Molecular mechanisms of the VBNC state induced by polyhexamethylene guanidine in biofilm-associated Staphylococcus aureus

Polyhexamethylene guanidine (PHMG) is widely used to eliminate biofilms in the dairy industry; however, sublethal chemical stress inadvertently forces pathogens into a viable but non-culturable (VBNC) state. This study examined PHMG-induced VBNC formation in biofilm-associated Staphylococcus aureus under laboratory and food-relevant conditions. PHMG at 4.0 mg/mL (0.4%) eliminated culturability within 3 h, whereas flow cytometry detected a viable fraction of 25.70 ± 3.83% in TSB-grown biofilms at 25 °C. This shift was primarily driven by acute oxidative stress, marked by a 1.56-fold accumulation of intracellular ROS levels and compensatory increases in catalase and superoxide dismutase activities. Concurrently, the cells underwent severe metabolic shutdown, characterized by intracellular ATP depletion, membrane depolarization, and cell shrinkage. Transcriptional profiling of energy- and virulence-related genes confirmed this systemic reprogramming, revealing altered expression in key energy metabolism and stress survival pathways. Crucially, this dormant state was highly unstable in food environments. When transferred back to milk, the VBNC cells rapidly resuscitated within 4 h and successfully resumed hemolysin production. Ultimately, these findings highlight the urgent need for the dairy industry to re-evaluate current PHMG disinfection strategies and closely monitor hidden VBNC risks to ensure food safety.