Addition of antifungal skin bacteria to salamanders ameliorates the effects of chytridiomycosis.

Chytridiomycosis, caused by the skin fungus Batrachochytrium dendrobatidis (Bd), has caused population declines of many amphibians in remote protected habitats. Progress has been made in understanding the pathogen's life cycle, documenting its devastating effects on individual amphibians and on populations, and understanding how and why disease outbreaks occur. No research has directly addressed the critical question of how to prevent declines and extinctions caused by outbreaks of the disease. We have identified a number of bacterial species of amphibian skin that inhibit Bd in vitro. Here, we demonstrate that a species of anti-Bd skin bacteria can be successfully added to skins of salamanders Plethodon cinereus, and that addition of this bacterium reduced the severity of a disease symptom in experimentally infected individuals. This is the first demonstration that manipulating the natural skin microbiota of an amphibian species can alter the pathogen's negative effects on infected amphibians and appears to be the first demonstration that an epibiotic manipulation of any wildlife species can lessen the effects of an emerging infectious disease. It suggests that probiotic or bio-augmentation manipulations of cutaneous microbiota could have the potential to reduce susceptibility of amphibians to the disease in nature. This is the first approach suggested that could slow or halt epidemic outbreaks and allow successful reintroductions of amphibian species that have become locally or globally extinct in the wild. Our results also suggest a mechanism for the association of climate change and the likelihood of chytridiomycosis outbreaks via the effects of the former on antifungal bacterial communities.

Antifungal skin bacteria, embryonic survival, and communal nesting in four-toed salamanders, Hemidactylium scutatum.

We examined a novel hypothesis for the maintenance of communal nesting in the salamander, Hemidactylium scutatum, namely that communal nests are more likely than solitary nests to be associated with cutaneous antifungal bacteria, which can inhibit fungal infections of embryos. A communal nest contains eggs of two or more females of the same species. The nesting behavior of H. scutatum females and survival of embryos were determined by frequent nest surveys at three ponds. For communal nests, embryonic survival tended to be higher and catastrophic nest failure was lower. Pure bacterial cultures of resident species were obtained from the salamanders' skins by swabbing and tested against a fungal pathogen of embryos (Mariannaea sp.) in laboratory assays. We found that 27% of females had skin bacteria inhibitory to Mariannaea sp. Communal nests were more likely to have at least one female with antifungal bacteria than were solitary nests. Using a culture-independent assay (denaturing gradient gel electrophoresis of 16S rRNA gene fragments), we found that bacterial species on females and embryos were more similar to each other than they were to bacterial species found in soil within the nest, suggesting that females transmitted skin bacteria to embryos. The presence of anti-Mariannaea skin bacteria identified from the laboratory assays did not prevent fungal presence in field nests. However, once a nest was visibly infected with fungi, presence of anti-Mariannaea bacteria was positively correlated with survival of embryos. Microbe transmission is usually thought to be a cost of group living, but communal nesting in H. scutatum may facilitate the transmission of antifungal bacteria to embryos.

Diversity of cutaneous bacteria with antifungal activity isolated from female four-toed salamanders.

Among the microbiota of amphibian skin are bacteria that produce antifungal compounds. We isolated cutaneous bacteria from the skins of three populations of the nest-attending plethodontid salamander Hemidactylium scutatum and subsequently tested the bacterial isolates against two different fungi (related to Mariannaea elegans and Rhizomucor variabilis) that were obtained from dead salamander eggs. The culturable antifungal bacteria were phylogenetically characterized based on 16S rRNA phylogeny, and belonged to four phyla, comprising 14 bacterial families, 16 genera and 48 species. We found that about half of the antifungal bacterial genera and families were shared with a related salamander species, but there was virtually no overlap at the species level. The proportion of culturable antifungal bacterial taxa shared between two large populations of H. scutatum was the same as the proportion of taxa shared between H. scutatum and Plethodon cinereus, suggesting that populations within a species have unique antifungal bacterial species. Approximately 30% of individuals from both salamander species carried anti-M. elegans cutaneous bacteria and almost 90% of P. cinereus and 100% of H. scutatum salamanders carried anti-R. variabilis cutaneous bacteria. A culture independent method (PCR/DGGE) revealed a shared resident bacterial community of about 25% of the entire resident bacterial community within and among populations of H. scutatum. Thus, the culturable antifungal microbiota was far more variable on salamander skins than was the bacterial microbiota detected by PCR/DGGE. The resident cutaneous antifungal bacteria may play an important role in amphibians' innate defense against pathogens, including the lethal chytrid fungus Batrachochytrium dendrobatidis.