Analysis and Exploration of Novel Antibiotic-Producing Streptomyces spp. in Spokane County, Washington

According to the Centers for Disease Control and Prevention, a US citizen is infected by an antibiotic-resistant pathogen every 11 seconds, and every 15 minutes, a patient dies as a result of these infections. Due to the increasing incidence of antibiotic-resistant pathogenic microbes, the study and exploration of novel antibiotics from novel environments are imperative as infectious diseases are the second leading cause of death in the United States. The purpose of this research is to investigate and analyze antibiotic-producing soil microbes in Spokane County, WA, with hopes of discovering novel antibiotic-producing microbes, specifically Streptomyces species, and explore some of the variables that influence the production of secondary metabolites. My hypotheses are as follows: Soil microbes existing in Spokane County will include Streptomyces spp. capable of producing secondary metabolites suitable to combat selected Gram-negative or Gram-positive bacterial ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and pathogenic fungi such as Candida albicans. Additionally, modifying laboratory variables such as incubation temperature, time in incubation, and the type of media will influence the production of metabolites produced by Streptomyces isolates. Modifying these variables will impact the inhibitory capabilities of these isolates against Gram-negative, Gram-positive, and pathogenic fungal microbes. Cell-free supernatants of secondary metabolites on disk diffusion and 96 well plate assays will be utilized to measure zones of inhibition and inhibitory capabilities with absorbance measured at 600nm using a spectrophotometer

Geography, Climate, and Habitat Shape the Microbiome of the Endangered Rock Gnome Lichen (Cetradonia linearis)

Bacterial symbionts are essential components of healthy biological systems. They are increasingly recognized as important factors in the study and management of threatened species and ecosystems. Despite management shifts at the ecosystem level, microbial communities are often neglected in discussions of holobiont conservation in favor of the primary members of a symbiosis. In this study, we addressed the bacterial community knowledge gap for one of two federally endangered lichen species in the United States, Cetradonia linearis (Cladoniaceae). We collected 28 samples of the endangered rock gnome lichen (Cetradonia linearis) from 13 sites and characterized bacterial communities in thalli using 16S rRNA metabarcoding to investigate the factors influencing the microbiome composition and diversity within the thallus. We found that Proteobacteria (37.8% ± 10.3) and Acidobacteria (25.9% ± 6.0) were the most abundant phyla recovered. Cyanobacteria were a major component of the microbiome in some individuals, despite this species associating with a green algal symbiont. Habitat, climate, and geography were all found to have significant influences on bacterial community composition. An analysis of the core microbiome at a 90% threshold revealed shared amplicon sequence variants in the microbiomes of other lichens in the family Cladoniaceae. We concluded that the bacterial microbiome of Cetradonia linearis is influenced by environmental factors and that some bacterial taxa may be core to this group. Further exploration into the microbiomes of rare lichen species is needed to understand the importance of bacterial symbionts to lichen diversity and distributions

Genetic variability and ontogeny predict microbiome structure in a disease-challenged montane amphibian

Amphibian populations worldwide are at risk of extinction from infectious diseases, including chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Amphibian cutaneous microbiomes interact with Bd and can confer protective benefits to the host. The composition of the microbiome itself is influenced by many environment- and host-related factors. However, little is known about the interacting effects of host population structure, genetic variation and developmental stage on microbiome composition and Bd prevalence across multiple sites. Here we explore these questions in Amietia hymenopus, a disease-affected frog in southern Africa. We use microsatellite genotyping and 16S amplicon sequencing to show that the microbiome associated with tadpole mouthparts is structured spatially, and is influenced by host genotype and developmental stage. We observed strong genetic structure in host populations based on rivers and geographic distances, but this did not correspond to spatial patterns in microbiome composition. These results indicate that demographic and host genetic factors affect microbiome composition within sites, but different factors are responsible for host population structure and microbiome structure at the between-site level. Our results help to elucidate complex within- and among- population drivers of microbiome structure in amphibian populations. That there is a genetic basis to microbiome composition in amphibians could help to inform amphibian conservation efforts against infectious diseases

Tetrodotoxin, fungal pathogen infection, and bacterial microbiome associations are variable in the skin microecosystems of two Taricha newt species

A diverse metabolome exists on amphibian skin that mediates interactions between hosts and skin microbiomes. Tetrodotoxin is one such metabolite that occurs across a variety of taxa, and is particularly well studied in newts of the genus Taricha that are susceptible to infection with chytrid fungi. The interaction of tetrodotoxin with the skin microbiome, including pathogenic fungi, is not well understood, and here we describe these patterns across 12 populations of Taricha granulosa and T. torosa in Washington, Oregon, and California. We found no correlation of TTX and Batrachochytrium dendrobatidis (Bd) infection in either T. granulosa or T. torosa, a pattern inconsistent with a previous study. In addition, TTX, but not Bd, was significantly correlated with the skin microbiome composition in T. granulosa. In T. torosa, however, Bd, but not TTX, was correlated with the skin microbiome structure. The relationship between TTX and skin microbiome composition differed between species, with significant correlations observed only in T. granulosa, which exhibited higher TTX concentrations. We also detected significantly higher abundances of bacterial taxa (e.g., Pseudomonadaceae) associated with TTX production in newts with higher skin TTX. These taxa (ASVs matching Aeromonas, Pseudomonas, Shewanella, and Sphingopyxis) were associated with all body sites of previously sampled T. granulosa, but not found in soil samples. Our results suggest that toxins can shape the newt skin microbiome and may influence pathogen infection through indirect mechanisms, as TTX showed no direct inhibition of Bd or B. salamandrivorans growth

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

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

Harnessing the Microbiome to Prevent Fungal Infections: Lessons from Amphibians

All multicellular organisms are host to microbial symbionts that constitute the microbiome and can have significant impacts on the host, including altering development, behavior, and health [1]. In turn, aspects of the host and their environment can influence the microbiome [2]. Here, we briefly summarize current knowledge of the amphibian skin microbiome and its role in heath and disease. Given the increase in fungal diseases that now threaten amphibians and other wildlife—including bees, bats, snakes, and corals, as well as a variety of economically important crops [3]—we hope that lessons learned from amphibian host–microbe interactions can also ultimately be applied in other systems (Fig 1).Funding was provided by the National Science Foundation (DEB-1136640). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Impact of Chytrid Fungus Pathogen on Skin Microbiome of Columbia Spotted Frogs in Northern Idaho

Chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), is associated with an estimated 501 population declines and 90 extinctions of amphibian species worldwide, the greatest documented loss of biodiversity attributed to a disease. Research on the amphibian skin microbiome may provide solutions to conservation of amphibian species by bettering our understanding of 1) Bd’s effect on the skin’s microbial community composition and 2) the effects of microbial community composition in protection against Bd. Our goal was to investigate differences in microbiome composition between infected and uninfected frogs. DNA samples from skins of 399 Columbia spotted frogs (Rana luteiventris) were obtained by the Idaho Department of Fish and Game in 2013-2014. Frogs were sampled from a total of 153 wetlands in northern Idaho, with Bd being detected on frogs in 80% (123) of the sampled wetlands. Of the 399 spotted frogs tested for Bd presence, 65% (261 frogs) tested positive. The average infection intensity was low, with zoospore equivalents ranging from 0 to 98.8 (mean = 0.93). The skin microbiomes of 92 frogs (47 infected and 45 uninfected) were characterized using amplicon barcoded sequencing of the V4-V5 region of the 16S rRNA gene. Infected and uninfected frogs had distinct microbiomes (p = .024, pseudo-F = 2.07, PERMANOVA, weighted UniFrac). A member of the order Burkholderiales, known to dominate the microbiome of highly infected frogs, increased in relative abundance in infected frogs. Pseudomonas spp., a known inhibitor of Bd, had greater relative abundance in uninfected frogs, potentially improving defenses against the disease

Classic Hoarding Cages Increase Gut Bacterial Abundance and Reduce the Individual Immune Response of Honey Bee (<i>Apis mellifera</i>) Workers

Abstract Laboratory experiments have advanced our understanding of honey bee (Apis mellifera) responses to environmental factors, but removal from the hive environment may also impact physiology. To examine whether the laboratory environment alters the honey bee gut bacterial community and immune responses, we compared bacterial community structure (based on amplicon sequence variant relative abundance), total bacterial abundance, and immune enzyme (phenoloxidase and glucose oxidase) activity of cohort honey bee workers kept under laboratory and hive conditions. Workers housed in the laboratory showed differences in the relative abundance of their core gut taxa, an increase in total gut bacterial abundance, and reduced phenoloxidase activity, compared to bees housed in hives.</jats:p

Journal of Insect Science

Laboratory experiments have advanced our understanding of honey bee (Apis mellifera) responses to environmental factors, but removal from the hive environment may also impact physiology. To examine whether the laboratory environment alters the honey bee gut bacterial community and immune responses, we compared bacterial community structure (based on amplicon sequence variant relative abundance), total bacterial abundance, and immune enzyme (phenoloxidase and glucose oxidase) activity of cohort honey bee workers kept under laboratory and hive conditions. Workers housed in the laboratory showed differences in the relative abundance of their core gut taxa, an increase in total gut bacterial abundance, and reduced phenoloxidase activity, compared to bees housed in hives.Virginia Agricultural Foundation [677]Published versionWe would like to thank Philene Vu for performing the immunological assays, and Hannah Miko for assisting with lab and field work. This work was supported by the Virginia Agricultural Foundation (Project number 677). We thank Virginia Tech's Open Access Subvention Fund for assistance with open access fees