Transferable mrkABCDF-bearing plasmids and benzalkonium bromide-lysozyme-based control of biofilms in multidrug-resistant Escherichia coli from food animals.

Strong biofilm-forming multidrug-resistant (MDR) Escherichia coli (E. coli) can persist on food-contact surfaces and contribute to contamination along the food production chain. In this study, we investigated the occurrence, genomic features, and plasmid transferability of strong biofilm-forming E. coli from food-producing animals and associated farm environments, and evaluated an approach for controlling their surface-associated biofilms. Among 536 isolates, 42 were classified as strong biofilm producers (7.84%, 42/536). The highest proportion was observed in pig-derived isolates (10.11%, 36/356), followed by duck-derived (3.70%, 3/81) and chicken-derived isolates (3.03%, 3/99). These isolates showed diverse sequence types and phylogroups. The biofilm-associated genes csgG, flgCD, and fliA were detected in all strong biofilm producers, whereas the adhesin gene cluster mrkABCDF was present in 24 isolates (57.1%, 24/42). Conjugation assays yielded 26 transconjugants, of which 14 retained strong biofilm-forming capacity. Among 13 sequenced transconjugants with analyzable plasmids, 11 carried complete mrkABCDF clusters, mainly on IncX1 plasmids. Public genome comparison further identified highly similar plasmid backbones across 550 public E. coli genomes from diverse host, geographic, and ecological sources, suggesting that biofilm-associated plasmids are not restricted to a single clonal lineage. Benzalkonium bromide combined with lysozyme reduced biofilm biomass, extracellular matrix production, bacterial aggregation, and surface colonization on glass, stainless steel, and polyvinyl chloride. Overall, these findings suggest that transferable mrkABCDF-bearing plasmids, particularly IncX1 plasmids, may serve as potential genetic vehicles for persistence-associated traits in MDR E. coli, and that benzalkonium bromide-lysozyme treatment may provide a candidate strategy for reducing biofilm persistence on food-contact surfaces.