Physical fields reverse FeSO4-induced VBNC state in Listeria monocytogenes and facilitate ferroptosis

Abstract

Non-thermal sterilization methods are effectively in eliminating foodborne pathogens while preserving the appearance and quality of food. In this study, three physical fields, magnetic field (MF), ultrasound (US), and blue light (BL), were introduced for their efficacy in sterilizing FeSO₄-induced viable but non-culturable (VBNC) Listeria monocytogenes (L. monocytogenes) cells over a 4 h treatment. The Fe-MF treatment induced L. monocytogenes cells to form VBNC state, resulting in an 80.13 % reduction in culturable cells, with a 90.58 % survival rate. The ultrasound-assisted FeSO₄ (Fe-US) treatment reduced the VBNC cell population by 52.63 %. In contrast, the blue light-assisted FeSO₄ (Fe-BL) treatment reversed the VBNC state toward culturable state at the node of 3 h, and induced irreversible 100 % cell death at 4 h, with a 94.73 % decrease in viability and 68.2 % membrane damage. Additionally, Fe-BL treatment led to cell wrinkling and secretion aggregation. BL treatment alone compromised membrane permeability and triggered intracellular protein aggregation. Mechanistic investigations revealed that BL-assisted treatment disrupted the protective mechanism of L. monocytogenes when induced by FeSO₄ to form VBNC state, compromised VBNC cell membranes, promoted intracellular Fe²⁺ accumulation, and induced a reactive oxygen species (ROS) burst and lipid peroxidation, ultimately leading to ferroptosis. Proteomic analysis identified 240 upregulated and 376 downregulated differentially expressed proteins, highlighting significant changes in pathways related to ribosome biosynthesis (related genes rplJ and hpf significant upregulated), intracellular iron homeostasis (ctaB, hemN, and lmo2590 downregulated), cellular morphology (mreB and tagH downregulated), oxidative stress response (lmo0720 and lmo0799 downregulated), and DNA synthesis (recA, dnaD, yidC2 and fruB downregulated). In conclusion, Fe-BL treatment effectively disrupted VBNC cell membranes, induced iron homeostasis imbalance, and triggered Fenton reaction-mediated ferroptosis. These findings provide a promising non-thermal sterilization strategy for inactivating VBNC L. monocytogenes, offering potential applications in food safety.