Lettuce surface enhances biofilm formation, AI-2 activity, and VBNC induction in mixed-species biofilms of Staphylococcus aureus and Escherichia coli O157:H7

Mixed-species biofilms on food-contact surfaces are persistent sources of cross-contamination in fresh-produce processing. This study compared mixed-species biofilms of Staphylococcus aureus and Escherichia coli O157:H7 on glass, polystyrene, stainless steel 304 (SS), and fresh lettuce, with in-depth analysis on SS and lettuce as abiotic and biotic representatives. Across the four surfaces, mixed-species biofilms exceeded single-species counterparts in viable counts, biomass (OD 595 , 1.30-3.24), and extracellular polymeric substance (EPS) yield (21.44-72.65 μg/coupon), with lettuce supporting the highest values. FTIR detected a lettuce-specific band at 1,126 cm -1 consistent with nucleic acid-associated vibrations, and HPLC showed a higher ribose proportion in lettuce-derived EPS (12.84%) than in SS-derived EPS (5.27%). Slightly acidic electrolyzed water (SAEW; 20 mg/L, pH 5.5) reduced mixed-species biofilm culturability below the detection limit in 10 h on lettuce and 12 h on SS, while propidium monoazide qPCR confirmed viable-but-non-culturable (VBNC) entry. Compared with SS, lettuce supported 1.83-fold higher autoinducer-2 (AI-2) activity and 2.0-4.0-fold upregulation of lsrA , lsrC , csgA , rpoS , eaeA , and stx1 . Under SAEW treatment, rpoS expression increased to 3.25-fold on lettuce, whereas the other five genes were downregulated. The biotic lettuce surface enhances AI-2 activity and accelerates VBNC formation, retaining virulence gene expression undetectable by standard culture.