Synergistic antibacterial and anti-biofilm mechanism of cold stress combined with plasma-activated water against Pseudomonas aeruginosa: A multi-target transcriptomic analysis.

The increasing resistance of Pseudomonas aeruginosa (P. aeruginosa) to conventional sanitizers demands novel multi-target intervention strategies. This study investigated the sequential synergistic antibacterial and anti-biofilm effects of cold stress (CS) and plasma-activated water (PAW) against P. aeruginosa. PAW generated at 750 W for 2 min exhibited acidic pH, high oxidation-reduction potential, and substantial levels of reactive oxygen and nitrogen species (RONS). Sequential CS-PAW treatment achieved a bacterial reduction rate of 88.20 ± 4.41% and reduced biofilm formation (P < 0.05), significantly outperforming CS or PAW alone. Mechanistically, CS pretreatment sensitized bacterial cells by compromising membrane integrity and depleting energy reserves, thereby enhancing their vulnerability to subsequent PAW attack. The combined treatment triggered excessive intracellular ROS accumulation, suppressed superoxide dismutase, catalase, and glutathione peroxidase activities, and induced severe lipid peroxidation. Key energy metabolism enzymes were inhibited, while alkaline phosphatase leakage confirmed cell wall damage. Furthermore, CS-PAW effectively interfered with quorum sensing (QS) by reducing C4-HSL signaling, downregulating QS-related genes (rhlI, rhlR, lasI, lasR), and suppressing the production of extracellular polymeric substances (EPS) and virulence factors. Transcriptomic analysis revealed 839 differentially expressed genes, with significant downregulation of pathways controlling QS, biofilm formation, bacterial chemotaxis, the type VI secretion system, and carbon metabolism. Notably, because the CS-PAW intervention targets highly conserved processes, including membrane integrity, central carbon metabolism, and QS signaling, the observed multi-target inhibition provides a robust mechanistic basis for its broad applicability against P. aeruginosa isolates; however, strict mathematical synergy was not quantified, and the current conclusions are explicitly limited to demonstrating statistically superior efficacy over single treatments. Collectively, these findings suggest that CS-PAW acts through a complementary multi-target mechanism rather than requiring strict mathematical synergy, representing a promising non-thermal strategy for controlling P. aeruginosa contamination in food processing environments.