Ligilactobacillus salivarius-driven metabolic alterations of Staphylococcus aureus amplifies virulence and disrupts intestinal barrier homeostasis.

Probiotics typically confer health benefits by inhibiting the adhesion of Staphylococcus aureus and reducing its adhesion and accumulation in the host. However, our previous studies have shown that Ligilactobacillus salivarius interacting with S. aureus, actually enhanced the adherence of S. aureus, but the underlying mechanisms are still unclear. In this study, using in vitro co-culture models, cell-based assays, and in vivo animal experiments, we compared the effects of viable L. salivarius (vLS), its supernatant (sLS), and inactivated L. salivarius (iLS) on S. aureus behavior. The results showed that co-culture of vLS, sLS and iLS with S. aureus promoted the formation of S. aureus biofilm and its soluble and non-soluble polysaccharides, and enhanced cell adhesion. Especially after co-culture with vLS, the adhesion number of S. aureus was increased by 57.40% compared to the SA group. The expression of adhesion genes (FnbpA, FnbpB, ClfA and ClfB) and virulence genes (agrA, agrB, agrC, agrD, hla and spa) were increased. In cell experiments, vLS, sLS, and iLS caused a decrease in Caco-2 cell viability and accelerated apoptosis through their virulence effects when co-cultured with S. aureus. Non-targeted metabolomics suggested that sLS upregulated the respiratory metabolism of S. aureus and increased metabolic intermediates associated with its biofilm and cell wall synthesis, thereby increasing the toxicity of S. aureus. In vivo studies, the co-culture of vLS, sLS, and iLS with S. aureus increased inflammation in mice and promoted the accumulation of S. aureus in the host, especially in the gut, and destroyed the structure of the gut microflora in mice, decreasing the abundance of Desulfovibrio and Limosilactobacillus, and promoting the abundance of Stenotrophomonas, which increased the intestinal barrier damage. Among the three LS preparations, vLS consistently exhibited the strongest pre-toxicity effect, followed by sLS, whereas iLS only showed partial activity. This indicated that both the live bacteria and the metabolites they secrete contribute to the phenotype. This study revealed for the first time the metabolic basis by which L. salivarius promotes the adhesion of S. aureus, providing a new perspective on the interaction between probiotics and pathogens, which is of great significance for elucidating the mechanism of maintaining intestinal homeostasis and guiding the safe application of probiotics.