Temporal Dynamics Dominates Microbiome and Resistome Assembly Despite Tetracycline Pressure in a Germfree Lettuce Model

Background Plant associated microbiomes, encompassing diverse microbial communities underpin plant health, stress adaptation, and agricultural productivity, but can also serve as environmental reservoirs of antibiotic resistance genes (ARGs). These bacterial communities and associated ARGs span the plant-soil-food interface that is central to One Health concept. Although many studies have characterized established plant microbiomes and resistomes, the mechanisms driving their initial formations and temporal dynamics remain poorly understood. Results We employed a germfree lettuce ( Lactuca sativa ) (GL) model to investigate core bacteria and ARGs in leaves, roots, and soils in early microbial populations subjected to tetracycline (TET) pressure and exposure to the enteric pathogen Salmonella enterica serotype Typhimurium. Our results showed that initial bacterial loads and temporal dynamics were the primary determinants of microbiome and resistome structures, independent of Salmonella inoculation of leaves, whereas TET exerted only transient and early selection. Burkholderiaceae and Pseudomonadaceae were the key early colonizers but persisted over time and dominated the roots and soils. These core bacteria were also the major hosts of multidrug resistance (MDR) determinants, particularly efflux pumps. Mex-pump family members remained abundant in the resistomes across all time points, whereas TET family of ARGs contributed minimally to the resistome. Notably, no antibiotic resistant Salmonella isolates were recovered from lettuce leaves, suggesting limited selective pressure in the phyllosphere. Germfree models offer the advantage of manipulating the early colonizers, providing valuable insights into the role of specific microbes in the formation of new resistomes. Therefore, our GL model can offer valuable insights into the role of specific microbes in the formation of new resistomes. Conclusions Our findings indicate that intrinsic MDR efflux systems constitute the backbone of plant resistomes and that priority effects and time outweigh antibiotic selection in directing resistome trajectories. Framed within One Health, our results suggest that interventions targeting early community assembly may be as consequential as reducing antibiotic inputs for limiting ARG propagation across agricultural ecosystems and downstream to food chains. The germfree plant platform offers a tractable testbed to evaluate mitigation strategies that align agricultural practice with One Health goals.