Biosurfactant-Mediated Inhibition of Salmonella Typhimurium Biofilms on Plastics: Influence of Lipopolysaccharide Structure

Abstract Salmonella enterica subsp. enterica serovar Typhimurium is a major foodborne pathogen whose ability to form biofilms contributes to persistent contamination in food-processing and clinical environments. This study investigated the anti-biofilm activity of the biosurfactant surfactin, produced by Bacillus subtilis, against S. Typhimurium wild type (LT2) and its lipopolysaccharide (LPS)-modified mutants on commonly used plastic surfaces such as polypropylene (PP) and polystyrene (PS). Biofilm formation was quantified using the crystal violet assay, revealing significantly higher biomass on PS compared to PP (p < 0.0001). Surfactin at 5 µg/mL was identified as the minimum biofilm inhibitory concentration (MBIC), significantly reducing biofilm formation in the wild-type and LPS mutants rfaL, rfaJ, rfaF (all p < 0.0001), and rfaI (p < 0.01). Further analysis using fluorescence microscopy and SYPRO® Ruby staining confirmed a significant reduction in extracellular polymeric substances (EPSs) on PP surfaces following surfactin treatment, particularly in strains LT2 (p < 0.0001), rfa (p < 0.01), rfaL (p < 0.0001), rfaG (p < 0.05), and rfaE (p < 0.0001). These findings highlight the influence of LPS structure on biofilm development and demonstrate surfactin’s potential as an eco-friendly antimicrobial agent for controlling S. Typhimurium biofilms on food-contact surfaces. Analysis of mutants revealed that disruption of the rfa gene, which is involved in the biosynthesis of the outermost region of the lipopolysaccharide (LPS), significantly reduced bacterial attachment to polypropylene. This suggests that interactions between the external LPS layer and the plastic surface are important for colonisation. In contrast, mutants in core LPS biosynthesis genes such as rfaE and rfaD did not show any notable differences in attachment compared to the wild-type strain. This highlights the specific importance of outer LPS components, particularly under surfactant conditions, in mediating interactions with plastic surfaces. This work supports the application of biosurfactants in food safety strategies to reduce the risk of biofilm-associated contamination.