Bacteria from the skin of salamanders inhibit Batrachochytrium dendrobatidis

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is causing a worldwide decline in salamander populations. Bd causes chytridiomycosis in salamanders, which is a lethal infection of keratinized epithelial cells. Previous studies have characterized many microbial species from the skin of amphibians and shown some of them can inhibit Bd by producing antifungal metabolites. In this research, the skin microbiota of four salamanders was sampled by separately swabbing their ventral and dorsal sides. Mixed microbial cultures were obtained on R2A media from the swabs and 64 unique colonies were isolated. Microorganisms were characterized by describing colony color, shape, size, margin, texture, elevation, and pictures were taken. The microorganisms were Gram stained to determine cellular morphologies and 52 were Gram-negative rods, 11 Gram positive (seven rods and four cocci), and one was a fungus. PCR was used to amplify portions of the 16S (prokaryotic) or 18S (eukaryotic) rRNA genes. The PCR products were purified, sequenced, and Basic Local Assignment Search Tool (BLAST) was used to identify the microorganisms, at least to the genus level. The most common genera were Chryseobacterium, Pseudomonas, Acinetobacter, and Bacillus, respectively. Competition assays were performed on tryptone agar plates to test the microorganisms for their ability to inhibit Bd. Nine microorganisms tested positive for inhibiting Bd, and each salamander had at least two isolates that tested positive. Pseudomonas species moraviensis and koreensis produced the largest zones of inhibition, and co-culturing the microorganisms increased the size of the zones by having an additive effect.