Efficacy and mechanism of cold plasma-activated lactic acid against Staphylococcus aureus biofilms

The resilient biofilms formed by Staphylococcus aureus (S. aureus) on food and food-processing surfaces pose serious risks of persistent contamination, product spoilage, and public health threats. This highlighted the urgent need for safe, nontoxic, and highly efficient antibiofilm agents. In this study, plasma-activated lactic acid (PALA) was evaluated for its efficacy in eradicating S. aureus biofilms and its underlying mechanism of action. Notably, a 20 min PALA treatment reduced viable biofilm cells by 4.10 log CFU/mL, increased propidium iodide fluorescence by 299.56%, and elevated supernatant OD₂₆₀/OD₂₈₀, indicating massive membrane disruption and intracellular leakage. Flow cytometry confirmed that 68.50% of cells suffered membrane damage. Moreover, PALA further induced severe oxidative stress, with intracellular ROS levels increasing by 163.09% after 15 min of treatment. During the 0-20 min treatment period, ATP levels in biofilm cells showed a transient increase from 0.094 to 0.79 μmol/L before declining, reflecting metabolic disruption. PALA exerted its antibiofilm effect through multiple synergistic mechanisms: structural disintegration of the biofilm matrix, severe membrane damage with leakage of intracellular components, significant ROS accumulation causing metabolic disruption and double-stranded DNA breaks, and reduction in secondary biofilm formation by impairing auto-aggregation, lowering zeta potential, and decreasing surface hydrophobicity. Collectively, these findings demonstrate that PALA can effectively remove S. aureus biofilms and represents a promising novel biofilm control agent.