Chlorine dioxide injures Shigella flexneri, weakens biofilms, and produces a VBNC-like phenotype under cold stress: matrix-limited efficacy in broccoli wash water.

Shigella flexneri (S. flexneri) is a foodborne pathogen that can persist after sanitation stress through biofilm formation and nonculturable survival in food-related environments, such as vegetable wash water. This study examined how chlorine dioxide (ClO2) affects S. flexneri biofilm behavior, cold-stress loss of culturability and VBNC-like physiological responses and short-term metabolic responses. The apparent MIC and MBC of ClO2 under LB broth conditions were 200 and 400 mg/L, respectively. Permeability and leakage assays showed increased outer-membrane permeability, electrolyte release and extracellular accumulation of protein, nucleic-acid-associated material and ATP, consistent with progressive downstream envelope injury. These changes were accompanied by stronger DCF fluorescence, lower intracellular ATP and reduced kit-defined membrane-associated ATPase activity, indicating linked oxidative, membrane and energy-related injury. ClO2 also inhibited biofilm formation, reduced mature biofilm biomass and weakened biofilm metabolic activity, surface adhesion, auto-aggregation, hydrophobicity and EPS components. Under combined stress of 4 °C and sub-MIC ClO2, S. flexneri lost culturability but retained residual physiological activity and altered morphology, supporting a VBNC-like phenotype rather than complete cell death. Acute untargeted metabolomics further showed broader metabolic-feature changes in biofilm-associated cells than in planktonic cells, mainly involving nucleotide metabolism, transport-related pathways and stress-associated metabolites. The practical application of ClO2 in broccoli wash water shows a significant inhibitory effect on S. flexneri. These findings suggest that ClO2 control of S. flexneri depends on coupled envelope, oxidative and energy-related injury, while cold sub-MIC exposure may increase the risk of nonculturable persistence in food environments.