Lactococcus cremoris decreases virulence‑associated properties, and modifies the protein profile of foodborne pathogen Campylobacter jejuni in biofilm

Campylobacter jejuni, the most common foodborne pathogen, relies on adhesion, invasion, and biofilm formation for successful virulence and persistence in the food-processing environments. In this study, we investigated the effects of the transient bacterium with probiotic properties Lactococcus cremoris on C. jejuni virulence via an adhesion and invasion assay using human colon adenocarcinoma Caco-2 cells, culturability via colony-forming units, and proteomic adaptation in co-culture via label-free quantitative mass spectrometry. Caco-2 cell adhesion and invasion assay revealed that L. cremoris decreases C. jejuni adhesion (by 50%) and invasion (by 92%), particularly when inoculated prior to the pathogen. In mature (48 h old) dual-species biofilms, C. jejuni culturability decreased by 88%, while L. cremoris culturability remained unaffected, compared to single-species biofilms. Proteomic analysis of biofilm cells showed metabolic reprogramming in C. jejuni, with increased levels of proteins related to the TCA cycle, stress response, amino acid and nucleotide metabolism, chemotaxis, and energy production. The most upregulated proteins included the tungsten transporter TupA, LuxS, Dps, and hydrogenase subunits HydAB, indicating adaptations to nutrient limitation and oxidative stress. Nevertheless, the decreased culturability of C. jejuni suggests it is overwhelmed under the competitive conditions of dual-species biofilms. Our findings demonstrate that L. cremoris exerts strong antagonistic effects on C. jejuni, restricting its virulence, and triggering significant metabolic shifts in dual-species biofilms. These results highlight the potential of L. cremoris as a preventative strategy against C. jejuni adhesion, colonisation, and transmission.