provide insights for developing compartment-specific microbiome management strategies that integrate with One Health approaches to enhance agricultural resilience under increasing pharmaceutical pressure in agroecosystems.