A cryptic plasmid is among the most numerous genetic elements in the human gut.

Plasmids are extrachromosomal genetic elements that often encode fitness-enhancing features. However, many bacteria carry "cryptic" plasmids that do not confer clear beneficial functions. We identified one such cryptic plasmid, pBI143, which is ubiquitous across industrialized gut microbiomes and is 14 times as numerous as crAssphage, currently established as the most abundant extrachromosomal genetic element in the human gut. The majority of mutations in pBI143 accumulate in specific positions across thousands of metagenomes, indicating strong purifying selection. pBI143 is monoclonal in most individuals, likely due to the priority effect of the version first acquired, often from one's mother. pBI143 can transfer between Bacteroidales, and although it does not appear to impact bacterial host fitness in vivo, it can transiently acquire additional genetic content. We identified important practical applications of pBI143, including its use in identifying human fecal contamination and its potential as an alternative approach to track human colonic inflammatory states.

Intestinal Blastocystis is linked to healthier diets and more favorable cardiometabolic outcomes in 56,989 individuals from 32 countries.

Diet impacts human health, influencing body adiposity and the risk of developing cardiometabolic diseases. The gut microbiome is a key player in the diet-health axis, but while its bacterial fraction is widely studied, the role of micro-eukaryotes, including Blastocystis, is underexplored. We performed a global-scale analysis on 56,989 metagenomes and showed that human Blastocystis exhibits distinct prevalence patterns linked to geography, lifestyle, and dietary habits. Blastocystis presence defined a specific bacterial signature and was positively associated with more favorable cardiometabolic profiles and negatively with obesity (p < 1e-16) and disorders linked to altered gut ecology (p < 1e-8). In a diet intervention study involving 1,124 individuals, improvements in dietary quality were linked to weight loss and increases in Blastocystis prevalence (p = 0.003) and abundance (p < 1e-7). Our findings suggest a potentially beneficial role for Blastocystis, which may help explain personalized host responses to diet and downstream disease etiopathogenesis.

Ultra-deep sequencing of Hadza hunter-gatherers recovers vanishing gut microbes.

The gut microbiome modulates immune and metabolic health. Human microbiome data are biased toward industrialized populations, limiting our understanding of non-industrialized microbiomes. Here, we performed ultra-deep metagenomic sequencing on 351 fecal samples from the Hadza hunter-gatherers of Tanzania and comparative populations in Nepal and California. We recovered 91,662 genomes of bacteria, archaea, bacteriophages, and eukaryotes, 44% of which are absent from existing unified datasets. We identified 124 gut-resident species vanishing in industrialized populations and highlighted distinct aspects of the Hadza gut microbiome related to in situ replication rates, signatures of selection, and strain sharing. Industrialized gut microbes were found to be enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. This unparalleled view of the Hadza gut microbiome provides a valuable resource, expands our understanding of microbes capable of colonizing the human gut, and clarifies the extensive perturbation induced by the industrialized lifestyle.

Massive expansion of human gut bacteriophage diversity.

Bacteriophages drive evolutionary change in bacterial communities by creating gene flow networks that fuel ecological adaptions. However, the extent of viral diversity and its prevalence in the human gut remains largely unknown. Here, we introduce the Gut Phage Database, a collection of ∼142,000 non-redundant viral genomes (>10 kb) obtained by mining a dataset of 28,060 globally distributed human gut metagenomes and 2,898 reference genomes of cultured gut bacteria. Host assignment revealed that viral diversity is highest in the Firmicutes phyla and that ∼36% of viral clusters (VCs) are not restricted to a single species, creating gene flow networks across phylogenetically distinct bacterial species. Epidemiological analysis uncovered 280 globally distributed VCs found in at least 5 continents and a highly prevalent phage clade with features reminiscent of p-crAssphage. This high-quality, large-scale catalog of phage genomes will improve future virome studies and enable ecological and evolutionary analysis of human gut bacteriophages.

Elevated rates of horizontal gene transfer in the industrialized human microbiome.

Industrialization has impacted the human gut ecosystem, resulting in altered microbiome composition and diversity. Whether bacterial genomes may also adapt to the industrialization of their host populations remains largely unexplored. Here, we investigate the extent to which the rates and targets of horizontal gene transfer (HGT) vary across thousands of bacterial strains from 15 human populations spanning a range of industrialization. We show that HGTs have accumulated in the microbiome over recent host generations and that HGT occurs at high frequency within individuals. Comparison across human populations reveals that industrialized lifestyles are associated with higher HGT rates and that the functions of HGTs are related to the level of host industrialization. Our results suggest that gut bacteria continuously acquire new functionality based on host lifestyle and that high rates of HGT may be a recent development in human history linked to industrialization.

The long-term genetic stability and individual specificity of the human gut microbiome.

By following up the gut microbiome, 51 human phenotypes and plasma levels of 1,183 metabolites in 338 individuals after 4 years, we characterize microbial stability and variation in relation to host physiology. Using these individual-specific and temporally stable microbial profiles, including bacterial SNPs and structural variations, we develop a microbial fingerprinting method that shows up to 85% accuracy in classifying metagenomic samples taken 4 years apart. Application of our fingerprinting method to the independent HMP cohort results in 95% accuracy for samples taken 1 year apart. We further observe temporal changes in the abundance of multiple bacterial species, metabolic pathways, and structural variation, as well as strain replacement. We report 190 longitudinal microbial associations with host phenotypes and 519 associations with plasma metabolites. These associations are enriched for cardiometabolic traits, vitamin B, and uremic toxins. Finally, mediation analysis suggests that the gut microbiome may influence cardiometabolic health through its metabolites.

Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity.

Gut microbial dysbioses are linked to aberrant immune responses, which are often accompanied by abnormal production of inflammatory cytokines. As part of the Human Functional Genomics Project (HFGP), we investigate how differences in composition and function of gut microbial communities may contribute to inter-individual variation in cytokine responses to microbial stimulations in healthy humans. We observe microbiome-cytokine interaction patterns that are stimulus specific, cytokine specific, and cytokine and stimulus specific. Validation of two predicted host-microbial interactions reveal that TNFα and IFNγ production are associated with specific microbial metabolic pathways: palmitoleic acid metabolism and tryptophan degradation to tryptophol. Besides providing a resource of predicted microbially derived mediators that influence immune phenotypes in response to common microorganisms, these data can help to define principles for understanding disease susceptibility. The three HFGP studies presented in this issue lay the groundwork for further studies aimed at understanding the interplay between microbial, genetic, and environmental factors in the regulation of the immune response in humans. PAPERCLIP.

Dinickel enzyme evolved to metabolize the pharmaceutical metformin and its implications for wastewater and human microbiomes.

Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug's direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmA and mfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 × 103 M-1s-1 and KM for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.

Enteropathway: the metabolic pathway database for the human gut microbiota.

The human gut microbiota produces diverse, extensive metabolites that have the potential to affect host physiology. Despite significant efforts to identify metabolic pathways for producing these microbial metabolites, a comprehensive metabolic pathway database for the human gut microbiota is still lacking. Here, we present Enteropathway, a metabolic pathway database that integrates 3269 compounds, 3677 reactions, and 876 modules that were obtained from 1012 manually curated scientific literature. Notably, 698 modules of these modules are new entries and cannot be found in any other databases. The database is accessible from a web application (https://enteropathway.org) that offers a metabolic diagram for graphical visualization of metabolic pathways, a customization interface, and an enrichment analysis feature for highlighting enriched modules on the metabolic diagram. Overall, Enteropathway is a comprehensive reference database that can complement widely used databases, and a tool for visual and statistical analysis in human gut microbiota studies and was designed to help researchers pinpoint new insights into the complex interplay between microbiota and host metabolism.

Culturing Human Gut Microbiomes in the Laboratory.

The human gut microbiota is a complex community of prokaryotic and eukaryotic microbes and viral particles that is increasingly associated with many aspects of host physiology and health. However, the classical microbiology approach of axenic culture cannot provide a complete picture of the complex interactions between microbes and their hosts in vivo. As such, recently there has been much interest in the culture of gut microbial ecosystems in the laboratory as a strategy to better understand their compositions and functions. In this review, we discuss the model platforms and methods available in the contemporary microbiology laboratory to study human gut microbiomes, as well as current knowledge surrounding the isolation of human gut microbes for the potential construction of defined communities for use in model systems.

The Landscape and Perspectives of the Human Gut Metaproteomics.

The human gut microbiome is closely associated with human health and diseases. Metaproteomics has emerged as a valuable tool for studying the functionality of the gut microbiome by analyzing the entire proteins present in microbial communities. Recent advancements in liquid chromatography and tandem mass spectrometry (LC-MS/MS) techniques have expanded the detection range of metaproteomics. However, the overall coverage of the proteome in metaproteomics is still limited. While metagenomics studies have revealed substantial microbial diversity and functional potential of the human gut microbiome, few studies have summarized and studied the human gut microbiome landscape revealed with metaproteomics. In this article, we present the current landscape of human gut metaproteomics studies by re-analyzing the identification results from 15 published studies. We quantified the limited proteome coverage in metaproteomics and revealed a high proportion of annotation coverage of metaproteomics-identified proteins. We conducted a preliminary comparison between the metaproteomics view and the metagenomics view of the human gut microbiome, identifying key areas of consistency and divergence. Based on the current landscape of human gut metaproteomics, we discuss the feasibility of using metaproteomics to study functionally unknown proteins and propose a whole workflow peptide-centric analysis. Additionally, we suggest enhancing metaproteomics analysis by refining taxonomic classification and calculating confidence scores, as well as developing tools for analyzing the interaction between taxonomy and function.

Inducible CRISPR-targeted "knockdown" of human gut Bacteroides in gnotobiotic mice discloses glycan utilization strategies.

Understanding how members of the human gut microbiota prioritize nutrient resources is one component of a larger effort to decipher the mechanisms defining microbial community robustness and resiliency in health and disease. This knowledge is foundational for development of microbiota-directed therapeutics. To model how bacteria prioritize glycans in the gut, germfree mice were colonized with 13 human gut bacterial strains, including seven saccharolytic Bacteroidaceae species. Animals were fed a Western diet supplemented with pea fiber. After community assembly, an inducible CRISPR-based system was used to selectively and temporarily reduce the absolute abundance of Bacteroides thetaiotaomicron or B. cellulosilyticus by 10- to 60-fold. Each knockdown resulted in specific, reproducible increases in the abundances of other Bacteroidaceae and dynamic alterations in their expression of genes involved in glycan utilization. Emergence of these "alternate consumers" was associated with preservation of community saccharolytic activity. Using an inducible system for CRISPR base editing in vitro, we disrupted translation of transporters critical for utilizing dietary polysaccharides in Phocaeicola vulgatus, a B. cellulosilyticus knockdown-responsive taxon. In vitro and in vivo tests of the resulting P. vulgatus mutants allowed us to further characterize mechanisms associated with its increased fitness after knockdown. In principle, the approach described can be applied to study utilization of a range of nutrients and to preclinical efforts designed to develop therapeutic strategies for precision manipulation of microbial communities.

The molecular basis of cereal mixed-linkage ß-glucan utilization by the human gut bacterium Segatella copri.

Mixed-linkage ß(1,3)/ß(1,4)-glucan (MLG) is abundant in the human diet through the ingestion of cereal grains and is widely associated with healthful effects on metabolism and cholesterol levels. MLG is also a major source of fermentable glucose for the human gut microbiota (HGM). Bacteria from the family Prevotellaceae are highly represented in the HGM of individuals who eat plant-rich diets, including certain indigenous people and vegetarians in postindustrial societies. Here, we have defined and functionally characterized an exemplar Prevotellaceae MLG polysaccharide utilization locus (MLG-PUL) in the type-strain Segatella copri (syn. Prevotella copri) DSM 18205 through transcriptomic, biochemical, and structural biological approaches. In particular, structure-function analysis of the cell-surface glycan-binding proteins and glycoside hydrolases of the S. copri MLG-PUL revealed the molecular basis for glycan capture and saccharification. Notably, syntenic MLG-PULs from human gut, human oral, and ruminant gut Prevotellaceae are distinguished from their counterparts in Bacteroidaceae by the presence of a ß(1,3)-specific endo-glucanase from glycoside hydrolase family 5, subfamily 4 (GH5_4) that initiates MLG backbone cleavage. The definition of a family of homologous MLG-PULs in individual species enabled a survey of nearly 2000 human fecal microbiomes using these genes as molecular markers, which revealed global population-specific distributions of Bacteroidaceae- and Prevotellaceae-mediated MLG utilization. Altogether, the data presented here provide new insight into the molecular basis of ß-glucan metabolism in the HGM, as a basis for informing the development of approaches to improve the nutrition and health of humans and other animals.

Action and cooperation in alginate degradation by three enzymes from the human gut bacterium Bacteroides eggerthii DSM 20697.

Alginate is a polysaccharide consumed by humans in edible seaweed and different foods where it is applied as a texturizing hydrocolloid or in encapsulations of drugs and probiotics. While gut bacteria are found to utilize and ferment alginate to health-beneficial short-chain fatty acids, knowledge on the details of the molecular reactions is sparse. Alginates are composed of mannuronic acid (M) and its C-5 epimer guluronic acid (G). An alginate-related polysaccharide utilization locus (PUL) has been identified in the gut bacterium Bacteroides eggerthii DSM 20697. The PUL encodes two polysaccharide lyases (PLs) from the PL6 (BePL6) and PL17 (BePL17) families as well as a KdgF-like metalloprotein (BeKdgF) known to catalyze ring-opening of 4,5-unsaturated monouronates yielding 4-deoxy-l-erythro-5-hexoseulose uronate (DEH). B. eggerthii DSM 20697 does not grow on alginate, but readily proliferates with a lag phase of a few hours in the presence of an endo-acting alginate lyase A1-I from the marine bacterium Sphingomonas sp. A1. The B. eggerthii lyases are both exo-acting and while BePL6 is strictly G-block specific, BePL17 prefers M-blocks. BeKdgF retained 10-27% activity in the presence of 0.1-1 mM EDTA. X-ray crystallography was used to investigate the three-dimensional structure of BeKdgF, based on which a catalytic mechanism was proposed to involve Asp102, acting as acid/base having pKa of 5.9 as determined by NMR pH titration. BePL6 and BePL17 cooperate in alginate degradation with BeKdgF linearizing producing 4,5-unsaturated monouronates. Their efficiency of alginate degradation was much enhanced by the addition of the A1-I alginate lyase.

Rapid turnover and short-term blooms of Escherichia coli in the human gut.

Escherichia coli (E. coli) is a common microorganism that is widely present in the environment and closely related to human health. The extent of E. coli presence in the human gut has been a subject of ongoing debate. Through whole-genome shotgun metagenomic sequencing, our study revealed that E. coli exists in the human body at a low abundance (average abundance 1.21%), with occasional short-term bursts leading to temporary increases in abundance, with the highest recorded at 50.91%. Further investigations into the factors contributing to these short-term blooms of E. coli showed significant variations in strain types and genomes within fecal samples collected from the same individuals at different time points. Evolutionary tree analysis indicated that samples from different individuals crossed, suggesting a change in the dominant E. coli strains within the human gut. Therefore, it can be inferred that E. coli in the human body are more likely to be transient bacteria rather than permanent residents in the gut. The rapid rate of turnover among months (87.5% within a month) and short-term blooms of E. coli in the human body can establish "latent infections" of nonpathogenic strains in healthy individuals while also posing a potential risk of introducing pathogenic strains, thereby impacting human health. In summary, our study revealed the variation in E. coli abundance and strains within the human gut, influenced by geographic area and temporal factors. These findings contribute to a better understanding of the relationship between E. coli, the gut microbiota, and human health. IMPORTANCE Escherichia coli (E. coli) is a microorganism closely linked to human health, and its presence in the human gut has been a topic of debate. Our study, using whole-genome shotgun metagenomic sequencing, revealed that E. coli exists at a low abundance in the human body, with occasional short-term bursts leading to temporary increases. Strain and genome variations were observed within fecal samples from the same individuals at different time points, suggesting transient rather than permanent residence of E. coli in the gut. The rapid turnover rate and short-term blooms of E. coli can establish latent infections while also posing a risk of introducing pathogenic strains. These findings enhance our understanding of the relationship between E. coli, the gut microbiota, and human health.

Simvastatin induces human gut bacterial cell surface genes.

Drugs intended to target mammalian cells can have broad off-target effects on the human gut microbiota with potential downstream consequences for drug efficacy and side effect profiles. Yet, despite a rich literature on antibiotic resistance, we still know very little about the mechanisms through which commensal bacteria evade non-antibiotic drugs. Here, we focus on statins, one of the most prescribed drug types in the world and an essential tool in the prevention and treatment of high circulating cholesterol levels. Prior work in humans, mice, and cell culture support an off-target effect of statins on human gut bacteria; however, the genetic determinants of statin sensitivity remain unknown. We confirmed that simvastatin inhibits the growth of diverse human gut bacterial strains grown in communities and in pure cultures. Drug sensitivity varied between phyla and was dose-dependent. We selected two representative simvastatin-sensitive species for more in-depth analysis: Eggerthella lenta (phylum: Actinobacteriota) and Bacteroides thetaiotaomicron (phylum: Bacteroidota). Transcriptomics revealed that both bacterial species upregulate genes in response to simvastatin that alter the cell membrane, including fatty acid biogenesis (E. lenta) and drug efflux systems (B. thetaiotaomicron). Transposon mutagenesis identified a key efflux system in B. thetaiotaomicron that enables growth in the presence of statins. Taken together, these results emphasize the importance of the bacterial cell membrane in countering the off-target effects of host-targeted drugs. Continued mechanistic dissection of the various mechanisms through which the human gut microbiota evades drugs will be essential to understand and predict the effects of drug administration in human cohorts and the potential downstream consequences for health and disease.

Human gut microbiome, diet, and mental disorders.

Diet is one of the most important external factor shaping the composition and metabolic activities of the gut microbiome. The gut microbiome plays a crucial role in host health, including immune system development, nutrients metabolism, and the synthesis of bioactive molecules. In addition, the gut microbiome has been described as critical for the development of several mental disorders. Nutritional psychiatry is an emerging field of research that may provide a link between diet, microbial function, and brain health. In this study, we have reviewed the influence of different diet types, such as Western, Mediterranean, vegetarian, and ketogenic, on the gut microbiota composition and function, and their implication in various neuropsychiatric and psychological disorders.

Characterisation of Indian gut microbiome for B-vitamin production and its comparison with Chinese cohort.

The human gut microbiota can biosynthesize essential micronutrients such as B-vitamins and is also known for its metabolic cooperative behaviour. The present study characterises such B-vitamin biosynthesizers, their biosynthetic pathways, explores their prevalence and abundance, examines how lifestyle or diet affects them in multiple Indian cohorts and compares it with the Chinese cohort. To achieve this, publicly available faecal metagenome data of healthy individuals from multiple Indian (two urban and three tribal populations) and a Chinese cohort were analysed. The distribution of prevalence and abundance of B-vitamin biosynthesizers showed similar profiles to that of the entire gut community of the Indian cohort, and there were 28 B-vitamin biosynthesizers that had modest or higher prevalence and abundance. The omnivorous diet affected only the prevalence of a few B-vitamin biosynthesizers; however, lifestyle and/or location affected both prevalence and abundance. A comparison with the Chinese cohort showed that fourteen B-vitamin biosynthesizers were significantly more prevalent and abundant in Chinese as compared with Indian samples (False Discovery Rate (FDR) <= 0·05). The metabolic potential of the entire gut community for B-vitamin production showed that within India, the tribal cohort has a higher abundance of B-vitamin biosynthesis pathways as compared with two urban cohorts namely, Bhopal and Kasargod, and comparison with the Chinese cohort revealed a higher abundance in the latter group. Potential metabolic cooperative behaviour of the Indian gut microbiome for biosynthesis of the B-vitamins showed multiple pairs of species showed theoretical complementarity for complete biosynthetic pathways genes of thiamine, riboflavin, niacin and pantothenate.

Structural characterization and Bacteroides proliferation promotion activity of a novel homogeneous arabinoglucuronoxylan from Commelina communis L.

Commelina communis L., a functional food and herbal plant in Asia, has been used against obesity, diabetes, and infections for centuries. A growing body of studies has demonstrated that indigestible polysaccharides are significant in obesity management. However, the structures and bioactivities of homogeneous polysaccharides from C. communis remain unclear. This study presented the structural characterization, simulated digestion, and human gut Bacteroides proliferation promotion activity of a novel homogeneous polysaccharide (CCB-3) from C. communis. The results showed that CCB-3 was an arabinoglucuronoxylan, primarily composed of arabinose, galactose, xylose, glucuronic acid (GlcA), and 4-O-methyl GlcA with a molecular weight (Mw) of 58.8 kDa. Following a 6-hour exposure to simulated gastrointestinal fluid, the Mw of CCB-3 remained unchanged, revealing that CCB-3 was an indigestible polysaccharide. Notably, CCB-3 could promote the proliferation of B. thetaiotaomicron, B. ovatus, and B. cellulosilyticus and produce short-chain fatty acids (SCFAs) and 1,2-propanediol. These findings might shed light on the discovery of polysaccharide-based leading compounds from C. communis against obesity.

Bioinformatic Analysis of Sulfotransferases from an Unexplored Gut Microbe, Sutterella wadsworthensis 3_1_45B: Possible Roles towards Detoxification via Sulfonation by Members of the Human Gut Microbiome.

Sulfonation, primarily facilitated by sulfotransferases, plays a crucial role in the detoxification pathways of endogenous substances and xenobiotics, promoting metabolism and elimination. Traditionally, this bioconversion has been attributed to a family of human cytosolic sulfotransferases (hSULTs) known for their high sequence similarity and dependence on 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfo donor. However, recent studies have revealed the presence of PAPS-dependent sulfotransferases within gut commensals, indicating that the gut microbiome may harbor a diverse array of sulfotransferase enzymes and contribute to detoxification processes via sulfation. In this study, we investigated the prevalence of sulfotransferases in members of the human gut microbiome. Interestingly, we stumbled upon PAPS-independent sulfotransferases, known as aryl-sulfate sulfotransferases (ASSTs). Our bioinformatics analyses revealed that members of the gut microbial genus Sutterella harbor multiple asst genes, possibly encoding multiple ASST enzymes within its members. Fluctuations in the microbes of the genus Sutterella have been associated with various health conditions. For this reason, we characterized 17 different ASSTs from Sutterella wadsworthensis 3_1_45B. Our findings reveal that SwASSTs share similarities with E. coli ASST but also exhibit significant structural variations and sequence diversity. These differences might drive potential functional diversification and likely reflect an evolutionary divergence from their PAPS-dependent counterparts.