Microbial Composition and Genes for Key Metabolic Attributes in the Gut Digesta of Sea Urchins Lytechinus variegatus and Strongylocentrotus purpuratus Using Shotgun Metagenomics.

This paper describes the microbial community composition and genes for key metabolic genes, particularly the nitrogen fixation of the mucous-enveloped gut digesta of green (Lytechinus variegatus) and purple (Strongylocentrotus purpuratus) sea urchins by using the shotgun metagenomics approach. Both green and purple urchins showed high relative abundances of Gammaproteobacteria at 30% and 60%, respectively. However, Alphaproteobacteria in the green urchins had higher relative abundances (20%) than the purple urchins (2%). At the genus level, Vibrio was dominant in both green (~9%) and purple (~10%) urchins, whereas Psychromonas was prevalent only in purple urchins (~24%). An enrichment of Roseobacter and Ruegeria was found in the green urchins, whereas purple urchins revealed a higher abundance of Shewanella, Photobacterium, and Bacteroides (q-value < 0.01). Analysis of key metabolic genes at the KEGG-Level-2 categories revealed genes for amino acids (~20%), nucleotides (~5%), cofactors and vitamins (~6%), energy (~5%), carbohydrates (~13%) metabolisms, and an abundance of genes for assimilatory nitrogen reduction pathway in both urchins. Overall, the results from this study revealed the differences in the microbial community and genes designated for the metabolic processes in the nutrient-rich sea urchin gut digesta, suggesting their likely importance to the host and their environment.

Human gut microbial communities dictate efficacy of anti-PD-1 therapy in a humanized microbiome mouse model of glioma.

BACKGROUND: Although immunotherapy works well in glioblastoma (GBM) preclinical mouse models, the therapy has not demonstrated efficacy in humans. To address this anomaly, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a preclinical mouse model of GBM. METHODS: We used 5 healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate 5 independent humanized mouse lines (HuM1-HuM5). RESULTS: Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. All HuM mouse lines were susceptible to GBM transplantation, and exhibited similar median survival ranging from 19 to 26 days. Interestingly, we found that HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM1, HuM4, and HuM5 mice are nonresponders to anti-PD-1, while HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls. Bray-Curtis cluster analysis of the 5 HuM gut microbial communities revealed that responders HuM2 and HuM3 were closely related, and detailed taxonomic comparison analysis revealed that Bacteroides cellulosilyticus was commonly found in HuM2 and HuM3 with high abundances. CONCLUSIONS: The results of our study establish the utility of humanized microbiome mice as avatars to delineate features of the host interaction with gut microbial communities needed for effective immunotherapy against GBM.

An individualized mosaic of maternal microbial strains is transmitted to the infant gut microbial community.

To understand the origins of the infant gut microbial community, we have used a published metagenomic dataset of the faecal microbiome of mothers and their related infants at early (4, 7 and 21 days) and late times (6-15 months) following birth. Using strain-tracking analysis, individual-specific patterns of microbial strain sharing were found between mothers and infants following vaginal birth. Overall, three mother-infant pairs showed only related strains, while 12 infants of mother-infant pairs contained a mosaic of maternal-related and unrelated microbes. Analysis of a second dataset from nine women taken at different times of pregnancy revealed individual-specific faecal microbial strain variation that occurred in seven women. To model transmission in the absence of environmental microbes, we analysed the microbial strain transmission to F1 progenies of human faecal transplanted gnotobiotic mice bred with gnotobiotic males. Strain-tracking analysis of five different dams and their F1 progeny revealed both related and unrelated microbial strains in the mother's faeces. The results of our analysis demonstrate that multiple strains of maternal microbes, some that are not abundant in the maternal faecal community, can be transmitted during birth to establish a diverse infant gut microbial community.

Sharing of gut microbial strains between selected individual sets of twins cohabitating for decades.

BACKGROUND: Given the increasing realization of the important functions of the gut microbial community in human health, it is important to determine whether the increased age of the host coupled with inevitable environmental changes can alter the stability of individual microbial strains of the gut microbial community. Since early studies demonstrated that pairs of twins possess the related gut microbial communities, to gain insights into the temporal stability of the reservoir of gut microbial strains in humans, we have assessed the strain relatedness of samples from two previously published data sets that were obtained from twin children and adults (36-80 years old) who have been either living together or apart for different times. METHODS: We analyzed the two data sets; twin children (n = 24) and adults (n = 50) using our previously developed strain-tracking program called Window-based Single Nucleotide Variant (SNV) Similarity (WSS) that can distinguish a related strain pair from a non-related strain pair based on the overall genome-wide SNV similarity. To independently substantiate the identification of distinct microbial genomic variants (herein strains) observed from WSS analysis, we used analysis by StrainPhlAn. RESULTS: Analysis of the twin children data set revealed a significantly (P-value <0.05) higher number of the shared strain pairs with a predominance of Bacteroides vulgatus between individual sets of twin pairs than the twin adult data set. Additional analysis on the adult twins showed that twins who have been living apart less than 10 years shared significantly more related strain pairs than twins living apart between 10 to 60 years. Eighty-year-old twins who had been living together for 79 years then separated for 1 year showed the highest number of related strain pairs consisting of B. vulgatus, Eubacterium eligens, and Bifidobacterium adolescentis. The next highest number of related strain pairs was found in 56-year-old twins who had been living together for 51 years then separated for 5 years (B. vulgatus and Coprococcus eutactus as related strains), 73-year-old twins living together for 66 years and then separated for 7 years (Bacteroides uniformis and Clostrium sp. L2-50 as related strains) and 36-year-old twins separated for 19 years (shared strains of Alistipes shahii and E. eligens). Finally, a sporadic appearance of a single shared strain that did not show a correlation with time of separation was observed in three twin sets that had separation times between 22 to 54 years. CONCLUSION: We conclude from our strain-tracking analysis of twins that certain gut microbial strains can be shared between individuals in some cases for decades. Changes in the host environmental conditions over time can impact the stability landscape of the gut microbial community resulting in the appearance of new strains that could potentially impact microbe interactions that are essential for function in human health.

Individualized recovery of gut microbial strains post antibiotics.

To further understand the impact of antibiotics on the gastrointestinal tract microbial community, the intra-individual recovery pattern of specific microbial strains was determined using metagenomic sequencing coupled with strain-tracking analyses. In a study where 18 individuals were administered a single antibiotic (cefprozil), new microbial genomic variants (herein strains) were transiently detected in 15 individuals, while in a second study that used a cocktail of three antibiotics (meropenem, gentamicin, and vancomycin), all 12 participants had either permanent or transient strain changes. The presence of distinct microbial genomic variants indicates a pattern of strain recovery that is intra-individual specific following disruption of the human gastrointestinal tract with antibiotics.

High-throughput amplicon sequencing datasets of the metacommunity DNA of the gut microbiota of naturally occurring and laboratory aquaculture green sea urchins Lytechinus variegatus.

We present high-throughput amplicon sequence (HTS) datasets of the microbial metacommunity DNA of the gut tissue and the gut digesta of naturally occurring (n = 3) and laboratory aquaculture (n = 2) green sea urchins, Lytechinus variegatus. The HTS datasets were generated on an Illumina MiSeq by targeting the amplicons of the V4 region of the 16S rRNA gene. After the raw sequences were quality checked and filtered, 88% of the sequence reads were subjected to bioinformatics analyses to generate operation taxonomic units (OTUs), which were then verified for saturation by using rarefaction analysis at a 3% sequence variation. Further, the OTUs were randomly subsampled to the minimum sequence count values. Then, the FASTA-formatted representative sequences of the microbiota were assigned taxonomic identities through multiple databases using the SILVA ACT: Alignment, Classification and Tree Service (www.arb-silva.de/aligner). The HTS datasets of this metagenome can be accessed from the BioSample Submission Portal (https://www.ncbi.nlm.nih.gov/bioproject/) under the BioProject IDs PRJNA291441 and PRJNA326427.

The Purple Sea Urchin Strongylocentrotus purpuratus Demonstrates a Compartmentalization of Gut Bacterial Microbiota, Predictive Functional Attributes, and Taxonomic Co-Occurrence.

The sea urchin Strongylocentrotus purpuratus (order Camarodonta, family Strongylocentrotidae) can be found dominating low intertidal pool biomass on the southern coast of Oregon, USA. In this case study, three adult sea urchins were collected from their shared intertidal pool, and the bacteriome of their pharynx, gut tissue, and gut digesta, including their tide pool water and algae, was determined using targeted high-throughput sequencing (HTS) of the 16S rRNA genes and bioinformatics tools. Overall, the gut tissue demonstrated Arcobacter and Sulfurimonas (Epsilonproteobacteria) to be abundant, whereas the gut digesta was dominated by Psychromonas (Gammaproteobacteria), Propionigenium (Fusobacteria), and Flavobacteriales (Bacteroidetes). Alpha and beta diversity analyses indicated low species richness and distinct microbial communities comprising the gut tissue and digesta, while the pharynx tissue had higher richness, more closely resembling the water microbiota. Predicted functional profiles showed Kyoto Encyclopedia of Genes and Genomes (KEGG) Level-2 categories of energy metabolism, membrane transport, cell motility, and signal transduction in the gut tissue, and the gut digesta represented amino acid, carbohydrate, vitamin and cofactor metabolisms, and replication and repair. Co-occurrence network analysis showed the potential relationships and key taxa, such as the highly abundant Arcobacter and Propionigenium, influencing population patterns and taxonomic organization between the gut tissue and digesta. These results demonstrate a trend of microbial community integration, allocation, predicted metabolic roles, and taxonomic co-occurrence patterns in the S. purpuratus gut ecosystem.

Metagenomic Analysis of Microbial Community Compositions and Cold-Responsive Stress Genes in Selected Antarctic Lacustrine and Soil Ecosystems.

This study describes microbial community compositions, and various cold-responsive stress genes, encompassing cold-induced proteins (CIPs) and cold-associated general stress-responsive proteins (CASPs) in selected Antarctic lake water, sediment, and soil metagenomes. Overall, Proteobacteria and Bacteroidetes were the major taxa in all metagenomes. Prochlorococcus and Thiomicrospira were highly abundant in waters, while Myxococcus, Anaeromyxobacter, Haliangium, and Gloeobacter were dominant in the soil and lake sediment metagenomes. Among CIPs, genes necessary for DNA replication, translation initiation, and transcription termination were highly abundant in all metagenomes. However, genes for fatty acid desaturase (FAD) and trehalose synthase (TS) were common in the soil and lake sediment metagenomes. Interestingly, the Lake Untersee water and sediment metagenome samples contained histone-like nucleoid structuring protein (H-NS) and all genes for CIPs. As for the CASPs, high abundances of a wide range of genes for cryo- and osmo-protectants (glutamate, glycine, choline, and betaine) were identified in all metagenomes. However, genes for exopolysaccharide biosynthesis were dominant in Lake Untersee water, sediment, and other soil metagenomes. The results from this study indicate that although diverse microbial communities are present in various metagenomes, they share common cold-responsive stress genes necessary for their survival and sustenance in the extreme Antarctic conditions.

Ocean warming alters predicted microbiome functionality in a common sea urchin.

The microbiome of sea urchins plays a role in maintaining digestive health and innate immunity. Here, we investigated the effects of long-term (90 day) exposure to elevated seawater temperatures on the microbiome of the common, subtropical sea urchin Lytechinus variegatus The community composition and diversity of microbes varied according to the type of sample collected from the sea urchin (seawater, feed, intestines, coelomic fluid, digested pellet and faeces), with the lowest microbial diversity (predominately the order Campylobacterales) located in the intestinal tissue. Sea urchins exposed to near-future seawater temperatures maintained the community structure and diversity of microbes associated with their tissues. However, marginal, non-significant shifts in microbial community structure with elevated temperature resulted in significant changes in predicted metagenomic functions such as membrane transport and amino acid and carbohydrate metabolism. The predicted changes in key metabolic categories suggest that near-future climate-induced increases in seawater temperature could shift microbial community function and impact sea urchin digestive and immune physiology.

Microbial Communities and Their Predicted Metabolic Functions in Growth Laminae of a Unique Large Conical Mat from Lake Untersee, East Antarctica.

In this study, we report the distribution of microbial taxa and their predicted metabolic functions observed in the top (U1), middle (U2), and inner (U3) decadal growth laminae of a unique large conical microbial mat from perennially ice-covered Lake Untersee of East Antarctica, using NextGen sequencing of the 16S rRNA gene and bioinformatics tools. The results showed that the U1 lamina was dominated by cyanobacteria, specifically Phormidium sp., Leptolyngbya sp., and Pseudanabaena sp. The U2 and U3 laminae had high abundances of Actinobacteria, Verrucomicrobia, Proteobacteria, and Bacteroidetes. Closely related taxa within each abundant bacterial taxon found in each lamina were further differentiated at the highest taxonomic resolution using the oligotyping method. PICRUSt analysis, which determines predicted KEGG functional categories from the gene contents and abundances among microbial communities, revealed a high number of sequences belonging to carbon fixation, energy metabolism, cyanophycin, chlorophyll, and photosynthesis proteins in the U1 lamina. The functional predictions of the microbial communities in U2 and U3 represented signal transduction, membrane transport, zinc transport and amino acid-, carbohydrate-, and arsenic- metabolisms. The Nearest Sequenced Taxon Index (NSTI) values processed through PICRUSt were 0.10, 0.13, and 0.11 for U1, U2, and U3 laminae, respectively. These values indicated a close correspondence with the reference microbial genome database, implying high confidence in the predicted metabolic functions of the microbial communities in each lamina. The distribution of microbial taxa observed in each lamina and their predicted metabolic functions provides additional insight into the complex microbial ecosystem at Lake Untersee, and lays the foundation for studies that will enhance our understanding of the mechanisms responsible for the formation of these unique mat structures and their evolutionary significance.

Comparison of two bioinformatics tools used to characterize the microbial diversity and predictive functional attributes of microbial mats from Lake Obersee, Antarctica.

In this study, using NextGen sequencing of the collective 16S rRNA genes obtained from two sets of samples collected from Lake Obersee, Antarctica, we compared and contrasted two bioinformatics tools, PICRUSt and Tax4Fun. We then developed an R script to assess the taxonomic and predictive functional profiles of the microbial communities within the samples. Taxa such as Pseudoxanthomonas, Planctomycetaceae, Cyanobacteria Subsection III, Nitrosomonadaceae, Leptothrix, and Rhodobacter were exclusively identified by Tax4Fun that uses SILVA database; whereas PICRUSt that uses Greengenes database uniquely identified Pirellulaceae, Gemmatimonadetes A1-B1, Pseudanabaena, Salinibacterium and Sinobacteraceae. Predictive functional profiling of the microbial communities using Tax4Fun and PICRUSt separately revealed common metabolic capabilities, while also showing specific functional IDs not shared between the two approaches. Combining these functional predictions using a customized R script revealed a more inclusive metabolic profile, such as hydrolases, oxidoreductases, transferases; enzymes involved in carbohydrate and amino acid metabolisms; and membrane transport proteins known for nutrient uptake from the surrounding environment. Our results present the first molecular-phylogenetic characterization and predictive functional profiles of the microbial mat communities in Lake Obersee, while demonstrating the efficacy of combining both the taxonomic assignment information and functional IDs using the R script created in this study for a more streamlined evaluation of predictive functional profiles of microbial communities.

Metagenomics approach to the study of the gut microbiome structure and function in zebrafish Danio rerio fed with gluten formulated diet.

In this study, we report the gut microbial composition and predictive functional profiles of zebrafish, Danio rerio, fed with a control formulated diet (CFD), and a gluten formulated diet (GFD) using a metagenomics approach and bioinformatics tools. The microbial communities of the GFD-fed D. rerio displayed heightened abundances of Legionellales, Rhizobiaceae, and Rhodobacter, as compared to the CFD-fed counterparts. Predicted metagenomics of microbial communities (PICRUSt) in GFD-fed D. rerio showed KEGG functional categories corresponding to bile secretion, secondary bile acid biosynthesis, and the metabolism of glycine, serine, and threonine. The CFD-fed D. rerio exhibited KEGG functional categories of bacteria-mediated cobalamin biosynthesis, which was supported by the presence of cobalamin synthesizers such as Bacteroides and Lactobacillus. Though these bacteria were absent in GFD-fed D. rerio, a comparable level of the cobalamin biosynthesis KEGG functional category was observed, which could be contributed by the compensatory enrichment of Cetobacterium. Based on these results, we conclude D. rerio to be a suitable alternative animal model for the use of a targeted metagenomics approach along with bioinformatics tools to further investigate the relationship between the gluten diet and microbiome profile in the gut ecosystem leading to gastrointestinal diseases and other undesired adverse health effects.

The gut microbiome of the sea urchin, Lytechinus variegatus, from its natural habitat demonstrates selective attributes of microbial taxa and predictive metabolic profiles.

In this paper, we describe the microbial composition and their predictive metabolic profile in the sea urchin Lytechinus variegatus gut ecosystem along with samples from its habitat by using NextGen amplicon sequencing and downstream bioinformatics analyses. The microbial communities of the gut tissue revealed a near-exclusive abundance of Campylobacteraceae, whereas the pharynx tissue consisted of Tenericutes, followed by Gamma-, Alpha- and Epsilonproteobacteria at approximately equal capacities. The gut digesta and egested fecal pellets exhibited a microbial profile comprised of Gammaproteobacteria, mainly Vibrio, and Bacteroidetes. Both the seagrass and surrounding sea water revealed Alpha- and Betaproteobacteria. Bray-Curtis distances of microbial communities indicated a clustering profile with low intrasample variation. Predictive metagenomics performed on the microbial communities revealed that the gut tissue had high relative abundances of metabolisms assigned to the KEGG-Level-2 designation of energy metabolisms compared to the gut digesta, which had higher carbohydrate, amino acid and lipid metabolisms. Overall, the results of this study elaborate the spatial distribution of microbial communities in the gut ecosystem of L. variegatus, and specifically a selective attribute for Campylobacteraceae in the gut tissue. Also, the predictive functional significance of bacterial communities in uniquely compartmentalized gut ecosystems of L. variegatus has been described.

Distribution of cold adaptation proteins in microbial mats in Lake Joyce, Antarctica: Analysis of metagenomic data by using two bioinformatics tools.

In this study, we report the distribution and abundance of cold-adaptation proteins in microbial mat communities in the perennially ice-covered Lake Joyce, located in the McMurdo Dry Valleys, Antarctica. We have used MG-RAST and R code bioinformatics tools on Illumina HiSeq2000 shotgun metagenomic data and compared the filtering efficacy of these two methods on cold-adaptation proteins. Overall, the abundance of cold-shock DEAD-box protein A (CSDA), antifreeze proteins (AFPs), fatty acid desaturase (FAD), trehalose synthase (TS), and cold-shock family of proteins (CSPs) were present in all mat samples at high, moderate, or low levels, whereas the ice nucleation protein (INP) was present only in the ice and bulbous mat samples at insignificant levels. Considering the near homogeneous temperature profile of Lake Joyce (0.08-0.29 °C), the distribution and abundance of these proteins across various mat samples predictively correlated with known functional attributes necessary for microbial communities to thrive in this ecosystem. The comparison of the MG-RAST and the R code methods showed dissimilar occurrences of the cold-adaptation protein sequences, though with insignificant ANOSIM (R = 0.357; p-value = 0.012), ADONIS (R(2) = 0.274; p-value = 0.03) and STAMP (p-values = 0.521-0.984) statistical analyses. Furthermore, filtering targeted sequences using the R code accounted for taxonomic groups by avoiding sequence redundancies, whereas the MG-RAST provided total counts resulting in a higher sequence output. The results from this study revealed for the first time the distribution of cold-adaptation proteins in six different types of microbial mats in Lake Joyce, while suggesting a simpler and more manageable user-defined method of R code, as compared to a web-based MG-RAST pipeline.

An abundance of Epsilonproteobacteria revealed in the gut microbiome of the laboratory cultured sea urchin, Lytechinus variegatus.

In this study, we have examined the bacterial community composition of the laboratory cultured sea urchin Lytechinus variegatus gut microbiome and its culture environment using NextGen amplicon sequencing of the V4 segment of the 16S rRNA gene, and downstream bioinformatics tools. Overall, the gut and tank water was dominated by Proteobacteria, whereas the feed consisted of a co-occurrence of Proteobacteria and Firmicutes at a high abundance. The gut tissue represented Epsilonproteobacteria as dominant, with order Campylobacterales at the highest relative abundance (>95%). However, the pharynx tissue was dominated by class Alphaproteobacteria. The gut digesta and egested fecal pellets had a high abundance of class Gammaproteobacteria, from which Vibrio was found to be the primary genus, and Epsilonproteobacteria, with genus Arcobacter occurring at a moderate level. At the class level, the tank water was dominated by Gammaproteobacteria, and the feed by Alphaproteobacteria. Multi-Dimensional Scaling analysis showed that the microbial community of the gut tissue clustered together, as did the pharynx tissue to the feed. The gut digesta and egested fecal pellets showed a similarity relationship to the tank water. Further analysis of Campylobacterales at a lower taxonomic level using the oligotyping method revealed 37 unique types across the 10 samples, where Oligotype 1 was primarily represented in the gut tissue. BLAST analysis identified Oligotype 1 to be Arcobacter sp., Sulfuricurvum sp., and Arcobacter bivalviorum at an identity level >90%. This study showed that although distinct microbial communities are evident across multiple components of the sea urchin gut ecosystem, there is a noticeable correlation between the overall microbial communities of the gut with the sea urchin L. variegatus culture environment.