Myra C. Hughey

Antifungal isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens

Microbial symbionts of vertebrate skin have an important function in defense of the host against pathogens. In particular, the emerging chytrid fungus Batrachochytrium dendrobatidis, causes widespread disease in amphibians but can be inhibited via secondary metabolites produced by many different skin-associated bacteria. Similarly, the fungal pathogens of terrestrial salamander eggs Mariannaea elegans and Rhizomucor variabilis are also inhibited by a variety of skin-associated bacteria. Indeed, probiotic therapy against fungal diseases is a recent approach in conservation medicine with growing experimental support. We present a comprehensive Antifungal Isolates Database of amphibian skin-associated bacteria that have been cultured, isolated, and tested for antifungal properties. At the start, this database includes nearly 2000 cultured bacterial isolates from 37 amphibian host species across 18 studies on five continents: Africa, Oceania, Europe, and North and South America. As the research community gathers information on additional isolates, the database will be updated periodically. The resulting database can serve as a conservation tool for amphibians and other organisms, and provides empirical data for comparative and bioinformatic studies. The database consists of a FASTA file containing 16S rRNA gene sequences of the bacterial isolates, and a metadata file containing information on the host species, life-stage, geographic region, and antifungal capacity and taxonomic identity of the isolate.

Skin bacterial diversity of Panamanian frogs is associated with host susceptibility and presence of Batrachochytrium dendrobatidis

Symbiotic bacteria on amphibian skin can inhibit growth of the fungus Batrachochytrium dendrobatidis (Bd) that has caused dramatic population declines and extinctions of amphibians in the Neotropics. It remains unclear how the amphibians’ skin microbiota is influenced by environmental bacterial reservoirs, host-associated factors such as susceptibility to pathogens, and pathogen presence in tropical amphibians. We sampled skin bacteria from five co-occurring frog species that differ in Bd susceptibility at one Bd-naive site, and sampled one of the non-susceptible species from Bd-endemic and Bd-naive sites in Panama. We hypothesized that skin bacterial communities (1) would be distinct from the surrounding environment regardless of the host habitat, (2) would differ between Bd susceptible and non-susceptible species and (3) would differ on hosts in Bd-naive and Bd-endemic sites. We found that skin bacterial communities were enriched in bacterial taxa that had low relative abundances in the environment. Non-susceptible species had very similar skin bacterial communities that were enriched in particular taxa such as the genera Pseudomonas and Acinetobacter. Bacterial communities of Craugastor fitzingeri in Bd-endemic sites were less diverse than in the naive site, and differences in community structure across sites were explained by changes in relative abundance of specific bacterial taxa. Our results indicate that skin microbial structure was associated with host susceptibility to Bd and might be associated to the history of Bd presence at different sites.

More than skin deep: functional genomic basis for resistance to amphibian chytridiomycosis.

The amphibian-killing chytrid fungus Batrachochytrium dendrobatidis (Bd) is one of the most generalist pathogens known, capable of infecting hundreds of species globally and causing widespread population declines and extinctions. However, some host species are seemingly unaffected by Bd, tolerating or clearing infections without clinical signs of disease. Variation in host immune responses is commonly evoked for these resistant or tolerant species, yet to date, we have no direct comparison of amphibian species responses to infection at the level of gene expression. In this study, we challenged four Central American frog species that vary in Bd susceptibility, with a sympatric virulent strain of the pathogen. We compared skin and spleen orthologous gene expression using differential expression tests and coexpression gene network analyses. We found that resistant species have reduced skin inflammatory responses and increased expression of genes involved in skin integrity. In contrast, only highly susceptible species exhibited suppression of splenic T-cell genes. We conclude that resistance to chytridiomycosis may be related to a species’ ability to escape the immunosuppressive activity of the fungus. Moreover, our results indicate that within-species differences in splenic proteolytic enzyme gene expression may contribute to intraspecific variation in survival. This first comparison of amphibian functional immunogenomic architecture in response to Bd provides insights into key genetic mechanisms underlying variation in disease outcomes among amphibian species.

Using "Omics" and Integrated Multi-Omics Approaches to Guide Probiotic Selection to Mitigate Chytridiomycosis and Other Emerging Infectious Diseases

Emerging infectious diseases in wildlife are responsible for massive population declines. In amphibians, chytridiomycosis caused by Batrachochytrium dendrobatidis, Bd, has severely affected many amphibian populations and species around the world. One promising management strategy is probiotic bioaugmentation of antifungal bacteria on amphibian skin. In vivo experimental trials using bioaugmentation strategies have had mixed results, and therefore a more informed strategy is needed to select successful probiotic candidates. Metagenomic, transcriptomic, and metabolomic methods, colloquially called “omics,” are approaches that can better inform probiotic selection and optimize selection protocols. The integration of multiple omic data using bioinformatic and statistical tools and in silico models that link bacterial community structure with bacterial defensive function can allow the identification of species involved in pathogen inhibition. We recommend using 16S rRNA gene amplicon sequencing and methods such as indicator species analysis, the Kolmogorov–Smirnov Measure, and co-occurrence networks to identify bacteria that are associated with pathogen resistance in field surveys and experimental trials. In addition to 16S amplicon sequencing, we recommend approaches that give insight into symbiont function such as shotgun metagenomics, metatranscriptomics, or metabolomics to maximize the probability of finding effective probiotic candidates, which can then be isolated in culture and tested in persistence and clinical trials. An effective mitigation strategy to ameliorate chytridiomycosis and other emerging infectious diseases is necessary; the advancement of omic methods and the integration of multiple omic data provide a promising avenue toward conservation of imperiled species.

Panamanian frog species host unique skin bacterial communities

Vertebrates, including amphibians, host diverse symbiotic microbes that contribute to host disease resistance. Globally, and especially in montane tropical systems, many amphibian species are threatened by a chytrid fungus, Batrachochytrium dendrobatidis (Bd), that causes a lethal skin disease. Bd therefore may be a strong selective agent on the diversity and function of the microbial communities inhabiting amphibian skin. In Panamá, amphibian population declines and the spread of Bd have been tracked. In 2012, we completed a field survey in Panamá to examine frog skin microbiota in the context of Bd infection. We focused on three frog species and collected two skin swabs per frog from a total of 136 frogs across four sites that varied from west to east in the time since Bd arrival. One swab was used to assess bacterial community structure using 16S rRNA amplicon sequencing and to determine Bd infection status, and one was used to assess metabolite diversity, as the bacterial production of anti-fungal metabolites is an important disease resistance function. The skin microbiota of the three Panamanian frog species differed in OTU (operational taxonomic unit, ~bacterial species) community composition and metabolite profiles, although the pattern was less strong for the metabolites. Comparisons between frog skin bacterial communities from Panamá and the US suggest broad similarities at the phylum level, but key differences at lower taxonomic levels. In our field survey in Panamá, across all four sites, only 35 individuals (~26%) were Bd infected. There was no clustering of OTUs or metabolite profiles based on Bd infection status and no clear pattern of west-east changes in OTUs or metabolite profiles across the four sites. Overall, our field survey data suggest that different bacterial communities might be producing broadly similar sets of metabolites across frog hosts and sites. Community structure and function may not be as tightly coupled in these skin symbiont microbial systems as it is in many macro-systems.

The Lethal Fungus Batrachochytrium dendrobatidis Is Present in Lowland Tropical Forests of Far Eastern Panamá

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is one of the main causes of amphibian population declines and extinctions all over the world. In the Neotropics, this fungal disease has caused catastrophic declines in the highlands as it has spread throughout Central America down to Panamá. In this study, we determined the prevalence and intensity of Bd infection in three species of frogs in one highland and four lowland tropical forests, including two lowland regions in eastern Panamá in which the pathogen had not been detected previously. Bd was present in all the sites sampled with a prevalence ranging from 15–34%, similar to other Neotropical lowland sites. The intensity of Bd infection on individual frogs was low, ranging from average values of 0.11–24 zoospore equivalents per site. Our work indicates that Bd is present in anuran communities in lowland Panamá, including the Darién province, and that the intensity of the infection may vary among species from different habitats and with different life histories. The population-level consequences of Bd infection in amphibian communities from the lowlands remain to be determined. Detailed studies of amphibian species from the lowlands will be essential to determine the reason why these species are persisting despite the presence of the pathogen.

Most of the dominant members of amphibian skin bacterial communities can be readily cultured

ABSTRACT Currently, it is estimated that only 0.001% to 15% of bacteria in any given system can be cultured by use of commonly used techniques and media, yet culturing is critically important for investigations of bacterial function. Despite this situation, few studies have attempted to link culture-dependent and culture-independent data for a single system to better understand which members of the microbial community are readily cultured. In amphibians, some cutaneous bacterial symbionts can inhibit establishment and growth of the fungal pathogen Batrachochytrium dendrobatidis, and thus there is great interest in using these symbionts as probiotics for the conservation of amphibians threatened by B. dendrobatidis. The present study examined the portion of the culture-independent bacterial community (based on Illumina amplicon sequencing of the 16S rRNA gene) that was cultured with R2A low-nutrient agar and whether the cultured bacteria represented rare or dominant members of the community in the following four amphibian species: bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), spring peepers (Pseudacris crucifer), and American toads (Anaxyrus americanus). To determine which percentage of the community was cultured, we clustered Illumina sequences at 97% similarity, using the culture sequences as a reference database. For each amphibian species, we cultured, on average, 0.59% to 1.12% of each individuals bacterial community. However, the average percentage of bacteria that were culturable for each amphibian species was higher, with averages ranging from 2.81% to 7.47%. Furthermore, most of the dominant operational taxonomic units (OTUs), families, and phyla were represented in our cultures. These results open up new research avenues for understanding the functional roles of these dominant bacteria in host health.

Variation in Metabolite Profiles of Amphibian Skin Bacterial Communities Across Elevations in the Neotropics

Both the structure and function of host-associated microbial communities are potentially impacted by environmental conditions, just as the outcomes of many free-living species interactions are context-dependent. Many amphibian populations have declined around the globe due to the fungal skin pathogen, Batrachochytrium dendrobatidis (Bd), but enivronmental conditions may influence disease dynamics. For instance, in Panamá, the most severe Bd outbreaks have occurred at high elevation sites. Some amphibian species harbor bacterial skin communities that can inhibit the growth of Bd, and therefore, there is interest in understanding whether environmental context could also alter these host-associated microbial communities in a way that might ultimately impact Bd dynamics. In a field survey in Panamá, we assessed skin bacterial communities (16S rRNA amplicon sequencing) and metabolite profiles (HPLC-UV/Vis) of Silverstoneia flotator from three high- and three low-elevation populations representing a range of environmental conditions. Across elevations, frogs had similar skin bacterial communities, although one lowland site appeared to differ. Interestingly, we found that bacterial richness decreased from west to east, coinciding with the direction of Bd spread through Panamá. Moreover, metabolite profiles suggested potential functional variation among frog populations and between elevations. While the frogs have similar bacterial community structure, the local environment might shape the metabolite profiles. Ultimately, host-associated community structure and function could be dependent on environmental conditions, which could ultimately influence host disease susceptibility across sites.

Short-Term Exposure to Coal Combustion Waste Has Little Impact on the Skin Microbiome of Adult Spring Peepers (Pseudacris crucifer)

ABSTRACT Disruptions to the microbiome can impact host health as can exposure to environmental contaminants. However, few studies have addressed how environmental contaminants impact the microbiome. We explored this question for frogs that breed in wetlands contaminated with fly ash, a by-product of coal combustion that is enriched in trace elements. We found differences in the bacterial communities among a fly ash-contaminated site and several reference wetlands. We then experimentally assessed the impacts of fly ash on the skin microbiome of adult spring peepers (Pseudacris crucifer). Frogs were exposed to fly ash in the laboratory for 12 h, the duration of a typical breeding event, and the skin microbiome was assessed after 5 days (experiment 1) or after 5 and 15 days (experiment 2). We examined bacterial community structure using 16S rRNA gene amplicon sequencing and metabolite profiles using high-pressure liquid chromatography-mass spectrometry (HPLC-MS). We found little impact as the result of acute exposure to fly ash on the bacterial communities or metabolite profiles in either experiment, suggesting that the bacterial symbiont communities of adults may be relatively resistant to brief contaminant exposure. However, housing frogs in the laboratory altered bacterial community structure in the two experiments, which supports prior research suggesting that environmental source pools are important for maintaining the amphibian skin microbiome. Therefore, for contaminants like fly ash that may alter the potential source pool of symbionts, we think it may be important to explore how contaminants affect the initial assembly of the amphibian skin microbiome in larval amphibians that develop within contaminated sites. IMPORTANCE Animals are hosts to many symbiotic microorganisms, collectively called the microbiome, that play critical roles in host health. Therefore, environmental contaminants that alter the microbiome may impact hosts. Some of the most widespread contaminants, produced worldwide, are derived from the mining, storage, and combustion of coal for energy. Fly ash, for example, is a by-product of coal combustion. It contains compounds such as arsenic, selenium, cadmium, and strontium and is a recognized source of ground and surface water contamination. Here, we experimentally investigated the impacts of short-term fly ash exposure on the skin microbiome of spring peepers, one of many species of amphibian that sometimes breed in open fly ash disposal ponds. This research provides a look into the potential impacts of fly ash on an animals microbiome and suggests important future directions for research on the effects of environmental contaminants on the microbiome.

Skin bacterial microbiome of a generalist Puerto Rican frog varies along elevation and land use gradients

Host-associated microbial communities are ubiquitous among animals, and serve important functions. For example, the bacterial skin microbiome of amphibians can play a role in preventing or reducing infection by the amphibian chytrid fungus, Batrachochytrium dendrobatidis. Evidence suggests that environmental bacteria likely serve as a source pool for at least some of the members of the amphibian skin bacterial community, underscoring the potential for local environmental changes to disrupt microbial community source pools that could be critical to the health of host organisms. However, few studies have assessed variation in the amphibian skin microbiome along clear environmental gradients, and so we know relatively little about how local environmental conditions influence microbiome diversity. We sampled the skin bacterial communities of Coqui frogs, Eleutherodactylus coqui (N = 77), along an elevational gradient in eastern Puerto Rico (0–875 m), with transects in two land use types: intact forest (N = 4 sites) and disturbed (N = 3 sites) forest. We found that alpha diversity (as assessed by Shannon, Simpson, and Phylogenetic Diversity indices) varied across sites, but this variation was not correlated with elevation or land use. Beta diversity (community structure), on the other hand, varied with site, elevation and land use, primarily due to changes in the relative abundance of certain bacterial OTUs (∼species) within these communities. Importantly, although microbiome diversity varied, E. coqui maintained a common core microbiota across all sites. Thus, our findings suggest that environmental conditions can influence the composition of the skin microbiome of terrestrial amphibians, but that some aspects of the microbiome remain consistent despite environmental variation.