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

Preparing for a Bsal invasion into North America has improved multi-sector readiness

Western palearctic salamander susceptibility to the skin disease caused by the amphibian chytrid fungus Batrachochytrium salamandrivorans (Bsal) was recognized in 2014, eliciting concerns for a potential novel wave of amphibian declines following the B. dendrobatidis (Bd) chytridiomycosis global pandemic. Although Bsal had not been detected in North America, initial experimental trials supported the heightened susceptibility of caudate amphibians to Bsal chytridiomycosis, recognizing the critical threat this pathogen poses to the North American salamander biodiversity hotspot. Here, we take stock of 10 years of research, collaboration, engagement, and outreach by the North American Bsal Task Force. We summarize main knowledge and conservation actions to both forestall and respond to Bsal invasion into North America. We address the questions: what have we learned; what are current challenges; and are we ready for a more effective reaction to Bsal’s eventual detection? We expect that the many contributions to preemptive planning accrued over the past decade will pay dividends in amphibian conservation effectiveness and can inform future responses to other novel wildlife diseases and extreme threats

The Salamander Bacterial Microbiome: Composition, Ecology, and Anti-Chytrid Utility

The chytrid fungi Batrachochytrium dendrobatidis and B. salamandrivorans recently emerged as epizootic pathogens with rapidly expanding geographic and host ranges, and they have caused mass mortality events in amphibian populations on five continents. Bioaugmentation of antifungal bacteria on the skin of amphibians to curb growth of the pathogens and mitigate disease has been attempted; however, few if any bioaugmentation-based treatments have been effective. This project aimed to pinpoint the best candidates for bioaugmentation by describing the structures of both the entire skin-bacterial community and the antifungal, culturable fraction of the skin-bacterial community of plethodontid salamanders. The entire bacterial community was characterized from skin swab samples (n = 249) using high-throughput sequencing of the 16S rRNA gene. Differences in community structures across three genera of plethodontid salamanders, three ecoregions of Tennessee, and two seasons were assessed. Core members of the communities were identified using indicator species analysis, core community analysis, and ecological interaction networks. The culturable, anti-chytrid fraction of the bacterial community was described using traditional microbiological techniques, Sanger sequencing of the 16S rRNA gene of isolates (n = 104), and co-culture challenge assays with a strain of B. dendrobatidis. Candidates for bioaugmentation-based treatments were evaluated based on 1) amenability to isolation from skin swabs, 2) ubiquity in skin communities of a broad range of salamander hosts, and 3) consistency in inhibition of B. dendrobatidis across multiple bacterial strains. Lastly, surveys for the presence of B. salamandrivorans in Appalachian plethodontid salamander populations were conducted at ten sites in Tennessee

*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