Bacteroidales species in the human gut are a reservoir of antibiotic resistance genes regulated by invertible promoters.

Antibiotic-resistance genes (ARGs) regulated by invertible promoters can mitigate the fitness cost of maintaining ARGs in the absence of antibiotics and could potentially prolong the persistence of ARGs in bacterial populations. However, the origin, prevalence, and distribution of these ARGs regulated by invertible promoters remains poorly understood. Here, we sought to assess the threat posed by ARGs regulated by invertible promoters by systematically searching for ARGs regulated by invertible promoters in the human gut microbiome and examining their origin, prevalence, and distribution. Through metagenomic assembly of 2227 human gut metagenomes and genomic analysis of the Unified Human Gastrointestinal Genome (UHGG) collection, we identified ARGs regulated by invertible promoters and categorized them into three classes based on the invertase-regulating phase variation. In the human gut microbiome, ARGs regulated by invertible promoters are exclusively found in Bacteroidales species. Through genomic analysis, we observed that ARGs regulated by invertible promoters have convergently originated from ARG insertions into glycan-synthesis loci that were regulated by invertible promoters at least three times. Moreover, all three classes of invertible promoters regulating ARGs are located within integrative conjugative elements (ICEs). Therefore, horizontal transfer via ICEs could explain the wide taxonomic distribution of ARGs regulated by invertible promoters. Overall, these findings reveal that glycan-synthesis loci regulated by invertible promoters in Bacteroidales species are an important hotspot for the emergence of clinically-relevant ARGs regulated by invertible promoters.

The Capacity to Produce Hydrogen Sulfide (H2S) via Cysteine Degradation Is Ubiquitous in the Human Gut Microbiome.

As one of the three mammalian gasotransmitters, hydrogen sulfide (H2S) plays a major role in maintaining physiological homeostasis. Endogenously produced H2S plays numerous beneficial roles including mediating vasodilation and conferring neuroprotection. Due to its high membrane permeability, exogenously produced H2S originating from the gut microbiota can also influence human physiology and is implicated in reducing intestinal mucosal integrity and potentiating genotoxicity and is therefore a potential target for therapeutic interventions. Gut microbial H2S production is often attributed to dissimilatory sulfate reducers such as Desulfovibrio and Bilophila species. However, an alternative source for H2S production, cysteine degradation, is present in some gut microbes, but the genes responsible for cysteine degradation have not been systematically annotated in all known gut microbes. We classify mechanisms of cysteine degradation into primary, secondary, and erroneous levels of H2S production and perform a comprehensive search for primary, secondary, and erroneous cysteine-degrading enzymes in 4,644 non-redundant bacterial genomes from the Unified Human Gastrointestinal Genome (UHGG) catalog. Of the 4,644 genomes we have putatively identified 2,046 primary, 1,951 secondary, and 5 erroneous cysteine-degrading species. We identified the presence of at least one putative cysteine-degrading bacteria in metagenomic data of 100% of 6,623 healthy subjects and the expression of cysteine-degrading genes in metatranscriptomic data of 100% of 736 samples taken from 318 individuals. Additionally, putative cysteine-degrading bacteria are more abundant than sulfate-reducing bacteria across healthy controls, IBD patients and CRC patients (p < 2.2e-16, Wilcoxon rank sum test). Although we have linked many taxa with the potential for cysteine degradation, experimental validation is required to establish the H2S production potential of the gut microbiome. Overall, this study improves our understanding of the capacity for H2S production by the human gut microbiome and may help to inform interventions to therapeutically modulate gut microbial H2S production.

The taxonomic distribution of histamine-secreting bacteria in the human gut microbiome.

BACKGROUND: Biogenic histamine plays an important role in immune response, neurotransmission, and allergic response. Although endogenous histamine production has been extensively studied, the contributions of histamine produced by the human gut microbiota have not been explored due to the absence of a systematic annotation of histamine-secreting bacteria. RESULTS: To identify the histamine-secreting bacteria from in the human gut microbiome, we conducted a systematic search for putative histamine-secreting bacteria in 36,554 genomes from the Genome Taxonomy Database and Unified Human Gastrointestinal Genome catalog. Using bioinformatic approaches, we identified 117 putative histamine-secreting bacteria species. A new three-component decarboxylation system including two colocalized decarboxylases and one transporter was observed in histamine-secreting bacteria among three different phyla. We found significant enrichment of histamine-secreting bacteria in patients with inflammatory bowel disease but not in patients with colorectal cancer suggesting a possible association between histamine-secreting bacteria and inflammatory bowel disease. CONCLUSIONS: The findings of this study expand our knowledge of the taxonomic distribution of putative histamine-secreting bacteria in the human gut.

Gut microbiome ADP-ribosyltransferases are widespread phage-encoded fitness factors.

Bacterial ADP-ribosyltransferases (ADPRTs) have been described as toxins involved in pathogenesis through the modification of host proteins. Here, we report that ADPRTs are not pathogen restricted but widely prevalent in the human gut microbiome and often associated with phage elements. We validated their biochemical activity in a large clinical isolate collection and further examined Bxa, a highly abundant ADPRT in Bacteroides. Bxa is expressed, secreted, and enzymatically active in Bacteroides and can ADP-ribosylate non-muscle myosin II proteins. Addition of Bxa to epithelial cells remodeled the actin cytoskeleton and induced secretion of inosine. Bxa-encoding B. stercoris can use inosine as a carbon source and colonizes the gut to significantly greater numbers than a bxa-deleted strain in germ-free and altered Schaedler flora (ASF) mice. Colonization correlated with increased inosine concentrations in the feces and tissues. Altogether, our results show that ADPRTs are abundant in the microbiome and act as bacterial fitness factors.

Characterizing Transcriptional Regulatory Sequences in Coronaviruses and Their Role in Recombination.

Novel coronaviruses, including SARS-CoV-2, SARS, and MERS, often originate from recombination events. The mechanism of recombination in RNA viruses is template switching. Coronavirus transcription also involves template switching at specific regions, called transcriptional regulatory sequences (TRS). It is hypothesized but not yet verified that TRS sites are prone to recombination events. Here, we developed a tool called SuPER to systematically identify TRS in coronavirus genomes and then investigated whether recombination is more common at TRS. We ran SuPER on 506 coronavirus genomes and identified 465 TRS-L and 3,509 TRS-B. We found that the TRS-L core sequence (CS) and the secondary structure of the leader sequence are generally conserved within coronavirus genera but different between genera. By examining the location of recombination breakpoints with respect to TRS-B CS, we observed that recombination hotspots are more frequently colocated with TRS-B sites than expected.

Cholesterol Metabolism by Uncultured Human Gut Bacteria Influences Host Cholesterol Level.

The human microbiome encodes extensive metabolic capabilities, but our understanding of the mechanisms linking gut microbes to human metabolism remains limited. Here, we focus on the conversion of cholesterol to the poorly absorbed sterol coprostanol by the gut microbiota to develop a framework for the identification of functional enzymes and microbes. By integrating paired metagenomics and metabolomics data from existing cohorts with biochemical knowledge and experimentation, we predict and validate a group of microbial cholesterol dehydrogenases that contribute to coprostanol formation. These enzymes are encoded by ismA genes in a clade of uncultured microorganisms, which are prevalent in geographically diverse human cohorts. Individuals harboring coprostanol-forming microbes have significantly lower fecal cholesterol levels and lower serum total cholesterol with effects comparable to those attributed to variations in lipid homeostasis genes. Thus, cholesterol metabolism by these microbes may play important roles in reducing intestinal and serum cholesterol concentrations, directly impacting human health.

Association Between Sulfur-Metabolizing Bacterial Communities in Stool and Risk of Distal Colorectal Cancer in Men.

BACKGROUND & AIMS: Sulfur-metabolizing microbes, which convert dietary sources of sulfur into genotoxic hydrogen sulfide (H2S), have been associated with development of colorectal cancer (CRC). We identified a dietary pattern associated with sulfur-metabolizing bacteria in stool and then investigated its association with risk of incident CRC using data from a large prospective study of men. METHODS: We collected data from 51,529 men enrolled in the Health Professionals Follow-up Study since 1986 to determine the association between sulfur-metabolizing bacteria in stool and risk of CRC over 26 years of follow-up. First, in a subcohort of 307 healthy men, we profiled serial stool metagenomes and metatranscriptomes and assessed diet using semiquantitative food frequency questionnaires to identify food groups associated with 43 bacterial species involved in sulfur metabolism. We used these data to develop a sulfur microbial dietary score. We then used Cox proportional hazards modeling to evaluate adherence to this pattern among eligible individuals (n = 48,246) from 1986 through 2012 with risk for incident CRC. RESULTS: Foods associated with higher sulfur microbial diet scores included increased consumption of processed meats and low-calorie drinks and lower consumption of vegetables and legumes. Increased sulfur microbial diet scores were associated with risk of distal colon and rectal cancers, after adjusting for other risk factors (multivariable relative risk, highest vs lowest quartile, 1.43; 95% confidence interval 1.14-1.81; P-trend = .002). In contrast, sulfur microbial diet scores were not associated with risk of proximal colon cancer (multivariable relative risk 0.86; 95% CI 0.65-1.14; P-trend = .31). CONCLUSIONS: In an analysis of participants in the Health Professionals Follow-up Study, we found that long-term adherence to a dietary pattern associated with sulfur-metabolizing bacteria in stool was associated with an increased risk of distal CRC. Further studies are needed to determine how sulfur-metabolizing bacteria might contribute to CRC pathogenesis.

Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases.

Inflammatory bowel diseases, which include Crohn's disease and ulcerative colitis, affect several million individuals worldwide. Crohn's disease and ulcerative colitis are complex diseases that are heterogeneous at the clinical, immunological, molecular, genetic, and microbial levels. Individual contributing factors have been the focus of extensive research. As part of the Integrative Human Microbiome Project (HMP2 or iHMP), we followed 132 subjects for one year each to generate integrated longitudinal molecular profiles of host and microbial activity during disease (up to 24 time points each; in total 2,965 stool, biopsy, and blood specimens). Here we present the results, which provide a comprehensive view of functional dysbiosis in the gut microbiome during inflammatory bowel disease activity. We demonstrate a characteristic increase in facultative anaerobes at the expense of obligate anaerobes, as well as molecular disruptions in microbial transcription (for example, among clostridia), metabolite pools (acylcarnitines, bile acids, and short-chain fatty acids), and levels of antibodies in host serum. Periods of disease activity were also marked by increases in temporal variability, with characteristic taxonomic, functional, and biochemical shifts. Finally, integrative analysis identified microbial, biochemical, and host factors central to this dysregulation. The study's infrastructure resources, results, and data, which are available through the Inflammatory Bowel Disease Multi'omics Database ( http://ibdmdb.org ), provide the most comprehensive description to date of host and microbial activities in inflammatory bowel diseases.

Author Correction: Gut microbiome structure and metabolic activity in inflammatory bowel disease.

In the Supplementary Tables 2, 4 and 6 originally published with this Article, the authors mistakenly included sample identifiers in the form of UMCGs rather than UMCG IBDs in the validation cohort; this has now been amended.

Invertible promoters mediate bacterial phase variation, antibiotic resistance, and host adaptation in the gut.

Phase variation, the reversible alternation between genetic states, enables infection by pathogens and colonization by commensals. However, the diversity of phase variation remains underexplored. We developed the PhaseFinder algorithm to quantify DNA inversion-mediated phase variation. A systematic search of 54,875 bacterial genomes identified 4686 intergenic invertible DNA regions (invertons), revealing an enrichment in host-associated bacteria. Invertons containing promoters often regulate extracellular products, underscoring the importance of surface diversity for gut colonization. We found invertons containing promoters regulating antibiotic resistance genes that shift to the ON orientation after antibiotic treatment in human metagenomic data and in vitro, thereby mitigating the cost of antibiotic resistance. We observed that the orientations of some invertons diverge after fecal microbiota transplant, potentially as a result of individual-specific selective forces.

Gut microbiome structure and metabolic activity in inflammatory bowel disease.

The inflammatory bowel diseases (IBDs), which include Crohn's disease (CD) and ulcerative colitis (UC), are multifactorial chronic conditions of the gastrointestinal tract. While IBD has been associated with dramatic changes in the gut microbiota, changes in the gut metabolome-the molecular interface between host and microbiota-are less well understood. To address this gap, we performed untargeted metabolomic and shotgun metagenomic profiling of cross-sectional stool samples from discovery (n = 155) and validation (n = 65) cohorts of CD, UC and non-IBD control patients. Metabolomic and metagenomic profiles were broadly correlated with faecal calprotectin levels (a measure of gut inflammation). Across >8,000 measured metabolite features, we identified chemicals and chemical classes that were differentially abundant in IBD, including enrichments for sphingolipids and bile acids, and depletions for triacylglycerols and tetrapyrroles. While > 50% of differentially abundant metabolite features were uncharacterized, many could be assigned putative roles through metabolomic 'guilt by association' (covariation with known metabolites). Differentially abundant species and functions from the metagenomic profiles reflected adaptation to oxidative stress in the IBD gut, and were individually consistent with previous findings. Integrating these data, however, we identified 122 robust associations between differentially abundant species and well-characterized differentially abundant metabolites, indicating possible mechanistic relationships that are perturbed in IBD. Finally, we found that metabolome- and metagenome-based classifiers of IBD status were highly accurate and, like the vast majority of individual trends, generalized well to the independent validation cohort. Our findings thus provide an improved understanding of perturbations of the microbiome-metabolome interface in IBD, including identification of many potential diagnostic and therapeutic targets.

Erratum: Strains, functions and dynamics in the expanded Human Microbiome Project.

This corrects the article DOI: 10.1038/nature23889.

Strains, functions and dynamics in the expanded Human Microbiome Project.

The characterization of baseline microbial and functional diversity in the human microbiome has enabled studies of microbiome-related disease, diversity, biogeography, and molecular function. The National Institutes of Health Human Microbiome Project has provided one of the broadest such characterizations so far. Here we introduce a second wave of data from the study, comprising 1,631 new metagenomes (2,355 total) targeting diverse body sites with multiple time points in 265 individuals. We applied updated profiling and assembly methods to provide new characterizations of microbiome personalization. Strain identification revealed subspecies clades specific to body sites; it also quantified species with phylogenetic diversity under-represented in isolate genomes. Body-wide functional profiling classified pathways into universal, human-enriched, and body site-enriched subsets. Finally, temporal analysis decomposed microbial variation into rapidly variable, moderately variable, and stable subsets. This study furthers our knowledge of baseline human microbial diversity and enables an understanding of personalized microbiome function and dynamics.

Genome analysis of a major urban malaria vector mosquito, Anopheles stephensi.

BACKGROUND: Anopheles stephensi is the key vector of malaria throughout the Indian subcontinent and Middle East and an emerging model for molecular and genetic studies of mosquito-parasite interactions. The type form of the species is responsible for the majority of urban malaria transmission across its range. RESULTS: Here, we report the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly represents more than 92% of the entire genome and was produced using a combination of 454, Illumina, and PacBio sequencing. Physical mapping assigned 62% of the genome onto chromosomes, enabling chromosome-based analysis. Comparisons between An. stephensi and An. gambiae reveal that the rate of gene order reshuffling on the X chromosome was three times higher than that on the autosomes. An. stephensi has more heterochromatin in pericentric regions but less repetitive DNA in chromosome arms than An. gambiae. We also identify a number of Y-chromosome contigs and BACs. Interspersed repeats constitute 7.1% of the assembled genome while LTR retrotransposons alone comprise more than 49% of the Y contigs. RNA-seq analyses provide new insights into mosquito innate immunity, development, and sexual dimorphism. CONCLUSIONS: The genome analysis described in this manuscript provides a resource and platform for fundamental and translational research into a major urban malaria vector. Chromosome-based investigations provide unique perspectives on Anopheles chromosome evolution. RNA-seq analysis and studies of immunity genes offer new insights into mosquito biology and mosquito-parasite interactions.