Abstract
Foodborne pathogenic biofilms pose significant challenges to food safety due to their enhanced resistance to conventional antimicrobial agents. In this study, we evaluated the synergistic antibiofilm activity of oregano essential oil (OEO) from Lippia graveolens and the lytic bacteriophage phiLLS against six foodborne bacteria. GC-MS analysis achieved a 100% identification ratio, revealing that OEO was mainly composed of carvacrol (58.9%), p-cymene (28.6%), γ-terpinene (2.9%), and caryophyllene (2.6%). The MIC and MBC of OEO were 1 and 2 mg/mL, respectively, for all strains except E. coli BALL1119 (MIC = 2 mg/mL). We assessed biofilm biomass by crystal violet (CV) staining and metabolic activity using the TTC assay under both individual and combined treatments, monitored 9-hour planktonic growth kinetics to calculate Bliss and HSA synergy indexes, and employed atomic force microscopy (AFM) to visualize nanoscale alterations in Staphylococcus aureus and Escherichia coli BALL1119 biofilms. Combined OEO (2 mg/mL) and phiLLS (MOI 1) treatments achieved significantly greater biofilm biomass reduction than single agents, notably yielding >70% inhibition of S. aureus biofilms (p < 0.05) and a Bliss synergy index of 10.8% in E. coli BALL1119 growth kinetics, whereas other strains were additive. In biofilm assays, S. aureus and Salmonella spp. showed the highest reductions in biomass (CV) (71.0% and 67.8%, ΔHSA = 27.0% and 17.4%; ΔBliss = 21.1% and 13.8%) and metabolic activity (TTC) (68.6% and 48.5%). AFM revealed that OEO alone smoothed the extracellular matrix (averaging a 35% reduction in roughness), whereas the combined treatment caused fracturing (≈68 nm roughness) and prominent lytic pits. Although variability in S. aureus biofilm architecture precluded statistically significant pairwise comparisons, AFM topography and consistent trends in Ra/Rz parameters provided clear visual corroboration of the significant reductions detected by CV and TTC assays. These complementary data indicate that OEO primes the biofilm matrix for enhanced phage-mediated collapse, offering a green, two-step strategy for controlling resilient foodborne biofilms.