Together or Alone: Evaluating the Pathogen Inhibition Potential of Bacterial Cocktails against an Amphibian Pathogen.

The amphibian fungal skin disease () has caused major biodiversity losses globally. Several experimental trials have tested the use of Janthinobacterium lividum to reduce mortality due to infections, usually in single-strain amendments. It is well-characterized in terms of its anti- activity mechanisms. However, there are many other microbes that inhibit , and not all experiments have demonstrated consistent results with J. lividum. We used a series of assays involving bacterial coculture with lawns, bacterial growth tests in liquid broth, and grown in bacterial cell-free supernatant (CFS) to determine: (i) which skin bacteria isolated from a locally endangered amphibian, namely, the Colorado boreal toad (), are able to inhibit growth; (ii) whether multistrain combinations are more effective than single-strains; and (iii) the mechanism behind microbe-microbe interactions. Our results indicate that there are some single strain and multistrain probiotics (especially including strains from Pseudomonas and ) that are potentially more -inhibitive than is J. lividum alone and that some combinations may lead to a loss of inhibition, potentially through antagonistic metabolite effects. Additionally, if J. lividum continues being developed as a wild boreal toad probiotic, we should investigate it in combination with as they inhibited additively and grew at a higher rate when combined than did either alone. This highlights the fact that combinations of probiotics function in variable and unpredictable ways as well as the importance of considering the potential for interactions among naturally resident host microbiota and probiotic additions. () is a pathogen that infects amphibians globally and is causing a biodiversity crisis. Our research group studies one of the species affected by , namely, the Colorado boreal toad (). Many researchers focus their studies on one probiotic bacterial isolate called Janthinobacterium lividum, which slows growth in lab cultures and is currently being field tested in Colorado boreal toads. Although promising, J. lividum is not consistently effective across all amphibian individuals or species. For Colorado boreal toads, we addressed whether there are other bacterial strains that also inhibit (potentially better than does J. lividum) and whether we can create two-strain probiotics that function better than do single-strain probiotics. In addition, we evaluate which types of interactions occur between two-strain combinations and what these results mean in the context of adding a probiotic to an existing amphibian skin microbiome.