An investigation of biosurfactant-mediated disruption and biological activity in mature biofilms formed on plastic surfaces by LPS mutants of Salmonella enterica serovar Typhimurium

Salmonella enterica subsp. enterica serovar Typhimurium is a major foodborne pathogen. Its ability to form biofilms (a community of microorganisms) contributes to contamination in the food industry and clinical environments, posing challenges for disinfection and increasing the risk of spread. Biosurfactants, compounds produced by microorganisms, can potentially control biofilm formation as eco-friendly antimicrobial agents and be applied to plastic food packaging to prevent biofilm formation and food spoilage. This study aimed to investigate the effect of biosurfactant, rhamnolipid (produced by Pseudomonas aeruginosa), and in particular surfactin (produced by Bacillus subtilis), on S. Typhimurium wild type (LT2) and its lipopolysaccharide (LPS)-modified mutants (with mutated rfa genes, critical for LPS biosynthesis). Furthermore, evaluate its effectiveness in S. Typhimurium cultivated on commonly used plastics (polypropylene and polystyrene) in food and medical industries.

Surfactin at 5 µg/mL and polypropylene (PP) plastic have been selected for further studies based on the Crystal Violet assay, which assesses the reduction of biofilm formation. The results revealed that surfactin treatments at 5 µg/mL significantly reduced biofilm mass in WT (p <0.0001) as well as in LPS-mutants: 228 (rfaL), 229 (rfaJ), 230 (rfaF) (all p <0.0001), and 227 (rfaI) (p <0.01). This indicates that LPS biosynthesis influences the cell surface of biofilm structure and adhesion. Surfactin appears to affect biofilm growth and reduce extracellular polymeric substance (EPS, a biofilm matrix protective barrier) on PP surfaces, which was also demonstrated by FilmTracerTM SYPRO® Ruby Biofilm Matrix staining using fluorescence microscopy. Significant reduction (p ≤0.05) of EPS was observed in S. Typhimurium (WT) and selected LPS-modified mutants (225 rfa, 228 rfaL, 231 rfaG, 258 rfaE). To understand the gene regulation involved in biofilm formation and its expression under surfactin treatments on PP coupons and planktonic conditions, the following genes csgD, flgK, cydA, bamA, ftsZ, murA, hisI, and dnaA from various clusters were investigated by RT-qPCR. Where surfactin up-regulates these genes, it could signify the adaptive stress response, and surfactin-related gene down-regulation suggests reduced survival. These effects are possibly due to the LPS chain variation between strains. There were distinct patterns between the PP coupon and planktonic conditions, which could be due to different synthesis mechanisms used in bacterial cell adhesion, cell replication and survival.

In conclusion, these findings have practical implications for designing biofilm control strategies in food processing environments. Targeting the LPS structure of S. Typhimurium by surfactin to disrupt biofilm formation may reduce the risks of foodborne infections. Further studies using the proteomic approach could confirm the surfactin mechanism and its effect on EPS and inhibition of biofilms.