Together or alone: Amphibian probiotic microbes are more successful against a skin pathogen in cocktails of related strains

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Alexandra Alexiev, Melissa Y. Chen, Andrew Weier, Timothy M. Korpita and Valerie J. McKenzie, Department of Ecology and Evolutionary Biology, University of Colorado Boulder, Boulder, CO

Background/Question/Methods
Batrachochytrium dendrobatidis (Bd) is a fungal emerging infectious disease that threatens amphibians globally, and has contributed to the decline of 501 species as of 2019. The typical treatment for a fungal infection is antifungal compounds, but these are problematic since they may increase pathogen resistance in pristine pond habitats and harm innate skin microbes that otherwise inhabit hosts. Many conservation researchers are employing intrinsic amphibian skin bacteria that inhibit Bd in culture as skin probiotics, the most heavily favored of these being strains of Janthinobacterium lividum. We studied the potential for other single strains and two-strain cocktails to inhibit Bd relative to J. lividum. Further, we designed the assay to differentiate between three possible mechanisms of inhibition (facilitative vs. additive or synergistic metabolite inhibition). We isolated bacteria from wild boreal toad (Anaxyrus boreas boreas) skin, a host that is locally endangered in Colorado due to Bd-related decline. We used co-culture assays to test all isolated toad skin bacteria for Bd inhibition, then chose five strong candidates for further tests in two-strain cocktails and to differentiate between mechanisms of inhibition. For the cocktail assay, we included one bacterial strain that did not inhibit Bd but is in the same order as Janthinobacterium and has, in the past, been found highly enriched on boreal toad tadpole skin.

Results/Conclusions
Microbial relatedness affected how metabolites interacted to inhibit the Bd pathogen. Most microbial cocktails functioned facultatively or lost inhibition, with one combination showing additive inhibition. Microbes of the Burkholdariales order, especially further closely related J. lividum strains, had increased Bd-inhibition when tested together as opposed to alone. Psuedomonas, Microbacterium, and Chryseobacterium were more inhibitive than these strains and even had decreased Bd-inhibition when combined with the Burkholdariales group. This suggest two things: (1) microbes in the Burkholdariales order, which includes the favored J. lividum strains used in past amphibian probiotic trials, can be more effective Bd-inhibitors in cocktails of different strains of the same species, and (2) other, less phylogenetically-related but still inhibitive, strains that live on toad skin may lose their inhibitive abilities if a probiotic Burkholdariales strain is applied, and therefore the context of the starting community may be a significant factor for future consideration and study of probiotic applications to amphibian skin.