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.

Staphylococcus aureus (S. aureus) is a prominent foodborne pathogen that poses a continuous threat to global public health and food safety due to its possession of a variety of toxins and its multidrug resistance. Medium-chain fatty acids (MCFAs), notably lauric acid (LA), exhibit strong antimicrobial properties, but their application is heavily constrained by poor water solubility. In this study, optimized LA emulsions stabilized by chitosan (CS) and polyvinyl alcohol (PVA) were evaluated for their antibacterial activity and detailed mode of action against S. aureus ATCC 6538. The antibacterial activities were evaluated by the maximum inhibition zone, with the 20 CS-PVA/DLTA-LA formulation exhibiting stable dispersion and potent antibacterial activity at 1%. The underlying antibacterial mechanisms against S. aureus were specifically focused on cell membranes and peptidoglycan. Therein, the binding of emulsion droplets to the anionic bacterial surface was driven by electrostatic attraction. Membrane degradation was also observed with membrane dysfunctions involving membrane depolarization, increased permeability, and fluidity reduction triggered by their subsequent insertion into the lipid bilayer, which may cause cell dysmetabolism, disintegration, and eventual cell death. Overall, these findings substantiate that LA emulsions disrupt S. aureus by operating potentially multi-targeted effects involving cell membrane disruption and peptidoglycan interference, offering a promising alternative approach warranting further investigation for foodborne pathogen control.