Synergistic Effect of Formate and Cell-Free Supernatant Fermented by Two Probiotics, <i>Bifidobacterium animalis</i> spp. <i>lactis</i> HN019 and <i>Lacticaseibacillus rhamnosus</i> HN001, against <i>Shigella flexneri</i> Growth

The interplay between probiotic metabolites, host health, and inhibition of pathogens has increasingly attracted interest but remains unresolved due to the complex molecular interactions among these factors. We investigated the action of cell-free supernatants (CFSs) from two probiotic bacteria, <i>Bifidobacterium animalis</i> spp. <i>lactis</i> (<i>B. lactis</i>) HN019 and <i>Lacticaseibacillus rhamnosus</i> (<i>L. rhamnosus</i>) HN001 and their effect against the bacterial pathogen <i>Shigella flexneri</i> 2457T (<i>S. flexneri</i>). The CFSs from <i>B. lactis</i> HN019 exhibited a higher antibacterial effect against <i>S. flexneri</i> growth than the CFSs from <i>L. rhamnosus</i> HN001, independent of the carbon source utilized. This effect correlated with higher formate within the CFSs from <i>B. lactis</i> HN019. As expected, the antimicrobial effects of CFSs were stronger against free <i>S. flexneri</i> cells than for <i>S. flexneri</i> infecting host cells. A synergistic effect of the CFSs with additional small organic molecules such as indole-3-lactate and formate was found. Such interplay between CFSs, indole-3-lactate, and formate was reflected by altered metabolic rates by <i>S. flexneri</i> in the presence of <i>B. lactis</i> HN019 CFSs both in the solution and under biofilm-forming conditions. The synergistic effect between different components acting on <i>S. flexneri</i> gives reasons to believe that suitably designed mixtures of probiotic metabolic products and small-molecule effectors bear promise for successfully combating pathogens.