Escherichia coli O157:H7 sublethally injured by freezing stress: Physiological characteristics and mechanisms underlying its formation and resuscitation.

Freezing stress can induce sublethal injury in foodborne pathogens, allowing injured cells to evade detection and subsequently resuscitate, thereby posing a hidden food safety risk. This study investigated the formation, physiological characteristics, and resuscitation of freezing-induced sublethally injured Escherichia coli O157:H7 at -20 °C and explored the underlying mechanisms using untargeted metabolomics. After 21 h of freezing, the proportion of sublethally injured cells exceeded 80%, accompanied by cell surface collapse, impaired membrane integrity, and depleted intracellular protein and ATP levels. Metabolomic analysis revealed distinct perturbations in pyruvate metabolism, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis. Resuscitation in tryptone soy broth at 25 °C for 8 h was accompanied by activation of purine, cysteine and methionine metabolism, glycolysis/gluconeogenesis, and riboflavin metabolism, alongside upregulation of phosphatidylethanolamine indicative of membrane remodeling. Notably, freezing-stressed E. coli cells exhibited significantly enhanced sensitivity to oregano essential oil (OEO), with the sublethal injury ratio decreasing from 79.73% to 22.70% upon OEO treatment at 1/2 × MIC (minimum inhibitory concentration), consistent with the membrane-damage mechanism. These findings provide integrated physiological and metabolomic insights into freezing‑induced sublethal injury and subsequent resuscitation, and support the rational design of membrane-targeting antimicrobials as effective hurdle strategies for frozen food safety.