Inactivation of foodborne pathogens with combined essential oil treatments and identification of antibacterial mechanism using transcriptomic analysis

Abstract

This study evaluated the antibacterial activity of thyme, clove, and oregano essential oils (EOs), individually and in combination, against major foodborne pathogens and investigated their synergistic mechanisms using transcriptomic analysis. Checkerboard analysis revealed that a combination of thyme and clove EOs (800 ppm each) exerted a synergistic effect, as indicated by a fractional inhibitory concentration index of 0.5, against inactivation of Escherichia coli O157:H7 and Salmonella Typhimurium. Transcriptomic profiling of E. coli O157:H7 under synergistic conditions identified a total of 245 differentially expressed genes (47 upregulated and 198 downregulated), with significant enrichment in pathways associated with redox processes, stress response, nitrogen metabolism, and two-component systems. Genes involved in carbon source conversion (dhaK, dhaL) and nitrate reduction (napA, napB) were upregulated, whereas those related to outer membrane porins (ompF, ompC), flagellar assembly (fliA, flgM), and chemotaxis (cheA, cheY, tar) were downregulated. Collectively, these results suggest that combined EO treatment activates cellular metabolic and stress response pathways while suppressing motility- and membrane-associated functions. RNA sequencing data were validated by quantitative reverse transcription PCR analysis of selected genes, which showed consistent patterns with the transcriptomic results. At the phenotypic level, a swimming motility assay demonstrated that EO treatment significantly reduced bacterial motility, as evidenced by a decrease in halo diameter from 55.62 ± 4.24 mm in the control to 46.05 ± 2.38 mm (p < 0.05). This study demonstrates that EO combinations exhibit enhanced antibacterial activity through synergistic interactions and provides molecular insights for the development of natural preservatives to improve food safety.