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ABSTRACT: Bacterial contamination of fresh produce is a growing concern for food safety, as apart from human pathogens, antibiotic-resistant bacteria (ARB) can persist on fresh leafy produce. A prominent persistence trait in bacteria is biofilm formation, as it provides increased tolerance to stressful conditions. We screened a comprehensive collection of 174 antibiotic-susceptible and -resistant Escherichia coli originating from fresh leafy produce and its production environment. We tested the ability of these strains to produce biofilms, ranging from none or weak to extreme biofilm-forming bacteria. Next, we tested the ability of selected antibiotic-resistant isolates to colonize gnotobiotic lamb's lettuce (Valerianella locusta) plants. We hypothesized that a higher in vitro biofilm formation capacity correlates with increased colonization of gnotobiotic plant leaves. Despite a marked difference in the ability to form in vitro biofilms for a number of E. coli strains, in vitro biofilm formation was not associated with increased survival on gnotobiotic V. locusta leaf surfaces. However, all tested strains persisted for at least 21 days, highlighting potential food safety risks through unwanted ingestion of resistant bacteria. Population densities of biofilm-forming E. coli exhibited a complex pattern, with subpopulations more successful in colonizing gnotobiotic V. locusta leaves. These findings emphasize the complex behavior of ARB on leaf surfaces and their implications for human safety.