Fighting a Deadly Fungus with Bacteria Harvested from Salamander Skin

Emerging fungal pathogens such as Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) have caused significant amphibian population declines worldwide. Healthy amphibian populations are very important indicator species, used in monitoring the health of ecosystems. Rapid decline of these creatures could therefore be highly detrimental to global environmental efforts. A probiotic treatment is the most feasible solution to combat the fungus on salamander skin. In an attempt to develop an efficient probiotic against these infections, we have been conducting research to see what effects the resident skin microbiomes of salamanders have against these pathogenic fungi. We collected 359 skin swabs from wild salamanders in the genera Plethodon, Desmognathus, and Eurycea. A total of 98 bacterial colonies were isolated from 11 skin swabs and grown in pure culture. Thirteen isolates were challenged against Bd on 1% tryptone agar plates. All isolates grew in the presence of Bd, but those species either did not form measurable zones of inhibition, or the zones produced were masked by bacterial motility. The isolates that did not form measurable zones of inhibition have been discounted as candidate probiotics. In future screens, we will retest the samples that were masked by bacterial motility. We expect other isolates will inhibit fungal growth, and we will recommend these isolates for therapeutic treatments of diseased amphibians. By identifying native salamander bacteria with antifungal properties, it is possible to give our salamander populations the means to resist this deadly threat

Inhibiting the causative agent of snake fungal disease, Ophidiomyces ophiodiicola, with common cutaneous bacteria isolated from snake skin.

Recent declines of North American snake populations have increased conservation concerns. These declines have been attributed in part to snake fungal disease (SFD) caused by mycotic infection of Ophidiomyces ophiodiicola (Oo). Multicellular organisms, including reptiles, have a beneficial microbial assemblage on their skin that defend the host from disease. We hypothesized that the snake skin microbiome may protect its host against SFD by production of antifungal metabolites, outcompeting Oo for space, and/or stabilizing the microbial community possibly increasing innate defensive immunity. The objectives of this project were to 1) obtain culturable resident bacterial isolates from snake skin, 2) genotype culturable isolates, and 3) challenge all isolates against Oo to determine if antifungal activity is present in the host microbiome which may translate to an innate protective response against SFD. From the twelve captured snakes, four black racers (Coluber constrictor) and eight timber rattlesnakes (Crotalus horridus), microbial samples were collected using a standardized swabbing technique. Those swabs from the 12 host snakes yielded a total of 58 bacterial colonies that were isolated, genotyped, and challenged against Oo. Sixteen of these bacterial isolates inhibited the growth of Oo in vitro. Species of antifungal bacteria occurred in the genera Aeromonas, Erwinia, Myroides, Morganella, and Stenotrophomonas, along with in the families Bacillaceae and Enterobacteriaceae. This study sheds light on the innate immune function of the culturable snake cutaneous microbiome and the resident probiotic members which may play a vital part in the fight against this emergent disease for both infected and uninfected hosts

*WINNER* Identifying patterns in the culturable skin microbiome of Appalachian salamanders

Batrachochytrium dendrobatidis (Bd) is a fungal pathogen that has devastated amphibian populations worldwide.  However, in the eastern United States, salamander populations persist in the presence of this fungus with relatively low rates of infection.  We hypothesize that probiotic species within the salamander cutaneous microbiome protect their host by inhibiting growth of Bd.  During previous work, we identified cutaneous defenses of salamanders by isolating bacteria from skin swabs and challenging them against Bd.  The objectives of our current work are to (1) characterize the microbiome through 16S sequencing techniques, and (2) determine whether the culturable microbiome differs based on host species and/or habitat. Salamanders belonging to the genera Desmognathus (n = 8), Eurycea (n = 7), and Plethodon (n = 7) were sampled in the Great Smoky Mountains National Park. From their skin swabs, we obtained and sequenced a total of 110 bacterial isolates. Bioinformatics analyses were conducted using the software mothur, and a two-way permutated multivariate analysis of variance was conducted in Primer7. Preliminary results indicate that differentiation exists between the microbiome of terrestrial and aquatic salamanders. In addition, 81% of inhibitory isolates occurred on Plethodon glutinosus salamanders, and Gram-positive bacilli accounted for 38% of inhibitory isolates. During future work, we will compare our findings to the high-throughput sequencing dataset to confirm if these patterns hold true.  We hope our work will pinpoint the best candidate probiotics and help establish a procedure for treating amphibians infected with Bd