Dynamics of mono and dual-species biofilms of Escherichia coli and Salmonella Typhimurium: Interspatial interactions and novel inhibition strategies.

Multi-species biofilms in the food industry present significant global health risks, contributing to frequent outbreaks, diseases, and fatalities. This study aimed to characterize the mono- and dual-species biofilms formed by invasive food isolates Escherichia coli EMC17 and Salmonella typhimurium SMC25, focusing on microbial load variations, interspecies interactions, and the exploration of a combinatorial inhibition strategy for biofilm disruption. The highest biofilm density was observed in dual-species cultures at equal inoculum after 96 h, as confirmed by Crystal Violet assay and exopolysaccharide (EPS) production (72 μg/ml). Dual-species biofilms showed total bacterial counts of 7.29 log10 CFU/cm2 for E. coli EMC17 and 5.95 log10 CFU/cm2 for S. Typhimurium SMC25, with both strains exhibiting strong multidrug resistance (MDR), as indicated by MAR indices of 0.46 and 0.38, respectively. Epifluorescence and confocal laser microscopy revealed synergistic interactions during dual-species biofilm development, further supported by principal component analysis, which highlighted distinct growth patterns between mono- and dual-species biofilms. Specifically, mono-species biofilms of E. coli EMC17 had a biovolume of 56.70 ± 1.36 μm3/μm2 at 96 h, whereas dual-species biofilms reached 62.61 ± 1.38 μm3/μm2. Additionally, dual-species biofilm-associated cells exhibited significantly enhanced adhesion (16.40 %) and invasion (2.07 %) potential compared to the mono-species biofilms. Furthermore, a combinatorial inhibition strategy using quercetin and citric acid demonstrated synergistic activity against the MDR strains, with fractional inhibitory concentration index (FIC) of <0.5. These findings offer new insights into the dynamics of dual-species biofilms, emphasizing the potential of phytochemical-citric acid combinations as promising alternatives to traditional antibiotics in biofilm management.