Inactivation of Salmonella Enteritidis by clove essential oil: Multi-pathway responses and gene-function analyses

Salmonella Enteritidis ( S. Enteritidis) is a primary foodborne pathogen whose stress resistance raises major food safety concerns. Clove essential oil (CEO), a natural antimicrobial agent, has garnered considerable attention due to its broad-spectrum antibacterial activity. However, the molecular mechanisms responsible for its antibacterial effect remain poorly elucidated. In this work, an integrated strategy combining proteomic profiling, bioinformatic analysis, and mutant analysis was adopted to explore the antibacterial mechanism of CEO against S. Enteritidis. The results showed that the minimum inhibitory and bactericidal concentrations of CEO were 2 g/L and 4 g/L, respectively. Proteomic and bioinformatic analyses revealed that CEO modulated the expression of 1437 proteins involved in cellular metabolism, ABC transporters, two-component systems, global regulation, flagellar assembly, and other cellular processes. Based on the proteomics results, three proteins from these pathways (HiuH, ProX, and PmrA) were selected for further investigation. The functional relevance of these proteins was subsequently validated by phenotypic comparisons between gene knockout mutants and the wild-type strain under CEO treatment. Intriguingly, the Δ hiuH , Δ proX , and Δ pmrA mutants displayed better in vitro growth yet greater susceptibility to CEO inactivation in chicken meat, highlighting the importance of assessing CEO efficacy in food matrices. Collectively, this work reveals the multi-pathway response of S. Enteritidis to CEO exposure, offering new mechanistic insights into the interplay between bacterial stress-defense systems and essential oil-mediated bacterial inactivation.