The growing emergence of multidrug-resistant bacterial pathogens drives the need for new antibacterial agents. Punicalagin exhibits efficacy against methicillin-resistant Staphylococcus aureus (MRSA), but its specific antibacterial mechanisms remain unclear. This study unveiled the specific antibacterial mechanism of punicalagin against MRSA via phenotypic and transcriptomic analyses. Punicalagin was found to induce severe cell wall damage and membrane disruption. Competitive binding assays identified lipoteichoic acid (LTA) as a potential target, and transcriptomic analysis further revealed that punicalagin downregulated key genes involved in cell wall synthesis (murA, murE) and LTA biosynthesis (dltA-D), consistent with the disruption of the cell wall. Additionally, punicalagin disrupted membrane homeostasis by inhibiting fatty acid synthesis (fabD, fabZ) and amino acid metabolism (dapA, dapB), leading to increased membrane permeability, which aligned with the phenotypic manifestations of membrane damage. Collectively, this work links phenotypic changes to specific gene expression patterns, unveiling that punicalagin inactivates MRSA via the multi-pathway regulation of the cell wall (LTA) and membrane function—providing insights for combating antibiotic-resistant pathogens in food safety and clinical settings.

Foodborne illness outbreaks from fresh produce underscore the urgent demand for sanitizing strategies that ensure safety while minimizing harmful by-products from high-dose chemical disinfectants such as sodium hypochlorite (NaOCl). Low-concentration combinations of organic acids and washing sanitizers were systematically evaluated to identify synergistic antibacterial effects, and citric acid (CA) was found to markedly potentiate the activity of NaOCl against Salmonella Typhimurium through a sequential assault on the cell envelope. A low-dose combination of sub-inhibitory concentrations (1/2 MIC of CA and 1/4 MIC of NaOCl) exhibited robust synergy, achieving a >6 log CFU/cm² reduction in the pathogen on a cherry tomato model within 3 min. Moreover, this synergistic entry leads to profound disruption of membrane integrity, resulting in leakage of nucleic acids and proteins, extensive oxidative damage, hyperpolarization, and cell lysis, as confirmed by electron and confocal microscopy together with physicochemical assays. Mechanistic investigation revealed that oxidative damage from NaOCl amplified CA-induced membrane acidification and permeability, facilitating deeper sanitizer penetration and accelerating envelope destruction. Collectively, these findings uncover a membrane-targeted synergistic mechanism, providing a solid scientific basis for the development of novel, low-residue, and high-efficacy food safety interventions.