Suppression of Escherichia coli O157:H7 by Pseudomonas fluorescens A506 on romaine lettuce leaves is independent of iron competition.

Contamination of romaine lettuce with Escherichia coli O157:H7 (EcO157) remains a persistent food safety concern due to the pathogen's ability to survive on leaf surfaces. Although Pseudomonas fluorescens A506 is well studied as a biocontrol agent in tree fruit production, its ability to suppress EcO157 on leafy greens has not been evaluated. In this study, romaine lettuce leaves were pre-inoculated with A506 and inoculated with EcO157 strain MD41 24 h later. Across all MD41 inoculation levels (3, 4, 5, or 6 log CFU/g lettuce), pre-colonization with A506 significantly reduced MD41 population sizes by 1-2 log CFU/g over 72 h compared with the control. A506 is a high producer of siderophores, allowing this strain to efficiently acquire iron under iron-limiting conditions and compete with other bacterial species. Cell-free supernatants from A506 grown under iron-limited conditions inhibited MD41 growth in minimal medium. This inhibition was reversed by iron repletion and not observed with the pyoverdine-deficient mutant A506-1, indicating an iron-dependent effect of A506 on MD41 in vitro. In contrast, on romaine lettuce, neither pre-inoculation with A506-1 nor iron supplementation re-established MD41 population sizes to levels observed in the control. Additionally, expression of MD41 entS, entB, and fepA, which are involved in siderophore production and iron acquisition, was not increased during co-colonization of leaves with A506. Overall, these results suggest that iron competition is unlikely the primary driver of A506-mediated inhibition of EcO157 on romaine lettuce and that other yet unknown mechanisms are at play.