Many of Earth's microbes remain uncultured and under-studied, limiting our understanding of the functional and evolutionary aspects of their genetic material, which remain largely overlooked in most metagenomic studies. Here, we analyzed 149,842 environmental genomes from multiple habitats and compiled a curated catalog of 404,085 functionally and evolutionarily significant novel (FESNov) gene families exclusive to uncultivated prokaryotic taxa. All FESNov families span multiple species, exhibit strong signals of purifying selection, and qualify as new orthologous groups, thus nearly tripling the number of bacterial and archaeal gene families described to date. The FESNov catalog is enriched in clade-specific traits, including 1,034 novel families that can distinguish entire uncultivated phyla, classes, and orders, likely representing synapomorphies that facilitated their evolutionary divergence. Using genomic context analysis and structural alignments, we predicted functional associations for 32.4% of FESNov families, including 4,349 high-confidence associations with important biological processes. These predictions provide a valuable hypothesis-driven framework, which we employed to experimentally validate a new gene family involved in cell motility and a novel set of antimicrobial peptides. We also demonstrate that the relative abundance profiles of novel families can discriminate between environments and clinical conditions, leading to the discovery of potentially new biomarkers associated with colorectal cancer. We expect this work to enhance future metagenomics studies and expand our knowledge of the genetic repertory of uncultivated organisms.

Meta'omic data on microbial diversity and function accrue exponentially in public repositories, but derived information is often siloed according to data type, study or sampled microbial environment. Here we present SPIRE, a Searchable Planetary-scale mIcrobiome REsource that integrates various consistently processed metagenome-derived microbial data modalities across habitats, geography and phylogeny. SPIRE encompasses 99 146 metagenomic samples from 739 studies covering a wide array of microbial environments and augmented with manually-curated contextual data. Across a total metagenomic assembly of 16 Tbp, SPIRE comprises 35 billion predicted protein sequences and 1.16 million newly constructed metagenome-assembled genomes (MAGs) of medium or high quality. Beyond mapping to the high-quality genome reference provided by proGenomes3 (http://progenomes.embl.de), these novel MAGs form 92 134 novel species-level clusters, the majority of which are unclassified at species level using current tools. SPIRE enables taxonomic profiling of these species clusters via an updated, custom mOTUs database (https://motu-tool.org/) and includes several layers of functional annotation, as well as crosslinks to several (micro-)biological databases. The resource is accessible, searchable and browsable via http://spire.embl.de.

The interpretation of genomic, transcriptomic and other microbial 'omics data is highly dependent on the availability of well-annotated genomes. As the number of publicly available microbial genomes continues to increase exponentially, the need for quality control and consistent annotation is becoming critical. We present proGenomes3, a database of 907 388 high-quality genomes containing 4 billion genes that passed stringent criteria and have been consistently annotated using multiple functional and taxonomic databases including mobile genetic elements and biosynthetic gene clusters. proGenomes3 encompasses 41 171 species-level clusters, defined based on universal single copy marker genes, for which pan-genomes and contextual habitat annotations are provided. The database is available at http://progenomes.embl.de/.

Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine learning-based approach to predict prokaryotic antimicrobial peptides (AMPs) by leveraging a vast dataset of 63,410 metagenomes and 87,920 microbial genomes. This led to the creation of AMPSphere, a comprehensive catalog comprising 863,498 non-redundant peptides, the majority of which were previously unknown. We observed that AMP production varies by habitat, with animal-associated samples displaying the highest proportion of AMPs compared to other habitats. Furthermore, within different human-associated microbiota, strain-level differences were evident. To validate our predictions, we synthesized and experimentally tested 50 AMPs, demonstrating their efficacy against clinically relevant drug-resistant pathogens both in vitro and in vivo. These AMPs exhibited antibacterial activity by targeting the bacterial membrane. Additionally, AMPSphere provides valuable insights into the evolutionary origins of peptides. In conclusion, our approach identified AMP sequences within prokaryotic microbiomes, opening up new avenues for the discovery of antibiotics.

Multi-omics analyses are used in microbiome studies to understand molecular changes in microbial communities exposed to different conditions. However, it is not always clear how much each omics data type contributes to our understanding and whether they are concordant with each other. Here, we map the molecular response of a synthetic community of 32 human gut bacteria to three non-antibiotic drugs by using five omics layers (16S rRNA gene profiling, metagenomics, metatranscriptomics, metaproteomics and metabolomics). We find that all the omics methods with species resolution are highly consistent in estimating relative species abundances. Furthermore, different omics methods complement each other for capturing functional changes. For example, while nearly all the omics data types captured that the antipsychotic drug chlorpromazine selectively inhibits Bacteroidota representatives in the community, the metatranscriptome and metaproteome suggested that the drug induces stress responses related to protein quality control. Metabolomics revealed a decrease in oligosaccharide uptake, likely caused by Bacteroidota depletion. Our study highlights how multi-omics datasets can be utilized to reveal complex molecular responses to external perturbations in microbial communities.

Microbial genes encode the majority of the functional repertoire of life on earth. However, despite increasing efforts in metagenomic sequencing of various habitats, 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.

Previous microbiome and metabolome analyses exploring non-communicable diseases have paid scant attention to major confounders of study outcomes, such as common, pre-morbid and co-morbid conditions, or polypharmacy. Here, in the context of ischemic heart disease (IHD), we used a study design that recapitulates disease initiation, escalation and response to treatment over time, mirroring a longitudinal study that would otherwise be difficult to perform given the protracted nature of IHD pathogenesis. We recruited 1,241 middle-aged Europeans, including healthy individuals, individuals with dysmetabolic morbidities (obesity and type 2 diabetes) but lacking overt IHD diagnosis and individuals with IHD at three distinct clinical stages-acute coronary syndrome, chronic IHD and IHD with heart failure-and characterized their phenome, gut metagenome and serum and urine metabolome. We found that about 75% of microbiome and metabolome features that distinguish individuals with IHD from healthy individuals after adjustment for effects of medication and lifestyle are present in individuals exhibiting dysmetabolism, suggesting that major alterations of the gut microbiome and metabolome might begin long before clinical onset of IHD. We further categorized microbiome and metabolome signatures related to prodromal dysmetabolism, specific to IHD in general or to each of its three subtypes or related to escalation or de-escalation of IHD. Discriminant analysis based on specific IHD microbiome and metabolome features could better differentiate individuals with IHD from healthy individuals or metabolically matched individuals as compared to the conventional risk markers, pointing to a pathophysiological relevance of these features.

Recent evidence suggests a role for the microbiome in pancreatic ductal adenocarcinoma (PDAC) aetiology and progression.

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.

Alterations to the gut microbiome have been linked to multiple chronic diseases. However, the drivers of such changes remain largely unknown. The oral cavity acts as a major route of exposure to exogenous factors including pathogens, and processes therein may affect the communities in the subsequent compartments of the gastrointestinal tract. Here, we perform strain-resolved, integrated meta-genomic, transcriptomic, and proteomic analyses of paired saliva and stool samples collected from 35 individuals from eight families with multiple cases of type 1 diabetes mellitus (T1DM).

To identify gut and oral metagenomic signatures that accurately predict pancreatic ductal carcinoma (PDAC) and to validate these signatures in independent cohorts.

Microorganisms play a role in the progression of various cancers. In this issue of Cancer Cell, Ghaddar et al. traced bacteria in pancreatic tumors at single-cell resolution and associated their intracellular presence with cell-type-specific transcriptional shifts, with links to clinical prognosis.

Fecal microbiota transplantation (FMT) is a therapeutic intervention for inflammatory diseases of the gastrointestinal tract, but its clinical mode of action and subsequent microbiome dynamics remain poorly understood. Here we analyzed metagenomes from 316 FMTs, sampled pre and post intervention, for the treatment of ten different disease indications. We quantified strain-level dynamics of 1,089 microbial species, complemented by 47,548 newly constructed metagenome-assembled genomes. Donor strain colonization and recipient strain resilience were mostly independent of clinical outcomes, but accurately predictable using LASSO-regularized regression models that accounted for host, microbiome and procedural variables. Recipient factors and donor-recipient complementarity, encompassing entire microbial communities to individual strains, were the main determinants of strain population dynamics, providing insights into the underlying processes that shape the post-FMT gut microbiome. Applying an ecology-based framework to our findings indicated parameters that may inform the development of more effective, targeted microbiome therapies in the future, and suggested how patient stratification can be used to enhance donor microbiota colonization or the displacement of recipient microbes in clinical practice.

During the transition from a healthy state to cardiometabolic disease, patients become heavily medicated, which leads to an increasingly aberrant gut microbiome and serum metabolome, and complicates biomarker discovery. Here, through integrated multi-omics analyses of 2,173 European residents from the MetaCardis cohort, we show that the explanatory power of drugs for the variability in both host and gut microbiome features exceeds that of disease. We quantify inferred effects of single medications, their combinations as well as additive effects, and show that the latter shift the metabolome and microbiome towards a healthier state, exemplified in synergistic reduction in serum atherogenic lipoproteins by statins combined with aspirin, or enrichment of intestinal Roseburia by diuretic agents combined with beta-blockers. Several antibiotics exhibit a quantitative relationship between the number of courses prescribed and progression towards a microbiome state that is associated with the severity of cardiometabolic disease. We also report a relationship between cardiometabolic drug dosage, improvement in clinical markers and microbiome composition, supporting direct drug effects. Taken together, our computational framework and resulting resources enable the disentanglement of the effects of drugs and disease on host and microbiome features in multimedicated individuals. Furthermore, the robust signatures identified using our framework provide new hypotheses for drug-host-microbiome interactions in cardiometabolic disease.

Genomes are critical units in microbiology, yet ascertaining quality in prokaryotic genome assemblies remains a formidable challenge. We present GUNC (the Genome UNClutterer), a tool that accurately detects and quantifies genome chimerism based on the lineage homogeneity of individual contigs using a genome's full complement of genes. GUNC complements existing approaches by targeting previously underdetected types of contamination: we conservatively estimate that 5.7% of genomes in GenBank, 5.2% in RefSeq, and 15-30% of pre-filtered "high-quality" metagenome-assembled genomes in recent studies are undetected chimeras. GUNC provides a fast and robust tool to substantially improve prokaryotic genome quality.

Microbial organisms inhabit virtually all environments and encompass a vast biological diversity. The pangenome concept aims to facilitate an understanding of diversity within defined phylogenetic groups. Hence, pangenomes are increasingly used to characterize the strain diversity of prokaryotic species. To understand the interdependence of pangenome features (such as the number of core and accessory genes) and to study the impact of environmental and phylogenetic constraints on the evolution of conspecific strains, we computed pangenomes for 155 phylogenetically diverse species (from ten phyla) using 7,000 high-quality genomes to each of which the respective habitats were assigned. Species habitat ubiquity was associated with several pangenome features. In particular, core-genome size was more important for ubiquity than accessory genome size. In general, environmental preferences had a stronger impact on pangenome evolution than phylogenetic inertia. Environmental preferences explained up to 49% of the variance for pangenome features, compared with 18% by phylogenetic inertia. This observation was robust when the dataset was extended to 10,100 species (59 phyla). The importance of environmental preferences was further accentuated by convergent evolution of pangenome features in a given habitat type across different phylogenetic clades. For example, the soil environment promotes expansion of pangenome size, while host-associated habitats lead to its reduction. Taken together, we explored the global principles of pangenome evolution, quantified the influence of habitat, and phylogenetic inertia on the evolution of pangenomes and identified criteria governing species ubiquity and habitat specificity.

Microbiology depends on the availability of annotated microbial genomes for many applications. Comparative genomics approaches have been a major advance, but consistent and accurate annotations of genomes can be hard to obtain. In addition, newer concepts such as the pan-genome concept are still being implemented to help answer biological questions. Hence, we present proGenomes2, which provides 87 920 high-quality genomes in a user-friendly and interactive manner. Genome sequences and annotations can be retrieved individually or by taxonomic clade. Every genome in the database has been assigned to a species cluster and most genomes could be accurately assigned to one or multiple habitats. In addition, general functional annotations and specific annotations of antibiotic resistance genes and single nucleotide variants are provided. In short, proGenomes2 provides threefold more genomes, enhanced habitat annotations, updated taxonomic and functional annotation and improved linkage to the NCBI BioSample database. The database is available at http://progenomes.embl.de/.

Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities. However, the dependency of most methods on reference genomes, which are currently unavailable for a substantial fraction of microbial species, introduces estimation biases. We present an updated and functionally extended tool based on universal (i.e., reference-independent), phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) enabling the profiling of >7700 microbial species. As more than 30% of them could not previously be quantified at this taxonomic resolution, relative abundance estimates based on mOTUs are more accurate compared to other methods. As a new feature, we show that mOTUs, which are based on essential housekeeping genes, are demonstrably well-suited for quantification of basal transcriptional activity of community members. Furthermore, single nucleotide variation profiles estimated using mOTUs reflect those from whole genomes, which allows for comparing microbial strain populations (e.g., across different human body sites).

The gastrointestinal tract is abundantly colonized by microbes, yet the translocation of oral species to the intestine is considered a rare aberrant event, and a hallmark of disease. By studying salivary and fecal microbial strain populations of 310 species in 470 individuals from five countries, we found that transmission to, and subsequent colonization of, the large intestine by oral microbes is common and extensive among healthy individuals. We found evidence for a vast majority of oral species to be transferable, with increased levels of transmission in colorectal cancer and rheumatoid arthritis patients and, more generally, for species described as opportunistic pathogens. This establishes the oral cavity as an endogenous reservoir for gut microbial strains, and oral-fecal transmission as an important process that shapes the gastrointestinal microbiome in health and disease.

Gut microbes influence their hosts in many ways, in particular by modulating the impact of diet. These effects have been studied most extensively in humans and mice. In this work, we used whole genome metagenomics to investigate the relationship between the gut metagenomes of dogs, humans, mice, and pigs.

Our understanding of the human gut microbiome continues to evolve at a rapid pace, but practical application of thisknowledge is still in its infancy. This review discusses the type of studies that will be essential for translating microbiome research into targeted modulations with dedicated benefits for the human host.

The identification of body site-specific microbial biomarkers and their use for classification tasks have promising applications in medicine, microbial ecology, and forensics. Previous studies have characterized site-specific microbiota and shown that sample origin can be accurately predicted by microbial content. However, these studies were usually restricted to single datasets with consistent experimental methods and conditions, as well as comparatively small sample numbers. The effects of study-specific biases and statistical power on classification performance and biomarker identification thus remain poorly understood. Furthermore, reliable detection in mixtures of different body sites or with noise from environmental contamination has rarely been investigated thus far. Finally, the impact of ecological associations between microbes on biomarker discovery was usually not considered in previous work.

Inflammatory bowel disease (IBD) may develop due to an immunogenic response to commensal gut microbiota triggered by environmental factors in the genetically susceptible host. Isotretinoin, applied in the treatment of severe acne, has been variably associated with IBD, but prior treatment with antibiotics, also associated with IBD development, confounds confirmation of this association. This study investigated the effects of doxycycline, metronidazole (frequently used in the treatment of acne and IBD, respectively) and isotretinoin on murine gut (faecal) microbiota after 2 weeks of treatment and after a 4-week recovery period. Faecal microbiota composition was assessed by 16S rRNA gene sequencing on the GS-FLX 454 platform with primers directed against the variable regions V1-V2. Doxycycline had a modest effect on bacterial richness and evenness, but had pronounced persistent and significant effects on the abundance of certain operational taxonomic units compared with the control group. In contrast, metronidazole induced a pronounced reduction in diversity after treatment, but these effects did not persist after the recovery period. This study demonstrates differential effects of antibiotics on the gut microbiota with doxycycline, unlike metronidazole, mediating long-term changes in the murine gut microbiota. Isotretinoin had no significant effect on the faecal microbiota.

Trichuris suis ova (TSO) have been tested for therapeutic application in inflammatory bowel diseases (IBD) yet understanding of the underlying mechanisms and safety in an immunocompromised host is limited due to lack of a suitable animal model. We used a recently established rabbit model of dextran sodium sulphate (DSS) induced colitis to study the efficacy, mechanisms and safety of TSO therapy in immunocompetent and immunosuppressed animals. TSO treatment prevented the DSS induced weight loss, delayed the onset of DSS induced symptoms by 2 days and significantly reduced the disease activity (DAI). TSO treatment protected caecal histology and prevented the colitis-associated loss in faecal microbiota diversity. Mainly the transcriptome of lamina propria mononuclear cells (LPMC) was affected by TSO treatment, showing dampened innate and adaptive inflammatory responses. The protective effect of TSO was lost in immunosuppressed rabbits, where TSO exacerbated colitis. Our data show that preventive TSO treatment ameliorates colitis severity in immunocompetent rabbits, modulates LPMC immune responses and reduces faecal dysbiosis. In contrast, the same TSO treatment exacerbates colitis in immunosuppressed animals. Our data provide further evidence for a therapeutic effect of TSO in IBD, yet caution is required with regard to TSO treatment in immunosuppressed patients.

Ribosomal RNA profiling has become crucial to studying microbial communities, but meaningful taxonomic analysis and inter-comparison of such data are still hampered by technical limitations, between-study design variability and inconsistencies between taxonomies used.

Interactions between taxa are essential drivers of ecological community structure and dynamics, but they are not taken into account by traditional indices of β diversity. In this study, we propose a novel family of indices that quantify community similarity in the context of taxa interaction networks. Using publicly available datasets, we assessed the performance of two specific indices that are Taxa INteraction-Adjusted (TINA, based on taxa co-occurrence networks), and Phylogenetic INteraction-Adjusted (PINA, based on phylogenetic similarities). TINA and PINA outperformed traditional indices when partitioning human-associated microbial communities according to habitat, even for extremely downsampled datasets, and when organising ocean micro-eukaryotic plankton diversity according to geographical and physicochemical gradients. We argue that interaction-adjusted indices capture novel aspects of diversity outside the scope of traditional approaches, highlighting the biological significance of ecological association networks in the interpretation of community similarity.