Bactericidal and anti-virulence effects of <i>Melaleuca alternifolia</i> essential oil against foodborne pathogens: <i>in vitro</i> and milk matrix assay

The increasing demand for natural antimicrobials has driven interest in plant-derived compounds for food safety applications. This study evaluated the antimicrobial activity of tea tree essential oil (Melaleuca alternifolia, TTO) against Escherichia coli and Salmonella spp., combining in vitro and in situ assays. Minimum inhibitory concentrations (MICs) ranged from 3.12 to 6.25 µg ml-1, and the minimum bactericidal concentrations were identical to the MICs, in media broth. Time-kill assays showed significant reductions in viable cells at 1× MIC, with complete inactivation of S. Enteritidis and S. Typhimurium within 24 h. Treatment with TTO resulted in significant changes in zeta potential and increased leakage of nucleic acids and proteins, indicating membrane destabilization. At sub-inhibitory concentrations, TTO reduced swimming motility and biofilm formation in a strain-dependent manner. In sterilized milk, MIC values were identical to those obtained in Brain Heart Infusion (BHI) for all Salmonella strains and for E. coli P2421, while E. coli ATCC 25922 exhibited a higher MIC (6.25 µg ml-1). Checkerboard assays with potassium sorbate showed an additive interaction for S. enteritidis (FICI 1.0), indifferent interactions for most strains, and antagonism for S. Typhi, resulting in a reduction in sorbate MIC from 5.0 to 2.5 mg ml-1 in selected strains.