Synergistic Inhibition of the Lethal Fungal Pathogen Batrachochytrium dendrobatidis : The Combined Effect of Symbiotic Bacterial Metabolites and Antimicrobial Peptides of the Frog Rana muscosa

A powerful mechanism for protection against disease in animals is synergy between metabolites present in the natural microbiota of the host and antimicrobial peptides (AMPs) produced by the host. We studied this method of protection in amphibians in regard to the lethal disease chytridiomycosis, which is caused by Batrachochytrium dendrobatidis ( Bd ). In this study, we show that the AMPs of Rana muscosa , as well as the metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) from Pseudomonas fluorescens , a bacterial species normally found on the skin of R. muscosa , were inhibitory to the growth of Bd in vitro . When both AMPs and 2,4-DAPG were used in growth inhibition assays, they worked synergistically to inhibit the growth of Bd . This synergy resulted in reduced minimum concentrations necessary for inhibition by either 2,4-DAPG or AMPs. This inhibitory concentration of AMPs did not inhibit the growth of a P. fluorescens strain that produced 2,4-DAPG in vitro , although its growth was inhibited at higher peptide concentrations. These data suggest that the AMPs secreted onto frog skin and the metabolites secreted by the resident beneficial bacteria may work synergistically to enhance protection against Bd infection on amphibian skin. These results may aid conservation efforts to augment amphibian skins’ resistance to chytridiomycosis by introducing anti- Bd bacterial species that work synergistically with amphibian AMPs.