Plasma-Activated Water: An Emerging Technology for the Control of Eggshell-Associated Biofilms

Biofilm formation on eggshell surfaces poses a significant food safety concern by promoting bacterial persistence and facilitating cross-contamination, contributing to foodborne illness outbreaks worldwide. Beyond public health impacts, bacterial contamination also drives spoilage, food loss, and waste, together placing substantial strain on healthcare systems and economic infrastructures. This study evaluated the anti-biofilm efficacy of plasma-activated water (PAW), a reactive oxygen and nitrogen species (RONS)-rich solution generated through the interaction of cold atmospheric plasma with water. 50 bacterial isolates were recovered from chicken eggshells, and the biofilm-forming capacities of eight representative species (Staphylococcus arlettae, Lysinibacillus fusiformis, Priestia megaterium, Peribacillus frigoritolerans, Acinetobacter lwoffii, Escherichia coli, Pseudomonas fulva, and Pseudomonas stutzeri) were characterised in vitro. All isolates formed biofilms, exhibiting variable culturable cell populations, biomass, and extracellular polymeric substance (EPS) matrix presence and composition. EPS analyses identified polysaccharides, extracellular DNA, and proteins as key matrix components, with relative abundance varying with biofilm maturity. PAW efficacy was assessed under short exposure times (1, 5, and 15 minutes) using freshly generated, 1-week-old, and 4-week-old PAW. Fresh PAW demonstrated potent anti-biofilm activity, achieving complete inactivation of E. coli and P. stutzeri biofilms below the limit of detection within 1 min, while biofilms of Gram-positive bacteria, L. fusiformis and P. megaterium, were generally more tolerant. Reduced efficacy of aged PAW was likely associated with RONS decay. Mechanistic analyses, including scanning electron microscopy and intracellular reactive oxygen species assays, linked PAW treatment to biofilm disruption and intracellular oxidative stress. This study provides the first detailed evaluation of biofilm formation by under-investigated eggshell-associated bacteria and elucidates mechanisms underlying PAW-mediated biofilm inactivation. Overall, PAW shows strong potential as a sanitising technology for the egg industry, supporting safer food production and reduced reliance on conventional antimicrobial agents amid rising global antimicrobial resistance rates.