The zebrafish gut microbiome influences benzo[a]pyrene developmental neurobehavioral toxicity.

Early-life exposure to environmental toxicants like Benzo[a]pyrene (BaP) is associated with several health consequences in vertebrates (i.e., impaired or altered neurophysiological and behavioral development). Although toxicant impacts were initially studied relative to host physiology, recent studies suggest that the gut microbiome is a possible target and/or mediator of behavioral responses to chemical exposure in organisms, via the gut-brain axis. However, the connection between BaP exposure, gut microbiota, and developmental neurotoxicity remains understudied. Using a zebrafish model, we determined whether the gut microbiome influences BaP impacts on behavior development. Embryonic zebrafish were treated with increasing concentrations of BaP and allowed to grow to the larval life stage, during which they underwent behavioral testing and intestinal dissection for gut microbiome profiling via high-throughput sequencing. We found that exposure affected larval zebrafish microbiome diversity and composition in a manner tied to behavioral development: increasing concentrations of BaP were associated with increased taxonomic diversity, exposure was associated with unweighted UniFrac distance, and microbiome diversity and exposure predicted larval behavior. Further, a gnotobiotic zebrafish experiment clarified whether microbiome presence was associated with BaP exposure response and behavioral changes. We found that gut microbiome state altered the relationship between BaP exposure concentration and behavioral response. These results support the idea that the zebrafish gut microbiome is a determinant of the developmental neurotoxicity that results from chemical exposure.

Together or Alone: Evaluating the Pathogen Inhibition Potential of Bacterial Cocktails against an Amphibian Pathogen.

The amphibian fungal skin disease Batrachochytrium dendrobatidis (Bd) has caused major biodiversity losses globally. Several experimental trials have tested the use of Janthinobacterium lividum to reduce mortality due to Bd infections, usually in single-strain amendments. It is well-characterized in terms of its anti-Bd activity mechanisms. However, there are many other microbes that inhibit Bd in vitro, and not all experiments have demonstrated consistent results with J. lividum. We used a series of in vitro assays involving bacterial coculture with Bd lawns, bacterial growth tests in liquid broth, and Bd grown in bacterial cell-free supernatant (CFS) to determine: (i) which skin bacteria isolated from a locally endangered amphibian, namely, the Colorado boreal toad (Anaxyrus boreas boreas), are able to inhibit Bd growth; (ii) whether multistrain combinations are more effective than single-strains; and (iii) the mechanism behind microbe-microbe interactions. Our results indicate that there are some single strain and multistrain probiotics (especially including strains from Pseudomonas, Chryseobacterium, and Microbacterium) that are potentially more Bd-inhibitive than is J. lividum alone and that some combinations may lead to a loss of inhibition, potentially through antagonistic metabolite effects. Additionally, if J. lividum continues being developed as a wild boreal toad probiotic, we should investigate it in combination with Curvibacter CW54D, as they inhibited Bd additively and grew at a higher rate when combined than did either alone. This highlights the fact that combinations of probiotics function in variable and unpredictable ways as well as the importance of considering the potential for interactions among naturally resident host microbiota and probiotic additions. IMPORTANCE Batrachochytrium dendrobatidis (Bd) is a pathogen that infects amphibians globally and is causing a biodiversity crisis. Our research group studies one of the species affected by Bd, namely, the Colorado boreal toad (Anaxyrus boreas boreas). Many researchers focus their studies on one probiotic bacterial isolate called Janthinobacterium lividum, which slows Bd growth in lab cultures and is currently being field tested in Colorado boreal toads. Although promising, J. lividum is not consistently effective across all amphibian individuals or species. For Colorado boreal toads, we addressed whether there are other bacterial strains that also inhibit Bd (potentially better than does J. lividum) and whether we can create two-strain probiotics that function better than do single-strain probiotics. In addition, we evaluate which types of interactions occur between two-strain combinations and what these results mean in the context of adding a probiotic to an existing amphibian skin microbiome.

Defining the environmental determinants of dysbiosis at scale with zebrafish.

The gut microbiome, critical to maintaining vertebrate homeostasis, is susceptible to a various exposures. In some cases, these exposures induce dysbiosis, wherein the microbiome changes into a state conducive to disease progression. To better prevent, manage, and treat health disorders, we need to define which exposures induce dysbiosis. Contemporary methods face challenges due to the immense diversity of the exposome and the restricted throughput of conventional experimental tools used for dysbiosis evaluation. We propose integrating high-throughput model systems as an augment to traditional techniques for rapid identification of dysbiosis-inducing agents. Although high-throughput screening tools revolutionized areas such as pharmacology and toxicology, their incorporation in gut microbiome research remains limited. One particularly powerful high-throughput model system is the zebrafish, which affords access to scalable in vivo experimentation involving a complex gut microbiome. Numerous studies have employed this model to identify potential dysbiosis triggers. However, its potential could be further harnessed via innovative study designs, such as evaluation of synergistic effects from combined exposures, expansions to the methodological toolkit to discern causal effects of microbiota, and efforts to assess and improve the translational relevance of the model. Ultimately, this burgeoning experimental resource can accelerate the discovery of agents that underlie dysbiotic disorders.

Mr. Toad's Wild Fungi: Fungal Isolate Diversity on Colorado Boreal Toads and their Capacity for Pathogen Inhibition.

The amphibian skin pathogen Batrachochytrium dendrobatidis (Bd) has caused an ongoing biodiversity crisis, including in the locally endangered Colorado boreal toad (Anaxyrus boreas boreas). Although researchers have investigated the bacteria living on amphibian skin and how they interact with Bd, there is less information about fungal community members. This study describes (1) the diversity of culturable fungi from boreal toad skin, (2) which subset of these isolates is Bd-inhibitory, and (3) how Bd affects these isolates' growth and morphology. Most isolates were from the orders Capnodiales, Helotiales, and Pleosporales. Of 16 isolates tested for Bd-inhibition, two from the genus Neobulgaria and three from Pseudeurotium inhibited Bd. Fungal growth in co-culture with Bd varied with weak statistical support for Neobulgaria sp. (isolate BTF_36) and cf Psychrophila (isolate BTF_60) (p-values = 0.076 and 0.092, respectively). Fungal morphology remained unchanged in co-culture with Bd, however, these results could be attributed to low replication per isolate. Nonetheless, two fungal isolates' growth may have been affected by Bd, implying that fungal growth changes in Bd co-culture could be a variable worth measuring in the future (with higher replication). These findings add to the sparse but growing literature on amphibian-associated fungi and suggest further study may uncover the relevance of fungi to amphibian health and Bd infection.

A Metagenomic Investigation of Spatial and Temporal Changes in Sewage Microbiomes across a University Campus.

Wastewater microbial communities are not static and can vary significantly across time and space, but this variation and the factors driving the observed spatiotemporal variation often remain undetermined. We used a shotgun metagenomic approach to investigate changes in wastewater microbial communities across 17 locations in a sewer network, with samples collected from each location over a 3-week period. Fecal material-derived bacteria constituted a relatively small fraction of the taxa found in the collected samples, highlighting the importance of environmental sources to the sewage microbiome. The prokaryotic communities were highly variable in composition depending on the location within the sampling network, and this spatial variation was most strongly associated with location-specific differences in sewage pH. However, we also observed substantial temporal variation in the composition of the prokaryotic communities at individual locations. This temporal variation was asynchronous across sampling locations, emphasizing the importance of independently considering both spatial and temporal variation when assessing the wastewater microbiome. The spatiotemporal patterns in viral community composition closely tracked those of the prokaryotic communities, allowing us to putatively identify the bacterial hosts of some of the dominant viruses in these systems. Finally, we found that antibiotic resistance gene profiles also exhibit a high degree of spatiotemporal variability, with most of these genes unlikely to be derived from fecal bacteria. Together, these results emphasize the dynamic nature of the wastewater microbiome, the challenges associated with studying these systems, and the utility of metagenomic approaches for building a multifaceted understanding of these microbial communities and their functional attributes. IMPORTANCE Sewage systems harbor extensive microbial diversity, including microbes derived from both human and environmental sources. Studies of the sewage microbiome are useful for monitoring public health and the health of our infrastructure, but the sewage microbiome can be highly variable in ways that are often unresolved. We sequenced DNA recovered from wastewater samples collected over a 3-week period at 17 locations in a single sewer system to determine how these communities vary across time and space. Most of the wastewater bacteria, and the antibiotic resistance genes they harbor, were not derived from human feces, but human usage patterns did impact how the amounts and types of bacteria and bacterial genes we found in these systems varied over time. Likewise, the wastewater communities, including both bacteria and their viruses, varied depending on location within the sewage network, highlighting the challenges and opportunities in efforts to monitor and understand the sewage microbiome.

Bacterial Biofilm Thickness and Fungal Inhibitory Bacterial Richness Both Prevent Establishment of the Amphibian Fungal Pathogen Batrachochytrium dendrobatidis.

Host-associated microbial biofilms can provide protection against pathogen establishment. In many host-microbe symbioses (including, but not limited to humans, plants, insects, and amphibians), there is a correlation between host-associated microbial diversity and pathogen infection risk. Diversity may prevent infection by pathogens through sampling effects and niche complementarity, but an alternative hypothesis may be that microbial biomass is confounded with diversity and that host-associated biofilms are deterring pathogen establishment through space preemption. In this study, we use the amphibian system as a model for host-microbe-pathogen interactions to ask two questions: (i) is bacterial richness confounded with biofilm thickness or cell density, and (ii) to what extent do biofilm thickness, cell density, and bacterial richness each deter the establishment of the amphibian fungal pathogen Batrachochytrium dendrobatidis? To answer these questions, we built a custom biofilm microcosm that mimics the host-environment interface by allowing nutrients to diffuse out of a fine-pore biofilm scaffolding. This created a competitive environment in which bacteria and the fungal pathogen compete for colonization space. We then challenged bacterial biofilms ranging in community richness, biofilm thickness, bacterial cell density, and B. dendrobatidis (also known as Bd)-inhibitory metabolite production with live B. dendrobatidis zoospores to determine how B. dendrobatidis establishment success on membranes varies. We found that biofilm thickness and B. dendrobatidis-inhibitory isolate richness work in complement to reduce B. dendrobatidis establishment success. This work underscores that physical aspects of biofilm communities can play a large role in pathogen inhibition, and in many studies, these traits are not studied. IMPORTANCE Our finding highlights the fact that diversity, as measured through 16S rRNA gene sequencing, may obscure the true mechanisms behind microbe-mediated pathogen defense and that physical space occupation by biofilm-forming symbionts may significantly contribute to pathogen protection. These findings have implications across a wide range of host-microbe systems since 16S rRNA gene sequencing is a standard tool used across many microbial systems. Further, our results are potentially relevant to many host-pathogen systems since host-associated bacterial biofilms are ubiquitous.

Identifying fungal-host associations in an amphibian host system.

Host-associated microbes can interact with macro-organisms in a number of ways that affect host health. Few studies of host-associated microbiomes, however, focus on fungi. In addition, it is difficult to discern whether a fungal organism found in or on an ectotherm host is associating with it in a durable, symbiotic interaction versus a transient one, and to what extent the habitat and host share microbes. We seek to identify these host-microbe interactions on an amphibian, the Colorado boreal toad (Anaxyrus boreas boreas). We sequenced the ITS1 region of the fungal community on the skin of wild toads (n = 124) from four sites in the Colorado Rocky Mountains, across its physiologically dynamic developmental life stages. We also sampled the common habitats used by boreal toads: water from their natal wetland and aquatic pond sediment. We then examined diversity patterns within different life stages, between host and habitat, and identified fungal taxa that could be putatively host-associated with toads by using an indicator species analysis on toad versus environmental samples. Host and habitat were strikingly similar, with the exception of toad eggs. Post-hatching toad life stages were distinct in their various fungal diversity measures. We identified eight fungal taxa that were significantly associated with eggs, but no other fungal taxa were associated with other toad life stages compared with their environmental habitat. This suggests that although pre- and post-metamorphic toad life stages differ from each other, the habitat and host fungal communities are so similar that identifying obligate host symbionts is difficult with the techniques used here. This approach does, however, leverage sequence data from host and habitat samples to predict which microbial taxa are host-associated versus transient microbes, thereby condensing a large set of sequence data into a smaller list of potential targets for further consideration.

Bacterial Community Sequences of Submerged Aquatic Vegetation in the Potomac River.

Here, we report results from PCR and sequencing of bacterial 16S rRNA genes from leaf and root surfaces from nine submerged aquatic vegetation (SAV) samples comprising five species. Samples were from four sites along the Potomac River.

Microbial Community Succession and Nutrient Cycling Responses following Perturbations of Experimental Saltwater Aquaria.

Although aquaria are common features of homes and other buildings, little is known about how environmental perturbations (i.e., tank cleaning, water changes, addition of habitat features) impact the diversity and succession of aquarium microbial communities. In this study, we sought to evaluate the hypotheses that newly established aquaria show clear microbial successional patterns over time and that common marine aquarium-conditioning practices, such as the addition of ocean-derived "live rocks" (defined as any "dead coral skeleton covered with crustose coralline algae" transferred into an aquarium from open ocean habitats) impact the diversity of microbial populations as well as nitrogen cycling in aquaria. We collected water chemistry data alongside water and sediment samples from two independent and newly established saltwater aquaria over a 3-month period. Microbial communities in samples were assessed by DNA extraction, amplification of the 16S rRNA gene, and Illumina MiSeq sequencing. Our results showed clear and replicable patterns of community succession in both aquaria, with the existence of multiple stable states for aquarium microbial assemblages. Notably, our results show that changes in aquarium microbial communities do not always correlate with water chemistry measurements and that operational taxonomic unit (OTU)-level patterns relevant to nitrogen cycling were not reported as statistically significant. Overall, our results demonstrate that aquarium perturbations have a substantial impact on microbial community profiles of aquarium water and sediment and that the addition of live rocks improves nutrient cycling by shifting aquarium communities toward a more typical saltwater assemblage of microbial taxa.IMPORTANCE Saltwater aquaria are living systems that support a complex biological community of fish, invertebrates, and microbes. The health and maintenance of saltwater tanks are pressing concerns for home hobbyists, zoos, and professionals in the aquarium trade; however, we do not yet understand the underlying microbial species interactions and community dynamics which contribute to tank setup and conditioning. This report provides a detailed view of ecological succession and changes in microbial community assemblages in two saltwater aquaria which were sampled over a 3-month period, from initial tank setup and conditioning with "live rocks" through subsequent tank cleanings and water replacement. Our results showed that microbial succession appeared to be consistent and replicable across both aquaria. However, changes in microbial communities did not always correlate with water chemistry measurements, and aquarium microbial communities appear to have shifted among multiple stable states without any obvious buildup of undesirable nitrogen compounds in the tank environment.

The Effects of Captivity on the Mammalian Gut Microbiome.

Recent studies increasingly note the effect of captivity or the built environment on the microbiome of humans and other animals. As symbiotic microbes are essential to many aspects of biology (e.g., digestive and immune functions), it is important to understand how lifestyle differences can impact the microbiome, and, consequently, the health of hosts. Animals living in captivity experience a range of changes that may influence the gut bacteria, such as diet changes, treatments, and reduced contact with other individuals, species and variable environmental substrates that act as sources of bacterial diversity. Thus far, initial results from previous studies point to a pattern of decreased bacterial diversity in captive animals. However, these studies are relatively limited in the scope of species that have been examined. Here we present a dataset that includes paired wild and captive samples from mammalian taxa across six Orders to investigate generalizable patterns of the effects captivity on mammalian gut bacteria. In comparing the wild to the captive condition, our results indicate that alpha diversity of the gut bacteria remains consistent in some mammalian hosts (bovids, giraffes, anteaters, and aardvarks), declines in the captive condition in some hosts (canids, primates, and equids), and increases in the captive condition in one host taxon (rhinoceros). Differences in gut bacterial beta diversity between the captive and wild state were observed for most of the taxa surveyed, except the even-toed ungulates (bovids and giraffes). Additionally, beta diversity variation was also strongly influenced by host taxonomic group, diet type, and gut fermentation physiology. Bacterial taxa that demonstrated larger shifts in relative abundance between the captive and wild states included members of the Firmicutes and Bacteroidetes. Overall, the patterns that we observe will inform a range of disciplines from veterinary practice to captive breeding efforts for biological conservation. Furthermore, bacterial taxa that persist in the captive state provide unique insight into symbiotic relationships with the host.

Draft Genome Sequence of Klebsiella pneumoniae UCD-JA29 Isolated from a Patient with Sepsis.

Here, we present the 6,155,188-bp draft genome sequence of Klebsiella pneumoniae UCD-JA29, isolated from blood cultures from a patient with sepsis at the University of California, Davis Medical Center in Sacramento, California, USA.

Draft Genome Sequence of Cobetia sp. UCD-24C, Isolated from Roots and Leaves of the Seagrass Zostera marina.

Here, we present the 4,230,758-bp draft genome for Cobetia sp. UCD-24C. This strain was isolated from Zostera marina roots collected in Woods Hole, Massachusetts, USA.

Draft Genome Sequences of Two Pseudoalteromonas Strains Isolated from Roots and Leaf Blades of the Seagrass Zostera marina.

Here, we present the draft genome sequences for Pseudoalteromonas sp. strain UCD-33C and Pseudoalteromonas lipolytica UCD-48B. Pseudoalteromonas sp. UCD-33C was isolated from Zostera marina roots and P. lipolytica UCD-48B from Z. marina leaf blades, both collected in Woods Hole, MA. These assemblies contain 4,479,285 bp and 4,592,435 bp, respectively.

Porphyrobacter mercurialis sp. nov., isolated from a stadium seat and emended description of the genus Porphyrobacter.

A novel, Gram-negative, non-spore-forming, pleomorphic yellow-orange bacterial strain was isolated from a stadium seat. Strain Coronado(T) falls within the Erythrobacteraceae family and the genus Porphyrobacter based on 16S rRNA phylogenetic analysis. This strain has Q-10 as the predominant respiratory lipoquinone, as do other members of the family. The fatty acid profile of this strain is similar to other Porphyrobacter, however Coronado(T) contains predominately C18:1ω7cis and C16:0, a high percentage of the latter not being observed in any other Erythrobacteraceae. This strain is catalase-positive and oxidase-negative, can grow from 4 to 28 °C, at NaCl concentrations 0.1-1.5%, and at pH 6.0-8.0. On the basis of phenotypic and phylogenetic data presented in this study, strain Coronado(T) represents a novel species in the Porphyrobacter genus for which the name Porphyrobacter mercurialis sp. nov. is proposed; the type strain is Coronado(T) (=DSMZ 29971, =LMG 28700).

Draft genome sequences of 26 porphyromonas strains isolated from the canine oral microbiome.

We present the draft genome sequences for 26 strains of Porphyromonas (P. canoris, P. gulae, P. cangingavalis, P. macacae, and 7 unidentified) and an unidentified member of the Porphyromonadaceae family. All of these strains were isolated from the canine oral cavity, from dogs with and without early periodontal disease.

Complete Genome Sequence of Coprothermobacter proteolyticus DSM 5265.

Here we present the complete 1,424,912-bp genome sequence of Coprothermobacter proteolyticus DSM 5265, isolated from a thermophilic digester fermenting tannery wastes and cattle manure.