Towards the biogeography of prokaryotic genes.

Microbial genes encode the majority of the functional repertoire of life on earth. However, despite increasing efforts in metagenomic sequencing of various habitats1-3, little is known about the distribution of genes across the global biosphere, with implications for human and planetary health. Here we constructed a non-redundant gene catalogue of 303 million species-level genes (clustered at 95% nucleotide identity) from 13,174 publicly available metagenomes across 14 major habitats and use it to show that most genes are specific to a single habitat. The small fraction of genes found in multiple habitats is enriched in antibiotic-resistance genes and markers for mobile genetic elements. By further clustering these species-level genes into 32 million protein families, we observed that a small fraction of these families contain the majority of the genes (0.6% of families account for 50% of the genes). The majority of species-level genes and protein families are rare. Furthermore, species-level genes, and in particular the rare ones, show low rates of positive (adaptive) selection, supporting a model in which most genetic variability observed within each protein family is neutral or nearly neutral.

adhesiomeR: a tool for Escherichia coli adhesin classification and analysis.

Adhesins are crucial factors in the virulence of bacterial pathogens such as Escherichia coli. However, to date no resources have been dedicated to the detailed analysis of E. coli adhesins. Here, we provide adhesiomeR software that enables characterization of the complete adhesin repertoire, termed the adhesiome. AdhesiomeR incorporates the most comprehensive database of E. coli adhesins and facilitates an extensive analysis of adhesiome. We demonstrate that adhesiomeR achieves 98% accuracy when compared with experimental analyses. Based on analysis of 15,000 E. coli genomes, we define novel adhesiome profiles and clusters, providing a nomenclature for a unified comparison of E. coli adhesiomes.

Structure and function of the global topsoil microbiome.

Soils harbour some of the most diverse microbiomes on Earth and are essential for both nutrient cycling and carbon storage. To understand soil functioning, it is necessary to model the global distribution patterns and functional gene repertoires of soil microorganisms, as well as the biotic and environmental associations between the diversity and structure of both bacterial and fungal soil communities1-4. Here we show, by leveraging metagenomics and metabarcoding of global topsoil samples (189 sites, 7,560 subsamples), that bacterial, but not fungal, genetic diversity is highest in temperate habitats and that microbial gene composition varies more strongly with environmental variables than with geographic distance. We demonstrate that fungi and bacteria show global niche differentiation that is associated with contrasting diversity responses to precipitation and soil pH. Furthermore, we provide evidence for strong bacterial-fungal antagonism, inferred from antibiotic-resistance genes, in topsoil and ocean habitats, indicating the substantial role of biotic interactions in shaping microbial communities. Our results suggest that both competition and environmental filtering affect the abundance, composition and encoded gene functions of bacterial and fungal communities, indicating that the relative contributions of these microorganisms to global nutrient cycling varies spatially.

Landscape of mobile genetic elements and their antibiotic resistance cargo in prokaryotic genomes.

Prokaryotic Mobile Genetic Elements (MGEs) such as transposons, integrons, phages and plasmids, play important roles in prokaryotic evolution and in the dispersal of cargo functions like antibiotic resistance. However, each of these MGE types is usually annotated and analysed individually, hampering a global understanding of phylogenetic and environmental patterns of MGE dispersal. We thus developed a computational framework that captures diverse MGE types, their cargos and MGE-mediated horizontal transfer events, using recombinases as ubiquitous MGE marker genes and pangenome information for MGE boundary estimation. Applied to ∼84k genomes with habitat annotation, we mapped 2.8 million MGE-specific recombinases to six operational MGE types, which together contain on average 13% of all the genes in a genome. Transposable elements (TEs) dominated across all taxa (∼1.7 million occurrences), outnumbering phages and phage-like elements (<0.4 million). We recorded numerous MGE-mediated horizontal transfer events across diverse phyla and habitats involving all MGE types, disentangled and quantified the extent of hitchhiking of TEs (17%) and integrons (63%) with other MGE categories, and established TEs as dominant carriers of antibiotic resistance genes. We integrated all these findings into a resource (proMGE.embl.de), which should facilitate future studies on the large mobile part of genomes and its horizontal dispersal.

Gut microbiota dysbiosis in Parkinson disease: A systematic review and pooled analysis.

BACKGROUND AND PURPOSE: The role of the gut microbiome in the pathogenesis of Parkinson disease (PD) is under intense investigation, and the results presented are still very heterogeneous. These discrepancies arise not only from the highly heterogeneous pathology of PD, but also from widely varying methodologies at all stages of the workflow, from sampling to final statistical analysis. The aim of the present work is to harmonize the workflow across studies to reduce the methodological heterogeneity and to perform a pooled analysis to account for other sources of heterogeneity. METHODS: We performed a systematic review to identify studies comparing the gut microbiota of PD patients to healthy controls. A workflow was designed to harmonize processing across all studies from bioinformatics processing to final statistical analysis using a Bayesian random-effects meta-analysis based on individual patient-level data. RESULTS: The results show that harmonizing workflows minimizes differences between statistical methods and reveals only a small set of taxa being associated with the pathogenesis of PD. Increased shares of the genera Akkermansia and Bifidobacterium and decreased shares of the genera Roseburia and Faecalibacterium were most characteristic for PD-associated microbiota. CONCLUSIONS: Our study summarizes evidence that reduced levels of butyrate-producing taxa in combination with possible degradation of the mucus layer by Akkermansia may promote intestinal inflammation and reduced permeability of the gut mucosal layer. This may allow potentially pathogenic metabolites to transit and enter the enteric nervous system.

mTAGs: taxonomic profiling using degenerate consensus reference sequences of ribosomal RNA genes.

Profiling the taxonomic composition of microbial communities commonly involves the classification of ribosomal RNA gene fragments. As a trade-off to maintain high classification accuracy, existing tools are typically limited to the genus level. Here, we present mTAGs, a taxonomic profiling tool that implements the alignment of metagenomic sequencing reads to degenerate consensus reference sequences of small subunit ribosomal RNA genes. It uses DNA fragments, that is, paired-end sequencing reads, as count units and provides relative abundance profiles at multiple taxonomic ranks, including operational taxonomic units based on a 97% sequence identity cutoff. At the genus rank, mTAGs outperformed other tools across several metrics, such as the F1 score by >11% across data from different environments, and achieved competitive (F1 score) or better results (Bray-Curtis dissimilarity) at the sub-genus level. AVAILABILITY AND IMPLEMENTATION: The software tool mTAGs is implemented in Python. The source code and binaries are freely available (https://github.com/SushiLab/mTAGs). The data underlying this article are available in Zenodo, at https://doi.org/10.5281/zenodo.4352762. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Human gut microbes impact host serum metabolome and insulin sensitivity.

Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin-resistant individuals is characterized by increased levels of branched-chain amino acids (BCAAs), which correlate with a gut microbiome that has an enriched biosynthetic potential for BCAAs and is deprived of genes encoding bacterial inward transporters for these amino acids. Prevotella copri and Bacteroides vulgatus are identified as the main species driving the association between biosynthesis of BCAAs and insulin resistance, and in mice we demonstrate that P. copri can induce insulin resistance, aggravate glucose intolerance and augment circulating levels of BCAAs. Our findings suggest that microbial targets may have the potential to diminish insulin resistance and reduce the incidence of common metabolic and cardiovascular disorders.

Microbiota Dysbiosis and Gut Barrier Dysfunction Associated with Non-Alcoholic Fatty Liver Disease Are Modulated by a Specific Metabolic Cofactors' Combination.

The gut is a selective barrier that not only allows the translocation of nutrients from food, but also microbe-derived metabolites to the systemic circulation that flows through the liver. Microbiota dysbiosis occurs when energy imbalances appear due to an unhealthy diet and a sedentary lifestyle. Dysbiosis has a critical impact on increasing intestinal permeability and epithelial barrier deterioration, contributing to bacterial and antigen translocation to the liver, triggering non-alcoholic fatty liver disease (NAFLD) progression. In this study, the potential therapeutic/beneficial effects of a combination of metabolic cofactors (a multi-ingredient; MI) (betaine, N-acetylcysteine, L-carnitine, and nicotinamide riboside) against NAFLD were evaluated. In addition, we investigated the effects of this metabolic cofactors' combination as a modulator of other players of the gut-liver axis during the disease, including gut barrier dysfunction and microbiota dysbiosis. Diet-induced NAFLD mice were distributed into two groups, treated with the vehicle (NAFLD group) or with a combination of metabolic cofactors (NAFLD-MI group), and small intestines were harvested from all animals for histological, molecular, and omics analysis. The MI treatment ameliorated gut morphological changes, decreased gut barrier permeability, and reduced gene expression of some proinflammatory cytokines. Moreover, epithelial cell proliferation and the number of goblet cells were increased after MI supplementation. In addition, supplementation with the MI combination promoted changes in the intestinal microbiota composition and diversity, as well as modulating short-chain fatty acids (SCFAs) concentrations in feces. Taken together, this specific combination of metabolic cofactors can reverse gut barrier disruption and microbiota dysbiosis contributing to the amelioration of NAFLD progression by modulating key players of the gut-liver axis.

Metagenomic assessment of the global diversity and distribution of bacteria and fungi.

Bacteria and fungi are of uttermost importance in determining environmental and host functioning. Despite close interactions between animals, plants, their associated microbiomes, and the environment they inhabit, the distribution and role of bacteria and especially fungi across host and environments as well as the cross-habitat determinants of their community compositions remain little investigated. Using a uniquely broad global dataset of 13 483 metagenomes, we analysed the microbiome structure and function of 25 host-associated and environmental habitats, focusing on potential interactions between bacteria and fungi. We found that the metagenomic relative abundance ratio of bacteria-to-fungi is a distinctive microbial feature of habitats. Compared with fungi, the cross-habitat distribution pattern of bacteria was more strongly driven by habitat type. Fungal diversity was depleted in host-associated communities compared with those in the environment, particularly terrestrial habitats, whereas this diversity pattern was less pronounced for bacteria. The relative gene functional potential of bacteria or fungi reflected their diversity patterns and appeared to depend on a balance between substrate availability and biotic interactions. Alongside helping to identify hotspots and sources of microbial diversity, our study provides support for differences in assembly patterns and processes between bacterial and fungal communities across different habitats.

Intestinal Microbiome-Macrophage Crosstalk Contributes to Cholestatic Liver Disease by Promoting Intestinal Permeability in Mice.

BACKGROUND AND AIMS: Mounting evidence supports an association between cholestatic liver disease and changes in the composition of the microbiome. Still, the role of the microbiome in the pathogenesis of this condition remains largely undefined. APPROACH AND RESULTS: To address this, we have used two experimental models, administering alpha-naphtylisocyanate or feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet, to induce cholestatic liver disease in germ-free mice and germ-free mice conventionalized with the microbiome from wild-type, specific pathogen-free animals. Next, we have inhibited macrophage activation by depleting these cells using clodronate liposomes and inhibiting the inflammasome with a specific inhibitor of NOD-, LRR-, and pyrin domain-containing protein 3. Our results demonstrate that cholestasis, the accumulation of bile acids in the liver, fails to promote liver injury in the absence of the microbiome in vivo. Additional in vitro studies supported that endotoxin sensitizes hepatocytes to bile-acid-induced cell death. We also demonstrate that during cholestasis, macrophages contribute to promoting intestinal permeability and to altered microbiome composition through activation of the inflammasome, overall leading to increased endotoxin flux into the cholestatic liver. CONCLUSIONS: We demonstrate that the intestinal microbiome contributes to cholestasis-mediated cell death and inflammation through mechanisms involving activation of the inflammasome in macrophages.

Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota.

In recent years, several associations between common chronic human disorders and altered gut microbiome composition and function have been reported. In most of these reports, treatment regimens were not controlled for and conclusions could thus be confounded by the effects of various drugs on the microbiota, which may obscure microbial causes, protective factors or diagnostically relevant signals. Our study addresses disease and drug signatures in the human gut microbiome of type 2 diabetes mellitus (T2D). Two previous quantitative gut metagenomics studies of T2D patients that were unstratified for treatment yielded divergent conclusions regarding its associated gut microbial dysbiosis. Here we show, using 784 available human gut metagenomes, how antidiabetic medication confounds these results, and analyse in detail the effects of the most widely used antidiabetic drug metformin. We provide support for microbial mediation of the therapeutic effects of metformin through short-chain fatty acid production, as well as for potential microbiota-mediated mechanisms behind known intestinal adverse effects in the form of a relative increase in abundance of Escherichia species. Controlling for metformin treatment, we report a unified signature of gut microbiome shifts in T2D with a depletion of butyrate-producing taxa. These in turn cause functional microbiome shifts, in part alleviated by metformin-induced changes. Overall, the present study emphasizes the need to disentangle gut microbiota signatures of specific human diseases from those of medication.

Plant nutrient-acquisition strategies drive topsoil microbiome structure and function.

Plant nutrient-acquisition strategies drive soil processes and vegetation performance, but their effect on the soil microbiome remains poorly understood. This knowledge is important to predict the shifts in microbial diversity and functions due to increasing changes in vegetation traits under global change. Here we documented the topsoil microbiomes of 145 boreal and temperate terrestrial sites in the Baltic region that broadly differed in vegetation type and nutritional traits, such as mycorrhizal types and symbiotic nitrogen-fixation. We found that sites dominated by arbuscular mycorrhizal (AM) vegetation harbor relatively more AM fungi, bacteria, fungal saprotrophs, and pathogens in the topsoil compared with sites dominated by ectomycorrhizal (EM) plants. These differences in microbiome composition reflect the rapid nutrient cycling and negative plant-soil feedback in AM soils. Lower fungal diversity and bacteria : fungi ratios in EM-dominated habitats are driven by monodominance of woody vegetation as well as soil acidification by EM fungi, which are associated with greater diversity and relative abundance of carbohydrate-active enzymes. Our study suggests that shifts in vegetation related to global change and land use may strongly alter the topsoil microbiome structure and function.

Population-level analysis of Blastocystis subtype prevalence and variation in the human gut microbiota.

OBJECTIVE: Human gut microbiome studies are mainly bacteria- and archaea-oriented, overlooking the presence of single-cell eukaryotes such as Blastocystis, an enteric stramenopiles with worldwide distribution. Here, we surveyed the prevalence and subtype variation of Blastocystis in faecal samples collected as part of the Flemish Gut Flora Project (FGFP), a Western population cohort. We assessed potential links between Blastocystis subtypes and identified microbiota-host covariates and quantified microbiota differentiation relative to subtype abundances. DESIGN: We profiled stool samples from 616 healthy individuals from the FGFP cohort as well as 107 patients with IBD using amplicon sequencing targeting the V4 variable region of the 16S rRNA and 18S rRNA genes. We evaluated associations of Blastocystis, and their subtypes, with host parameters, diversity and composition of bacterial and archaeal communities. RESULTS: Blastocystis prevalence in the non-clinical population cohort was 30% compared with 4% among Flemish patients with IBD. Within the FGFP cohort, out of 69 previously identified gut microbiota covariates, only age was associated with Blastocystis subtype carrier status. In contrast, a strong association between microbiota community composition and Blastocystis subtypes was observed, with effect sizes larger than that of host covariates. Microbial richness and diversity were linked to both Blastocystis prevalence and subtype variation. All Blastocystis subtypes detected in this cohort were found to be less prevalent in Bacteroides enterotyped samples. Interestingly, Blastocystis subtypes 3 and 4 were inversely correlated with Akkermansia, suggesting differential associations of subtypes with host health. CONCLUSIONS: These results emphasise the role of Blastocystis as a common constituent of the healthy gut microbiota. We show its prevalence is reduced in patients with active IBD and demonstrate that subtype characterisation is essential for assessing the relationship between Blastocystis, microbiota profile and host health. These findings have direct clinical applications, especially in donor selection for faecal transplantation.

Antibiotics-induced monodominance of a novel gut bacterial order.

OBJECTIVE: The composition of the healthy human adult gut microbiome is relatively stable over prolonged periods, and representatives of the most highly abundant and prevalent species have been cultured and described. However, microbial abundances can change on perturbations, such as antibiotics intake, enabling the identification and characterisation of otherwise low abundant species. DESIGN: Analysing gut microbial time-series data, we used shotgun metagenomics to create strain level taxonomic and functional profiles. Community dynamics were modelled postintervention with a focus on conditionally rare taxa and previously unknown bacteria. RESULTS: In response to a commonly prescribed cephalosporin (ceftriaxone), we observe a strong compositional shift in one subject, in which a previously unknown species, UBorkfalki ceftriaxensis, was identified, blooming to 92% relative abundance. The genome assembly reveals that this species (1) belongs to a so far undescribed order of Firmicutes, (2) is ubiquitously present at low abundances in at least one third of adults, (3) is opportunistically growing, being ecologically similar to typical probiotic species and (4) is stably associated to healthy hosts as determined by single nucleotide variation analysis. It was the first coloniser after the antibiotic intervention that led to a long-lasting microbial community shift and likely permanent loss of nine commensals. CONCLUSION: The bloom of UB. ceftriaxensis and a subsequent one of Parabacteroides distasonis demonstrate the existence of monodominance community states in the gut. Our study points to an undiscovered wealth of low abundant but common taxa in the human gut and calls for more highly resolved longitudinal studies, in particular on ecosystem perturbations.

Richness of human gut microbiome correlates with metabolic markers.

We are facing a global metabolic health crisis provoked by an obesity epidemic. Here we report the human gut microbial composition in a population sample of 123 non-obese and 169 obese Danish individuals. We find two groups of individuals that differ by the number of gut microbial genes and thus gut bacterial richness. They contain known and previously unknown bacterial species at different proportions; individuals with a low bacterial richness (23% of the population) are characterized by more marked overall adiposity, insulin resistance and dyslipidaemia and a more pronounced inflammatory phenotype when compared with high bacterial richness individuals. The obese individuals among the lower bacterial richness group also gain more weight over time. Only a few bacterial species are sufficient to distinguish between individuals with high and low bacterial richness, and even between lean and obese participants. Our classifications based on variation in the gut microbiome identify subsets of individuals in the general white adult population who may be at increased risk of progressing to adiposity-associated co-morbidities.

Host genetic variation strongly influences the microbiome structure and function in fungal fruiting-bodies.

Despite increasing knowledge on host-associated microbiomes, little is known about mechanisms underlying fungus-microbiome interactions. This study aimed to examine the relative importance of host genetic, geographic and environmental variations in structuring fungus-associated microbiomes. We analyzed the taxonomic composition and function of microbiomes inhabiting fungal fruiting-bodies in relation to host genetic variation, soil pH and geographic distance between samples. For this, we sequenced the metagenomes of 40 fruiting-bodies collected from six fairy rings (i.e., genets) of a saprotrophic fungus Marasmius oreades. Our analyses revealed that fine genetic variations between host fungi could strongly affect their associated microbiome, explaining, respectively, 25% and 37% of the variation in microbiome structure and function, whereas geographic distance and soil pH remained of secondary importance. These results, together with the smaller genome size of fungi compared to other eukaryotes, suggest that fruiting-bodies are suitable for further genome-centric studies on host-microbiome interactions.

RTK: efficient rarefaction analysis of large datasets.

MOTIVATION: The rapidly expanding microbiomics field is generating increasingly larger datasets, characterizing the microbiota in diverse environments. Although classical numerical ecology methods provide a robust statistical framework for their analysis, software currently available is inadequate for large datasets and some computationally intensive tasks, like rarefaction and associated analysis. RESULTS: Here we present a software package for rarefaction analysis of large count matrices, as well as estimation and visualization of diversity, richness and evenness. Our software is designed for ease of use, operating at least 7x faster than existing solutions, despite requiring 10x less memory. AVAILABILITY AND IMPLEMENTATION: C ++ and R source code (GPL v.2) as well as binaries are available from https://github.com/hildebra/Rarefaction and from CRAN (https://cran.r-project.org/). CONTACT: bork@embl.de or falk.hildebrand@embl.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Subspecies in the global human gut microbiome.

Population genomics of prokaryotes has been studied in depth in only a small number of primarily pathogenic bacteria, as genome sequences of isolates of diverse origin are lacking for most species. Here, we conducted a large-scale survey of population structure in prevalent human gut microbial species, sampled from their natural environment, with a culture-independent metagenomic approach. We examined the variation landscape of 71 species in 2,144 human fecal metagenomes and found that in 44 of these, accounting for 72% of the total assigned microbial abundance, single-nucleotide variation clearly indicates the existence of sub-populations (here termed subspecies). A single subspecies (per species) usually dominates within each host, as expected from ecological theory. At the global scale, geographic distributions of subspecies differ between phyla, with Firmicutes subspecies being significantly more geographically restricted. To investigate the functional significance of the delineated subspecies, we identified genes that consistently distinguish them in a manner that is independent of reference genomes. We further associated these subspecies-specific genes with properties of the microbial community and the host. For example, two of the three Eubacterium rectale subspecies consistently harbor an accessory pro-inflammatory flagellum operon that is associated with lower gut community diversity, higher host BMI, and higher blood fasting insulin levels. Using an additional 676 human oral samples, we further demonstrate the existence of niche specialized subspecies in the different parts of the oral cavity. Taken together, we provide evidence for subspecies in the majority of abundant gut prokaryotes, leading to a better functional and ecological understanding of the human gut microbiome in conjunction with its host.