The inhibitory mechanisms of plasma-activated water on biofilm formation of Pseudomonas fluorescens by disrupting quorum sensing

The use of plasma-activated water (PAW) to inactivate microorganisms in the food industry has attracted wide attention, while the research of PAW as a quorum sensing inhibitor (QSI) to mitigate biofilm is quite limited, and the molecular mechanisms have not been reported. In this study, PAW treatments under sub-inhibitory conditions of Pseudomonas fluorescens were selected, and no inhibitory effects on bacterial viable counts or growth curves were observed. Whereas biofilm biomass was significantly reduced by 28.38 % to 43.53 % during the 12 to 72 h incubation after PAW-60 treatment ( P  < 0.05). Extracellular polymeric substances (EPS) production was decreased, with a maximal reduction of 57.38 % in extracellular proteins, 53.43 % in polysaccharides, and 86.71 % in extracellular DNA compared to the control. Microscopic analyses evidenced that the biofilm became looser and thinner after PAW treatment. The surface properties of P. fluorescens , including hydrophobicity, auto-aggregation, and surface potential, were significantly declined by 31.15 %, 25.67 %, and 47.3 % respectively, after PAW-60 treatment at 12 h. Both swarming and swimming abilities of P. fluorescens were completely repressed by PAW-60 at 12 h. Moreover, PAW-60 significantly reduced QS signaling molecules production of AHLs and AI-2 by 46.98 % and 35.72 %, respectively ( P  < 0.05). Transcriptomic analysis and RT-qPCR demonstrated that PAW-60 suppressed chemotaxis and flagella assembly gene expression levels and downregulated genes related to the QS pathway, including the synthesis ( FadD1 ) and receptor ( LuxR ) of AHLs, and transporter protein genes of AI-2 ( RbsA , RbsC ). This study revealed the molecular mechanisms of PAW inhibition on biofilm formation of P. fluorescens by interfering with the QS pathway.