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p-Cresol sulfate (pCS) and indoxyl sulfate (IS), gut microbiome-derived metabolites, are traditionally associated with cardiovascular disease (CVD) risks in the setting of impaired kidney function. While pharmacologic provision of pCS or IS can promote pro-thrombotic phenotypes, neither the microbial enzymes involved nor direct gut microbial production have been linked to CVD. Untargeted metabolomics was performed on a discovery cohort (n = 1,149) with relatively preserved kidney function, followed by stable isotope-dilution mass spectrometry quantification of pCS and IS in an independent validation cohort (n = 3,954). Genetic engineering of human commensals to produce p-cresol and indole gain-of-function and loss-of-function mutants, followed by colonization of germ-free mice, and studies on host thrombosis were performed. Systemic pCS and IS levels were independently associated with all-cause mortality. Both in vitro and within colonized germ-free mice p-cresol productions were recapitulated by collaboration of two organisms: a Bacteroides strain that converts tyrosine to 4-hydroxyphenylacetate, and a Clostridium strain that decarboxylates 4-hydroxyphenylacetate to p-cresol. We then engineered a single organism, Bacteroides thetaiotaomicron, to produce p-cresol, indole, or both metabolites. Colonizing germ-free mice with engineered strains, we show the gut microbial genes for p-cresol (hpdBCA) and indole (tryptophanase) are sufficient to confer a pro-thrombotic phenotype in vivo. Moreover, human fecal metagenomics analyses show that abundances of hpdBCA and tryptophanase are associated with CVD. These studies show that pCS and IS, two abundant microbiome-derived metabolites, play a broader potential role in CVD than was previously known. They also suggest that therapeutic targeting of gut microbial p-cresol- and indole-producing pathways represent rational targets for CVD.IMPORTANCEAlterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.

Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here, we show that gut microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (Lepob/ob). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake but is instead associated with alterations in the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that gut microbial CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have potential as anti-obesity therapeutics.

The heightened cardiovascular disease (CVD) risk observed among omnivores is thought to be linked, in part, to gut microbiota-dependent generation of trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the interrelationship of γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. In the present study, we show that plasma γBB levels in individuals from a clinical cohort (n = 2,918) are strongly associated with incident CVD event risks. Culture of human faecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine → γBB → TMA transformation, elevate TMAO levels and enhance thrombosis potential in recipients after arterial injury. RNA-sequencing analyses of E. timonensis identified a six-gene cluster, herein named the γBB utilization (gbu) gene cluster, which is upregulated in response to γBB. Combinatorial cloning and functional studies identified four genes (gbuA, gbuB, gbuC and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when coexpressed in Escherichia coli. Finally, reanalysis of samples (n = 113) from a clinical, randomized diet, intervention study showed that the abundance of faecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine → γBB → TMA → TMAO transformation in hosts and contributes to CVD risk.

Death due to sepsis remains a persistent threat to critically ill patients confined to the intensive care unit and is characterized by colonization with multi-drug-resistant healthcare-associated pathogens. Here we report that sepsis in mice caused by a defined four-member pathogen community isolated from a patient with lethal sepsis is associated with the systemic suppression of key elements of the host transcriptome required for pathogen clearance and decreased butyrate expression. More specifically, these pathogens directly suppress interferon regulatory factor 3. Fecal microbiota transplant (FMT) reverses the course of otherwise lethal sepsis by enhancing pathogen clearance via the restoration of host immunity in an interferon regulatory factor 3-dependent manner. This protective effect is linked to the expansion of butyrate-producing Bacteroidetes. Taken together these results suggest that fecal microbiota transplantation may be a treatment option in sepsis associated with immunosuppression.

The cecum is a unique region in the mammalian intestinal tract in which the microbiome is localized to two compartments, the lumen and the crypts. The microbiome within crypts is particularly important as it is in direct contact with lining epithelial cells including stem cells. Here, we analyzed the microbiome in cecum of mice using multiple techniques including metagenomics. The lumen microbiome comprised Firmicutes and Bacteroidetes whereas the crypts were dominated by Proteobacteria and Deferribacteres, and the mucus comprised a mixture of these 4 phyla. The lumen microbial functional potential comprised mainly carbon metabolism, while the crypt microbiome was enriched for genes encoding stress resistance. In order to determine how this structure, assembly, and function are altered under provocative conditions, we exposed mice to overnight starvation (S), antibiotics (A), and a major surgical injury (partial hepatectomy [H]), as occurs with major surgery in humans. We have previously demonstrated that the combined effect of this "SAH" treatment leads to a major disturbance of the cecal microbiota at the bottom of crypts in a manner that disrupts crypt cell homeostasis. Here, we applied the SAH conditions and observed a loss of compartmentalization in both composition and function of the cecal microbiome associated with major shifts in local physicochemical cues including decrease of hypoxia, increase of pH, and loss of butyrate production. Taken together, these studies demonstrated a defined order, structure, and function of the cecal microbiome that can be disrupted under provocative conditions such as major surgery and its attendant exposures.IMPORTANCE The proximal colon and cecum are two intestinal regions in which the microbiome localizes to two spatially distinct compartments, the lumen and crypts. The differences in composition and function of luminal and crypt microbiome in the cecum and the effect of physiological stress on their compartmentalization remain poorly characterized. Here, we characterized the composition and function of the lumen-, mucus-, and crypt-associated microbiome in the cecum of mice. We observed a highly ordered microbial architecture within the cecum whose assembly and function become markedly disrupted when provoked by physiological stress such as surgery and its attendant preoperative treatments (i.e., overnight fasting and antibiotics). Major shifts in local physicochemical cues including a decrease in hypoxia levels, an increase in pH, and a loss of butyrate production were associated with the loss of compositional and functional compartmentalization of the cecal microbiome.

BackgroundGlaciers cover ~ 10% of land but are among the least explored environments on Earth. The basal portion of glaciers often harbors unique aquatic microbial ecosystems in the absence of sunlight, and knowledge on the microbial community structures and their metabolic potential is very limited. Here, we provide insights into the microbial lifestyle present at the base of the Matanuska Glacier, Alaska.ResultsDNA and RNA were extracted from samples of the Matanuska Glacier basal ice. Using Illumina MiSeq and HiSeq sequencing, we investigated the microbial diversity with the metagenomic shotgun reads and 16S ribosomal RNA data. We further assembled 9 partial and draft bacterial genomes from the metagenomic assembly, and identified key metabolic pathways such as sulfur oxidation and nitrification. Collectively, our analyses suggest a prevalence of lithotrophic and heterotrophic metabolisms in the subglacial microbiome.ConclusionOur results present the first metagenomic assembly and bacterial draft genomes for a subglacial environment. These results extend our understanding of the chemical and biological processes in subglacial environments critically influenced by global climate change.

Although much work has linked the human microbiome to specific phenotypes and lifestyle variables, data from different projects have been challenging to integrate and the extent of microbial and molecular diversity in human stool remains unknown. Using standardized protocols from the Earth Microbiome Project and sample contributions from over 10,000 citizen-scientists, together with an open research network, we compare human microbiome specimens primarily from the United States, United Kingdom, and Australia to one another and to environmental samples. Our results show an unexpected range of beta-diversity in human stool microbiomes compared to environmental samples; demonstrate the utility of procedures for removing the effects of overgrowth during room-temperature shipping for revealing phenotype correlations; uncover new molecules and kinds of molecular communities in the human stool metabolome; and examine emergent associations among the microbiome, metabolome, and the diversity of plants that are consumed (rather than relying on reductive categorical variables such as veganism, which have little or no explanatory power). We also demonstrate the utility of the living data resource and cross-cohort comparison to confirm existing associations between the microbiome and psychiatric illness and to reveal the extent of microbiome change within one individual during surgery, providing a paradigm for open microbiome research and education. IMPORTANCE We show that a citizen science, self-selected cohort shipping samples through the mail at room temperature recaptures many known microbiome results from clinically collected cohorts and reveals new ones. Of particular interest is integrating n = 1 study data with the population data, showing that the extent of microbiome change after events such as surgery can exceed differences between distinct environmental biomes, and the effect of diverse plants in the diet, which we confirm with untargeted metabolomics on hundreds of samples.

The microorganisms that inhabit hospitals may influence patient recovery and outcome, although the complexity and diversity of these bacterial communities can confound our ability to focus on potential pathogens in isolation. To develop a community-level understanding of how microorganisms colonize and move through the hospital environment, we characterized the bacterial dynamics among hospital surfaces, patients, and staff over the course of 1 year as a new hospital became operational. The bacteria in patient rooms, particularly on bedrails, consistently resembled the skin microbiota of the patient occupying the room. Bacterial communities on patients and room surfaces became increasingly similar over the course of a patient's stay. Temporal correlations in community structure demonstrated that patients initially acquired room-associated taxa that predated their stay but that their own microbial signatures began to influence the room community structure over time. The α- and β-diversity of patient skin samples were only weakly or nonsignificantly associated with clinical factors such as chemotherapy, antibiotic usage, and surgical recovery, and no factor except for ambulatory status affected microbial similarity between the microbiotas of a patient and their room. Metagenomic analyses revealed that genes conferring antimicrobial resistance were consistently more abundant on room surfaces than on the skin of the patients inhabiting those rooms. In addition, persistent unique genotypes of Staphylococcus and Propionibacterium were identified. Dynamic Bayesian network analysis suggested that hospital staff were more likely to be a source of bacteria on the skin of patients than the reverse but that there were no universal patterns of transmission across patient rooms.

Plant invasions often reduce native plant diversity and increase net primary productivity. Invaded soils appear to differ from surrounding soils in ways that impede restoration of diverse native plant communities. We hypothesize that invader-mediated shifts in edaphic properties reproducibly alter soil microbial community structure and function. Here, we take a holistic approach, characterizing plant, prokaryotic, and fungal communities and soil physicochemical properties in field sites, invasion gradients, and experimental plots for three invasive plant species that cooccur in the Rocky Mountain West. Each invader had a unique impact on soil physicochemical properties. We found that invasions drove shifts in the abundances of specific microbial taxa, while overall belowground community structure and functional potential were fairly constant. Forb invaders were generally enriched in copiotrophic bacteria with higher 16S rRNA gene copy numbers and showed greater microbial carbohydrate and nitrogen metabolic potential. Older invasions had stronger effects on abiotic soil properties, indicative of multiyear successions. Overall, we show that plant invasions are idiosyncratic in their impact on soils and are directly responsible for driving reproducible shifts in the soil environment over multiyear time scales. IMPORTANCE In this study, we show how invasive plant species drive rapid shifts in the soil environment from surrounding native communities. Each of the three plant invaders had different but consistent effects on soils. Thus, there does not appear to be a one-size-fits-all strategy for how plant invaders alter grassland soil environments. This work represents a crucial step toward understanding how invaders might be able to prevent or impair native reestablishment by changing soil biotic and abiotic properties.

[This corrects the article DOI: 10.1128/mSystems.00105-16.].

Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem of chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution.

Nitrates, such as cardiac therapeutics and food additives, are common headache triggers, with nitric oxide playing an important role. Facultative anaerobic bacteria in the oral cavity may contribute migraine-triggering levels of nitric oxide through the salivary nitrate-nitrite-nitric oxide pathway. Using high-throughput sequencing technologies, we detected observable and significantly higher abundances of nitrate, nitrite, and nitric oxide reductase genes in migraineurs versus nonmigraineurs in samples collected from the oral cavity and a slight but significant difference in fecal samples. IMPORTANCE Recent work has demonstrated a potentially symbiotic relationship between oral commensal bacteria and humans through the salivary nitrate-nitrite-nitric oxide pathway (C. Duncan et al., Nat Med 1:546-551, 1995, http://dx.doi.org/10.1038/nm0695-546). Oral nitrate-reducing bacteria contribute physiologically relevant levels of nitrite and nitric oxide to the human host that may have positive downstream effects on cardiovascular health (V. Kapil et al., Free Radic Biol Med 55:93-100, 2013, http://dx.doi.org/10.1016/j.freeradbiomed.2012.11.013). In the work presented here, we used 16S rRNA Illumina sequencing to determine whether a connection exists between oral nitrate-reducing bacteria, nitrates for cardiovascular disease, and migraines, which are a common side effect of nitrate medications (U. Thadani and T. Rodgers, Expert Opin Drug Saf 5:667-674, 2006, http://dx.doi.org/10.1517/14740338.5.5.667).

The adaptation of bacterial lineages to local environmental conditions creates the potential for broader genotypic diversity within a species, which can enable a species to dominate across ecological gradients because of niche flexibility. The genus Polynucleobacter maintains both free-living and symbiotic ecotypes and maintains an apparently ubiquitous distribution in freshwater ecosystems. Subspecies-level resolution supplemented with metagenome-derived genotype analysis revealed that differential functional constraints, not geographic distance, produce and maintain strain-level genetic conservation in Polynucleobacter populations across three geographically proximal riverine environments. Genes associated with cofactor biosynthesis and one-carbon metabolism showed habitat specificity, and protein-coding genes of unknown function and membrane transport proteins were under positive selection across each habitat. Characterized by different median ratios of nonsynonymous to synonymous evolutionary changes (dN/dS ratios) and a limited but statistically significant negative correlation between the dN/dS ratio and codon usage bias between habitats, the free-living and core genotypes were observed to be evolving under strong purifying selection pressure. Highlighting the potential role of genetic adaptation to the local environment, the two-component system protein-coding genes were highly stable (dN/dS ratio, < 0.03). These results suggest that despite the impact of the habitat on genetic diversity, and hence niche partition, strong environmental selection pressure maintains a conserved core genome for Polynucleobacter populations. IMPORTANCE Understanding the biological factors influencing habitat-wide genetic endemism is important for explaining observed biogeographic patterns. Polynucleobacter is a genus of bacteria that seems to have found a way to colonize myriad freshwater ecosystems and by doing so has become one of the most abundant bacteria in these environments. We sequenced metagenomes from locations across the Chicago River system and assembled Polynucleobacter genomes from different sites and compared how the nucleotide composition, gene codon usage, and the ratio of synonymous (codes for the same amino acid) to nonsynonymous (codes for a different amino acid) mutations varied across these population genomes at each site. The environmental pressures at each site drove purifying selection for functional traits that maintained a streamlined core genome across the Chicago River Polynucleobacter population while allowing for site-specific genomic adaptation. These adaptations enable Polynucleobacter to become dominant across different riverine environmental gradients.

Dietary intervention with extensively hydrolyzed casein formula supplemented with Lactobacillus rhamnosus GG (EHCF+LGG) accelerates tolerance acquisition in infants with cow's milk allergy (CMA). We examined whether this effect is attributable, at least in part, to an influence on the gut microbiota. Fecal samples from healthy controls (n=20) and from CMA infants (n=19) before and after treatment with EHCF with (n=12) and without (n=7) supplementation with LGG were compared by 16S rRNA-based operational taxonomic unit clustering and oligotyping. Differential feature selection and generalized linear model fitting revealed that the CMA infants have a diverse gut microbial community structure dominated by Lachnospiraceae (20.5±9.7%) and Ruminococcaceae (16.2±9.1%). Blautia, Roseburia and Coprococcus were significantly enriched following treatment with EHCF and LGG, but only one genus, Oscillospira, was significantly different between infants that became tolerant and those that remained allergic. However, most tolerant infants showed a significant increase in fecal butyrate levels, and those taxa that were significantly enriched in these samples, Blautia and Roseburia, exhibited specific strain-level demarcations between tolerant and allergic infants. Our data suggest that EHCF+LGG promotes tolerance in infants with CMA, in part, by influencing the strain-level bacterial community structure of the infant gut.

BackgroundSphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes (S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6).ResultsEfficient HCH degraders phylogenetically clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. Additionally, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity.ConclusionThe bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their arrangement with respect to IS6100 and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. This study further opens the horizon for selection of bacterial strains for inclusion in an HCH bioremediation consortium and suggests that HDIPO4, IP26 and B90A would be appropriate candidates for inclusion.

Over the last 60 years, the use of hexachlorocyclohexane (HCH) as a pesticide has resulted in the production of >4 million tons of HCH waste, which has been dumped in open sinks across the globe. Here, the combination of the genomes of two genetic subspecies (Sphingobium japonicum UT26 and Sphingobium indicum B90A; isolated from two discrete geographical locations, Japan and India, respectively) capable of degrading HCH, with metagenomic data from an HCH dumpsite (∼450 mg HCH per g soil), enabled the reconstruction and validation of the last-common ancestor (LCA) genotype. Mapping the LCA genotype (3128 genes) to the subspecies genomes demonstrated that >20% of the genes in each subspecies were absent in the LCA. This includes two enzymes from the 'upper' HCH degradation pathway, suggesting that the ancestor was unable to degrade HCH isomers, but descendants acquired lin genes by transposon-mediated lateral gene transfer. In addition, anthranilate and homogentisate degradation traits were found to be strain (selectively retained only by UT26) and environment (absent in the LCA and subspecies, but prevalent in the metagenome) specific, respectively. One draft secondary chromosome, two near complete plasmids and eight complete lin transposons were assembled from the metagenomic DNA. Collectively, these results reinforce the elastic nature of the genus Sphingobium, and describe the evolutionary acquisition mechanism of a xenobiotic degradation phenotype in response to environmental pollution. This also demonstrates for the first time the use of metagenomic data in ancestral genotype reconstruction, highlighting its potential to provide significant insight into the development of such phenotypes.