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To investigate a prebiotic fiber-enriched nutritional formula on health-related quality of life and metabolic control in type 2 diabetes.This was a 12-week, double-blind, placebo-controlled study with an unblinded dietary advice only comparator arm. Participants were randomized 2:1:1 to a prebiotic fiber-enriched nutritional formula (Active), a placebo fiber-absent nutritional formula (Placebo), or non-blinded dietary advice alone (Diet). Primary endpoint was change in core Type 2 Diabetes Distress Assessment System (cT2-DDAS) at week 12. HbA1c change was a key secondary endpoint.192 participants were randomized. Mean age was 54.3 years, HbA1c 7.8%, and BMI 35.9 kg/mA microbiome-targeting nutritional formula significantly improved cT2-DDAS and HbA1c, suggesting the potential for prebiotic fiber as a complement to lifestyle and/or pharmaceutical interventions for managing type 2 diabetes. This article is protected by copyright. All rights reserved.

Tools to measure in vivo redox activity of the gut microbiome and its influence on host health are lacking. In this paper, we present the design of new in vivo gut oxidation-reduction potential (ORP) sensors for rodents, to study host-microbe and microbe-environment interactions throughout the gut. These are the first in vivo sensors to combine ultrasonic wake-up and galvanic coupling telemetry, allowing for sensor miniaturization, experiment flexibility, and robust wireless measurements in live rodents. A novel study of in situ ORP along the intestine reveals biogeographical redox features that the ORP sensors can uniquely access in future gut microbiome studies.

Infant microbiome assembly is intensely studied in infants from industrialized nations, but little is known about this process in populations living non-industrialized lifestyles. In this study we deeply sequenced infant stool samples from the Hadza hunter-gatherers of Tanzania and analyzed them in a global meta-analysis. Infant microbiomes develop along lifestyle-associated trajectories, with over twenty percent of genomes detected in the Hadza infant gut representing phylogenetically diverse novel species. Industrialized infants, even those who are breastfed, have microbiomes characterized by a paucity of Bifidobacterium infantis and gene cassettes involved in human milk utilization. Strains within lifestyle-associated taxonomic groups are shared between mother-infant dyads, consistent with early-life inheritance of lifestyle-shaped microbiomes. The population-specific differences in infant microbiome composition and function underscore the importance of studying microbiomes from people outside of wealthy, industrialized nations.Recognition of work on indigenous communitiesResearch involving indigenous communities is needed for a variety of reasons including to ensure that scientific discoveries and understanding appropriately represent all populations and do not only benefit those living in industrialized nations. Special considerations must be made to ensure that this research is conducted ethically and in a non-exploitative manner. In this study we performed deep metagenomic sequencing on fecal samples that were collected from Hadza hunter-gatherers in 2013/2014 and were analyzed in previous publications using different methods (1, 2). A material transfer agreement with the National Institute for Medical Research in Tanzania ensures that stool samples collected are used solely for academic purposes, permission for the study was obtained from the National Institute of Medical Research (MR/53i 100/83, NIMR/HQ/R.8a/Vol.IX/1542) and the Tanzania Commission for Science and Technology, and verbal consent was obtained from the Hadza after the study’s intent and scope was described with the help of a translator. The publications that first described these samples included several scientists and Tanzanian field-guides as co-authors for the critical roles they played in sample collection, but as no new samples were collected in this study, only scientists who contributed to the analyses described here were included as co-authors in this publication. It is currently not possible for us to travel to Tanzania and present our results to the Hadza people, however we intend to do so once the conditions of the COVID-19 pandemic allow it.

The enteric pathogen Clostridioides difficile (Cd) is responsible for a toxin-mediated infection that causes more than 200,000 recorded hospitalizations and 13,000 deaths in the United States every year1. However, Cd can colonize the gut in the absence of disease symptoms. Prevalence of asymptomatic colonization by toxigenic Cd in healthy populations is high; asymptomatic carriers are at increased risk of infection compared to noncolonized individuals and may be a reservoir for transmission of Cd infection2,3. Elucidating the molecular mechanisms by which Cd persists in the absence of disease is necessary for understanding pathogenesis and developing refined therapeutic strategies. Here, we show with gut microbiome metatranscriptomic analysis that mice recalcitrant to Cd infection and inflammation exhibit increased community-wide expression of arginine and ornithine metabolic pathways. To query Cd metabolism specifically, we leverage RNA sequencing in gnotobiotic mice infected with two wild-type strains (630 and R20291) and isogenic toxin-deficient mutants of these strains to differentiate inflammation-dependent versus -independent transcriptional states. A single operon encoding oxidative ornithine degradation is consistently upregulated across non-toxigenic Cd strains. Combining untargeted and targeted metabolomics with bacterial and host genetics, we demonstrate that both diet- and host-derived sources of ornithine provide a competitive advantage to Cd, suggesting a mechanism for Cd persistence within a non-inflammatory, healthy gut., Using a combination of metabolomics and bacterial and host genetics, Pruss et al. show that upregulated oxidative ornithine metabolism in Clostridioides difficile promotes its persistence within the gastrointestinal tract under non-inflammatory conditions.

A disrupted “dysbiotic” gut microbiome engenders susceptibility to the diarrheal pathogen Clostridioides difficile by impacting the metabolic milieu of the gut. Diet, in particular the microbiota accessible carbohydrates (MACs) found in dietary fiber, is one of the most powerful ways to affect the composition and metabolic output of the gut microbiome. As such, diet is a powerful tool for understanding the biology of C. difficile and for developing alternative approaches for coping with this pathogen. One prominent class of metabolites produced by the gut microbiome are short chain fatty acids (SCFAs), the major metabolic end products of MAC metabolism. SCFAs are known decrease the fitness of C. difficile in vitro and that high intestinal SCFA concentrations are associated with reduced fitness of C. difficile in animal models of C. difficile infection (CDI). Here, we use controlled dietary conditions (8 diets that differ only by MAC composition) to show that C. difficile fitness is most consistently impacted by butyrate, rather than the other two prominent SCFAs (acetate and propionate), during murine model CDI. We similarly show that butyrate concentrations are lower in fecal samples from humans with CDI relative to healthy controls. Finally, we demonstrate that butyrate impacts growth in diverse C. difficile isolates. These findings provide a foundation for future work which will dissect how butyrate directly impacts C. difficile fitness and will lead to the development of diverse approaches distinct from antibiotics or fecal transplant, such as dietary interventions, for mitigating CDI in at-risk human populations.IMPORTANCEClostridioides difficile is a leading cause of infectious diarrhea in humans and it imposes a tremendous burden on the healthcare system. Current treatments for C. difficile infection (CDI) include antibiotics and fecal microbiota transplant, which contribute to recurrent CDIs and face major regulatory hurdles, respectively. Therefore, there is an ongoing need to develop new ways to cope with CDI. Notably, a disrupted “dysbiotic” gut microbiota is the primary risk factor for CDI but we incompletely understand how a healthy microbiota resists CDI. Here, we show that a specific molecule produced by the gut microbiota, butyrate, is negatively associated with C. difficile burdens in humans and in a mouse model of CDI and that butyrate impedes the growth of diverse C. difficile strains in pure culture. These findings help to build a foundation for designing alternative, possibly diet-based, strategies for mitigating CDI in humans.

The human gut microbiota produces dozens of small molecules that circulate in blood, accumulate to comparable levels as pharmaceutical drugs, and influence host physiology. Despite the importance of these metabolites to human health and disease, the origin of most microbially-produced molecules and their fate in the host remains largely unknown. Here, we uncover a host-microbe co-metabolic pathway for generation of hippuric acid, one of the most abundant organic acids in mammalian urine. Combining stable isotope tracing with bacterial and host genetics, we demonstrate reduction of phenylalanine to phenylpropionic acid by gut bacteria; the host re-oxidizes phenylpropionic acid involving medium-chain acyl-CoA dehydrogenase (MCAD). Generation of germ-free male and female MCAD−/− mice enabled gnotobiotic colonization combined with untargeted metabolomics to identify additional microbial metabolites processed by MCAD in host circulation. Our findings uncover a host-microbe pathway for the abundant, non-toxic phenylalanine metabolite hippurate and identify β-oxidation via MCAD as a novel mechanism by which mammals metabolize microbiota-derived metabolites.

A significant hurdle that has limited progress in microbiome science has been identifying and studying the diversity of metabolites produced by the gut microbes. Gut microbial metabolism produces thousands of difficult-to-identify metabolites, which present a challenge to study their roles in host biology. Over the recent years, mass spectrometry-based metabolomics has become one of the core technologies for identifying small metabolites. However, metabolomics expertise, ranging from sample preparation, instrument use, to data analysis, is often lacking in academic labs. Most targeted metabolomics methods provide high levels of sensitivity and quantification, while they are limited to a panel of predefined molecules that may not be informative to microbiome-focused studies. Here we have developed a gut microbe-focused, wide-spectrum, targeted metabolomic protocol using Liquid Chromatography-Mass Spectrometry (LC-MS) and bioinformatic analysis. This protocol enables users to carry out experiments from sample collection to data analysis, only requiring access to a LC-MS instrument, which is often available at local core facilities. By applying this protocol to samples containing human gut microbial metabolites, spanning from culture supernatant to human biospecimens, our approach enables high confidence identification of >800 metabolites. We expect this protocol will lower the barrier in tracking gut bacterial metabolism in vitro and in mammalian hosts, propelling hypothesis-driven mechanistic studies and accelerating our understanding of the gut microbiome at the chemical level.

Gut microorganisms modulate host phenotypes and are associated with numerous health effects in humans, ranging from host responses to cancer immunotherapy to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microorganisms has hindered efforts to define mechanistic connections between individual microbial strains and host phenotypes. One key way in which the gut microbiome influences host physiology is through the production of small molecules1–3, yet progress in elucidating this chemical interplay has been hindered by limited tools calibrated to detect the products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites in diverse sample types. We report the metabolic profiles of 178 gut microorganism strains using our library of 833 metabolites. Using this metabolomics resource, we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover a previously undescribed type of metabolism in Bacteroides, and reveal candidate biochemical pathways using comparative genomics. Microbiota-dependent metabolites can be detected in diverse biological fluids from gnotobiotic and conventionally colonized mice and traced back to the corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microorganisms and interactions between microorganisms and their host. A microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for the characterization of gut-resident microorganisms and the interactions between microorganisms and their host.

Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year1; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation—including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection. RNA-sequencing experiments determine that sorbitol, a metabolite produced by the host enzyme aldose reductase, is exploited by Clostridium difficile in its adaptation to inflammatory conditions in the gut.

SummaryProtists in the gut microbiota influence intestinal immunity and epithelial composition, yet the mechanisms used by these protists to shape the gut environment remain poorly understood. Here, we identify a new protist species, Tritrichomonas casperi, and show that metabolic differences between closely related commensal protists modulate their effects on host immunity and determine their ecological niche within the murine microbiota. Genomic and metabolomic analysis of commensal tritrichomonads reveal species-level differences in excretion of the tuft cell activating metabolite succinate, leading to differential induction of Th1, Th2, and Th17 cells in the small intestine. Using defined diets and in vitro growth assays, we show that different tritrichomonad species preferentially rely on dietary polysaccharides or mucus glycans, which leads to trans-kingdom competition with specific bacteria in the microbiota. Our findings elucidate differences in commensal tritrichomonad metabolism and suggest how dietary interventions could regulate the impact of these protists on gut health.

BACKGROUND Despite the rising popularity of plant-based alternative meats, there is limited evidence of the health effects of these products. OBJECTIVES We aimed to compare the effect of consuming plant-based alternative meat (Plant) as opposed to animal meat (Animal) on health factors. The primary outcome was fasting serum trimethylamine-N-oxide (TMAO). Secondary outcomes included fasting insulin-like growth factor 1, lipids, glucose, insulin, blood pressure, and weight. METHODS SWAP-MEAT (The Study With Appetizing Plantfood-Meat Eating Alternatives Trial) was a single-site, randomized crossover trial with no washout period. Participants received Plant and Animal products, dietary counseling, lab assessments, microbiome assessments (16S), and anthropometric measurements. Participants were instructed to consume ≥2 servings/d of Plant compared with Animal for 8 wk each, while keeping all other foods and beverages as similar as possible between the 2 phases. RESULTS The 36 participants who provided complete data for both crossover phases included 67% women, were 69% Caucasian, had a mean ± SD age 50 ± 14 y, and BMI 28 ± 5 kg/m2. Mean ± SD servings per day were not different by intervention sequence: 2.5 ± 0.6 compared with 2.6 ± 0.7 for Plant and Animal, respectively (P = 0.76). Mean ± SEM TMAO concentrations were significantly lower overall for Plant (2.7 ± 0.3) than for Animal (4.7 ± 0.9) (P = 0.012), but a significant order effect was observed (P = 0.023). TMAO concentrations were significantly lower for Plant among the n = 18 who received Plant second (2.9 ± 0.4 compared with 6.4 ± 1.5, Plant compared with Animal, P = 0.007), but not for the n = 18 who received Plant first (2.5 ± 0.4 compared with 3.0 ± 0.6, Plant compared with Animal, P = 0.23). Exploratory analyses of the microbiome failed to reveal possible responder compared with nonresponder factors. Mean ± SEM LDL-cholesterol concentrations (109.9 ± 4.5 compared with 120.7 ± 4.5 mg/dL, P = 0.002) and weight (78.7 ± 3.0 compared with 79.6 ± 3.0 kg, P

So much more to mucus Mammals accommodate a dense community of metabolically active microorganisms in their gut. This is not a passive relationship, and host and microbe have antagonistic as well as mutualistic responses to each other. Using a whole-colon imaging method in mice, Bergstrom et al. looked at the role of colonic mucus in segregating the microbiota from host cells during elimination of feces (see the Perspective by Birchenough and Johansson). Host goblet cells synthesize two forms of mucin that differ in branched chain O-glycosylation and the site of production in the colon. A “thick” mucus in the proximal, ascending colon wraps the microbiota to form fecal pellets. Transit along the distal, descending colon is lubricated by “thin” mucus that transiently links with the thick mucus. Normal mucus encapsulation prevents inflammation and hyperplasia and thus is important for maintenance of a healthy gut. Science , this issue p. 467 ; see also p. 402

A variety of cell surface structures dictate interactions between bacteria and their environment, including their viruses (bacteriophages). Members of the human gut Bacteroidetes characteristically produce several phase-variable capsular polysaccharides (CPSs), but their contributions to bacteriophage interactions are unknown. To begin to understand how CPSs have an impact on Bacteroides–phage interactions, we isolated 71 Bacteroides thetaiotaomicron-infecting bacteriophages from two locations in the United States. Using B. thetaiotaomicron strains that express defined subsets of CPSs, we show that CPSs dictate host tropism for these phages and that expression of non-permissive CPS variants is selected under phage predation, enabling survival. In the absence of CPSs, B. thetaiotaomicron escapes bacteriophage predation by altering expression of eight distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Our results reveal important roles for Bacteroides CPSs and other cell surface structures that allow these bacteria to persist under bacteriophage predation, and hold important implications for using bacteriophages therapeutically to target gut symbionts. Isolation of phages associated with the gut commensal Bacteroides thetaiotaomicron reveals a link between cell surface structures, including phase-variable capsular polysaccharides, lipoproteins and S-layer proteins, and susceptibility to phage infection.

BACKGROUND With the rising rates of obesity and associated metabolic disorders, there is a growing need for effective long-term weight-loss strategies, coupled with an understanding of how they interface with human physiology. Interest is growing in the potential role of gut microbes as they pertain to responses to different weight-loss diets; however, the ways that diet, the gut microbiota, and long-term weight loss influence one another is not well understood. OBJECTIVES Our primary objective was to determine if baseline microbiota composition or diversity was associated with weight-loss success. A secondary objective was to track the longitudinal associations of changes to lower-carbohydrate or lower-fat diets and concomitant weight loss with the composition and diversity of the gut microbiota. METHODS We used 16S ribosomal RNA gene amplicon sequencing to profile microbiota composition over a 12-mo period in 49 participants as part of a larger randomized dietary intervention study of participants consuming either a healthy low-carbohydrate or a healthy low-fat diet. RESULTS While baseline microbiota composition was not predictive of weight loss, each diet resulted in substantial changes in the microbiota 3-mo after the start of the intervention; some of these changes were diet specific (14 taxonomic changes specific to the healthy low-carbohydrate diet, 12 taxonomic changes specific to the healthy low-fat diet) and others tracked with weight loss (7 taxonomic changes in both diets). After these initial shifts, the microbiota returned near its original baseline state for the remainder of the intervention, despite participants maintaining their diet and weight loss for the entire study. CONCLUSIONS These results suggest a resilience to perturbation of the microbiota's starting profile. When considering the established contribution of obesity-associated microbiotas to weight gain in animal models, microbiota resilience may need to be overcome for long-term alterations to human physiology. This trial was registered at clinicaltrials.gov as NCT01826591.

INTRODUCTION: Although the genome is often called the blueprint of an organism, it is perhaps more accurate to describe it as a parts list composed of the various genes that may or may not be used in the different cell types of a multicellular organism. Although nearly every cell in the body has essentially the same genome, each cell type makes different use of that genome and expresses a subset of all possible genes. This has motivated efforts to characterize the molecular composition of various cell types within humans and multiple model organisms, both by transcriptional and proteomic approaches. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. RATIONALE: One caveat to current approaches to make cell atlases is that individual organs are often collected at different locations, collected from different donors, and processed using different protocols. Controlled comparisons of cell types between different tissues and organs are especially difficult when donors differ in genetic background, age, environmental exposure, and epigenetic effects. To address this, we developed an approach to analyzing large numbers of organs from the same individual. RESULTS: We collected multiple tissues from individual human donors and performed coordinated single-cell transcriptome analyses on live cells. The donors come from a range of ethnicities, are balanced by gender, have a mean age of 51 years, and have a variety of medical backgrounds. Tissue experts used a defined cell ontology terminology to annotate cell types consistently across the different tissues, leading to a total of 475 distinct cell types with reference transcriptome profiles. The full dataset can be explored online with the cellxgene tool. Data were collected for the bladder, blood, bone marrow, eye, fat, heart, kidney, large intestine, liver, lung, lymph node, mammary, muscle, pancreas, prostate, salivary gland, skin, small intestine, spleen, thymus, tongue, trachea, uterus, and vasculature. Fifty-nine separate specimens in total were collected, processed, and analyzed, and 483,152 cells passed quality control filtering. On a per-compartment basis, the dataset includes 264,824 immune cells, 104,148 epithelial cells, 31,691 endothelial cells, and 82,478 stromal cells. Working with live cells, as opposed to isolated nuclei, ensured that the dataset includes all mRNA transcripts within the cell, including transcripts that have been processed by the cell’s splicing machinery, thereby enabling insight into variation in alternative splicing. The Tabula Sapiens also provided an opportunity to densely and directly sample the human microbiome throughout the gastrointestinal tract. The intestines from two donors were sectioned into five regions: the duodenum, jejunum, ileum, and ascending and sigmoid colon. Each section was transected, and three to nine samples were collected from each location, followed by amplification and sequencing of the 16S ribosomal RNA gene. CONCLUSION: The Tabula Sapiens has revealed discoveries relating to shared behavior and subtle, organ-specific differences across cell types. We found T cell clones shared between organs and characterized organ-dependent hypermutation rates among B cells. Endothelial cells and macrophages are shared across tissues, often showing subtle but clear differences in gene expression. We found an unexpectedly large and diverse amount of cell type–specific RNA splice variant usage and discovered and validated many previously undefined splices. The intestinal microbiome was revealed to have nonuniform species distributions down to the 3-inch (7.62-cm) length scale. These are but a few examples of how the Tabula Sapiens represents a broadly useful reference to deeply understand and explore human biology at cellular resolution.

Ha and colleagues describe a previously unappreciated diversity of microbes in the mesenteric adipose tissue (MAT) surrounding the GI tract. Viable bacteria that are mislocalized from the gut microbiota and metabolically adapted to the MAT contribute to the “creeping fat” of Crohn’s disease.

Intestinal microbiotas contain beneficial microorganisms that protect against pathogen colonization; treatment with antibiotics disrupts the microbiota and compromises colonization resistance. Here, we determine the impact of exchanging microorganisms between hosts on resilience to the colonization of invaders after antibiotic-induced dysbiosis. We assess the functional consequences of dysbiosis using a mouse model of colonization resistance against Escherichia coli. Antibiotics caused stochastic loss of members of the microbiota, but the microbiotas of co-housed mice remained more similar to each other compared with the microbiotas among singly housed animals. Strikingly, co-housed mice maintained colonization resistance after treatment with antibiotics, whereas most singly housed mice were susceptible to E. coli. The ability to retain or share the commensal Klebsiella michiganensis, a member of the Enterobacteriaceae family, was sufficient for colonization resistance after treatment with antibiotics. K. michiganensis generally outcompeted E. coli in vitro, but in vivo administration of galactitol-a nutrient that supports the growth of only E. coli-to bi-colonized gnotobiotic mice abolished the colonization-resistance capacity of K. michiganensis against E. coli, supporting the idea that nutrient competition is the primary interaction mechanism. K. michiganensis also hampered colonization of the pathogen Salmonella, prolonging host survival. Our results address functional consequences of the stochastic effects of microbiota perturbations, whereby microbial transmission through host interactions can facilitate reacquisition of beneficial commensals, minimizing the negative impact of antibiotics. info:eu-repo/semantics/publishedVersion

Human-associated microbial communities have adapted to environmental pressures. Doses of antibiotics select for a community with increased antibiotic resistance, inflammation is accompanied by expansion of community members equipped to flourish in the presence of immune effectors and Western diets shift the microbiota away from fibre degraders in favour of species that thrive on mucus. Recent data suggest that the microbiota of industrialized societies differs substantially from the recent ancestral microbiota of humans. Rapid modernization, including medical practices and dietary changes, is causing progressive deterioration of the microbiota, and we hypothesize that this may contribute to various diseases prevalent in industrialized societies. In this Opinion article, we explore whether individuals in the industrialized world may be harbouring a microbial community that, while compatible with our environment, is now incompatible with our human biology. In this Opinion article, Sonnenburg and Sonnenburg explore whether individuals in the industrialized world may be harbouring a microbial community that is now incompatible with human biology, and they hypothesize that the modern, industrial lifestyle has contributed to alterations in the microbiota that may be linked to the deterioration of human health.

Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize in vitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex but not both, limiting their utility. In accompanying work, we constructed a complex synthetic community (104 strains, hCom1) containing the most common taxa in the human gut microbiome. Here, we used an iterative experimental process to improve hCom1 by filling open metabolic and/or anatomical niches. When we colonized germ-free mice with hCom1 and then challenged it with a human fecal sample, the consortium exhibited surprising stability; 89% of the cells and 58% of the taxa derive from the original community, and the pre- and post-challenge communities share a similar overall structure. We used these data to construct a second version of the community, adding 22 strains that engrafted following fecal challenge and omitting 7 that dropped out (119 strains, hCom2). In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by hCom2 versus human feces are similar in terms of microbiota-derived metabolites, immune cell profile, and bacterial density in the gut, suggesting that this consortium is a prototype of a model system for the human gut microbiome.

Gut microbes modulate host phenotypes and are associated with numerous health effects in humans, ranging from cancer immunotherapy response to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microbes has hindered defining mechanistic connections between individual microbial strains and host phenotypes. One key way the gut microbiome influences host physiology is through the production of small molecules1–3, yet progress in elucidating this chemical interplay has been hindered by limited tools calibrated to detect products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites (MDMs) in diverse sample types. We report the metabolic profiles of 178 gut microbe strains using our library of 833 metabolites. Leveraging this metabolomics resource we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover novel metabolism in Bacteroides, and employ comparative genomics-based discovery of candidate biochemical pathways. MDMs can be detected in diverse biofluids in gnotobiotic and conventional mice and traced back to corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microbe and microbe-host interactions.

SummaryMetabolism of dietary compounds by the gut microbiota generates a vast array of microbiome-dependent metabolites (MDMs), which are highly variable between individuals. The uremic MDMs (uMDMs) phenylacetylglutamine (PAG), p-cresol sulfate (PCS) and indoxyl sulfate (IS) accumulate during renal failure and are associated with poor outcomes. Targeted dietary interventions may reduce toxic MDMs generation; however, it is unclear if interindividual differences in diet or gut microbiome dominantly contribute to MDM variance. Here we use a 7-day homogeneous average American diet to standardize dietary precursor availability in 21 healthy individuals. Notably, the coefficient of variation in three uMDMs of interest, PAG, PCS, and IS (primary outcome), did not significantly decrease. The majority of circulating MDMs maintained variation despite identical diets. These results highlight the highly personalized profile of MDMs and limited contribution of short-term dietary heterogeneity, suggesting that dietary modification may need to be paired with microbial therapies to control MDM profiles.

Dietary fibers act through the microbiome to improve cardiovascular health and prevent metabolic disorders and cancer. To understand the health benefits of dietary fiber supplementation, we investigated two popular purified fibers, arabinoxylan (AX) and long-chain inulin (LCI), and a mixture of five fibers. We present multiomic signatures of metabolomics, lipidomics, proteomics, metagenomics, a cytokine panel, and clinical measurements on healthy and insulin-resistant participants. Each fiber is associated with fiber-dependent biochemical and microbial responses. AX consumption associates with a significant reduction in LDL and an increase in bile acids, contributing to its observed cholesterol reduction. LCI is associated with an increase in Bifidobacterium. However, at the highest LCI dose, there is increased inflammation and elevation in the liver enzyme alanine aminotransferase. This study yields insights into the effects of fiber supplementation and the mechanisms behind fiber-induced cholesterol reduction, and it shows effects of individual, purified fibers on the microbiome.

Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile (Cd) is responsible for a toxin-mediated colitis that causes 15,000 deaths in the U.S. yearly1, yet the molecular mechanisms by which Cd benefits from toxin-induced colitis remain understudied. Up to 21% of healthy adults are asymptomatic carriers of toxigenic Cd2, indicating that Cd can persist as part of a healthy microbiota; antibiotic-induced perturbation of the gut ecosystem is associated with transition to toxin-mediated disease. To understand how Cd metabolism adapts from a healthy gut to the inflamed conditions its toxins induce, we used RNA-seq to define the metabolic state of wild-type Cd versus an isogenic mutant lacking toxins in a mouse model. Combining bacterial and mouse genetics, we demonstrate that Cd utilizes sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation, including during Cd toxin-mediated disease. This work highlights a mechanism by which Cd can utilize a host-derived nutrient generated during toxin-induced disease by an enzyme not previously associated with infection.

SummaryDue to limitations on high-resolution strain tracking, selection dynamics during gut-microbiota colonization and transmission between hosts remain mostly mysterious. Here, we introduced hundreds of barcoded Escherichia coli strains into germ-free mice and quantified strain-level dynamics and metagenomic changes. Mutants involved in motility and utilization of abundant metabolites were reproducibly selected within days. Even with rapid selection, coprophagy enforced similar barcode distributions across co-housed mice. Whole-genome sequencing of hundreds of isolates quantified evolutionary dynamics and revealed linked alleles. A population-genetics model predicted substantial fitness advantages for certain mutants and that migration accounted for ~10% of the resident microbiota each day. Treatment with ciprofloxacin demonstrated the interplay between selection and transmission. While initial colonization was mostly uniform, in two mice a bottleneck reduced diversity and selected for ciprofloxacin resistance in the absence of drug. These findings highlight the interplay between environmental transmission and rapid, deterministic selection during evolution of the intestinal microbiota.

Diet modulates the gut microbiome, and gut microbes, in turn, can impact the immune system. Here, we used two gut microbiota-targeted dietary interventions, plant-based fiber or fermented foods, to determine how each influences the human microbiome and immune system in healthy adults. Using a 17-week randomized, prospective study design combined with -omics measurements of microbiome and host, including extensive immune profiling, we found distinct effects of each diet. High-fiber consumers showed increased gut microbiome-encoded glycan-degrading CAZymes despite stable community diversity. Three distinct immunological trajectories in high fiber-consumers corresponded to baseline microbiota diversity. Alternatively, the high-fermented food diet steadily increased microbiota diversity and decreased inflammatory markers. The data highlight how coupling dietary interventions to deep and longitudinal immune and microbiome profiling can provide individualized and population-wide insight. Our results indicate that fermented foods may be valuable in countering the decreased microbiome diversity and increased inflammation pervasive in the industrialized society.

SummaryMechanistic understanding of the impacts of the gut microbiota on human health has been hampered by limited throughput in animal models. To enable systematic interrogation of gut-relevant microbial communities, here we generated hundreds of in vitro communities cultured from diverse stool samples in various media. Species composition revealed stool-derived communities that are phylogenetically complex, diverse, stable, and highly reproducible. Community membership depended on both medium and initial inoculum, with certain media preserving inoculum compositions. Different inocula yielded different community compositions, indicating their potential for personalized therapeutics. Communities were robust to freezing and large-volume culturing, enabling future translational applications. Defined communities were generated from isolates and reconstituted growth and composition similar to those of communities derived from stool inocula. Finally, in vitro experiments probing the response to ciprofloxacin successfully predicted many changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. Thus, stool-derived in vitro communities constitute a powerful resource for microbiota research.

Summary Recent studies emphasize the role of microbial metabolites in regulating gastrointestinal (GI) physiology through activation of host receptors, highlighting the potential for inter-kingdom signaling in treating GI disorders. In this study, we show that tryptamine, a tryptophan-derived bacterial metabolite, stimulates mucus release from goblet cells via activation of G-protein-coupled receptor (GPCR) 5-HT4R. Germ-free mice colonized with engineered Bacteroides thetaiotaomicron optimized to produce tryptamine (Trp D+) exhibit decreased weight loss and increased mucus release following dextran sodium sulfate treatment when compared with mice colonized with control B. thetaiotaomicron (Trp D-). Additional beneficial effects in preventing barrier disruption and lower disease activity index were seen only in female mice, highlighting sex-specific effects of the bacterial metabolite. This study demonstrates potential for the precise modulation of mucus release by microbially produced 5-HT4 GPCR agonist as a therapeutic strategy to treat inflammatory conditions of the GI tract., Graphical Abstract, Highlights • Tryptamine increases serotonin-receptor4-dependent colonic mucus release • Bacterially derived tryptamine attenuates weight loss in DSS colitis mouse model • Protective effect of tryptamine in DSS colitis is more pronounced in female mice • Tryptamine reduces colitis severity and barrier disruption specifically in female mice, Rodent Gastroenterology; Microbiology

SUMMARY Clostridium difficile infection (CDI) is an enteric bacterial disease that is increasing in prevalence worldwide. C. difficile capitalizes on gut inflammation and microbiome dysbiosis to establish infection, with symptoms ranging from watery diarrhea to toxic megacolon. We reported that the safe-in-human clinical drug ebselen (ClinicalTrials.gov: NCT03013400, NCT01452607, NCT00762671, and NCT02603081) has biochemical, cell-based, and in vivo efficacy against the toxins of C. difficile. Here, we show that ebselen treatment reduces recurrence rates and decreases colitis in a hamster model of relapsing CDI. Furthermore, ebselen treatment does not alter microbiome diversity and promotes recovery back to that of healthy controls after antibiotic-induced dysbiosis in healthy and C. difficile-infected mice. This increased microbiome recovery upon ebselen treatment correlates with a decrease in host-derived inflammatory markers, suggesting that the anti-inflammatory properties of ebselen, combined with its anti-toxin function, help to mitigate the major clinical challenges of CDI, including recurrence, microbial dysbiosis, and colitis., In Brief Garland et al. show in a hamster model that the safe-in-human molecule ebselen, with its anti-toxin and anti-inflammatory activity, decreases colitis, and prevents reoccurrence of C. difficile infection. Ebselen also helps to reduce inflammation and promote recovery of microbiome diversity after antibiotic treatment in mice., Graphical Abstract

Sepsis is a deleterious immune response to infection that leads to organ failure and is the 11th most common cause of death worldwide. Despite plaguing humanity for thousands of years, the host factors that regulate this immunological response and subsequent sepsis severity and outcome are not fully understood. Here we describe how the Western diet (WD), a diet high in fat and sucrose and low in fiber, found rampant in industrialized countries, leads to worse disease and poorer outcomes in an LPS-driven sepsis model in WD-fed mice compared with mice fed standard fiber-rich chow (SC). We find that WD-fed mice have higher baseline inflammation (metaflammation) and signs of sepsis-associated immunoparalysis compared with SC-fed mice. WD mice also have an increased frequency of neutrophils, some with an “aged” phenotype, in the blood during sepsis compared with SC mice. Importantly, we found that the WD-dependent increase in sepsis severity and higher mortality is independent of the microbiome, suggesting that the diet may be directly regulating the innate immune system through an unknown mechanism. Strikingly, we could predict LPS-driven sepsis outcome by tracking specific WD-dependent disease factors (e.g., hypothermia and frequency of neutrophils in the blood) during disease progression and recovery. We conclude that the WD is reprogramming the basal immune status and acute response to LPS-driven sepsis and that this correlates with alternative disease paths that lead to more severe disease and poorer outcomes.

SummaryOur emerging view of the gut microbiome largely focuses on bacteria and less is known about other microbial components such as of bacteriophages (phages). Though phages are abundant in the gut, very few phages have been isolated from this ecosystem. Here, we report the genomes of 27 phages from the United States and Bangladesh that infect the prevalent human gut bacterium Bacteroides thetaiotaomicron. These phages are mostly distinct from previously sequenced phages with the exception of two, which are crAss-like phages. We compare these isolates to existing human gut metagenomes, revealing similarities to previously inferred phages and additional unexplored phage diversity. Finally, we use host tropisms of these phages to identify alleles of phage structural genes associated with infectivity. This work provides a detailed view of the gut’s “viral dark matter” and a framework for future efforts to further integrate isolation- and sequencing-focused efforts to understand gut-resident phages.

Efforts to probe the role of the gut microbiota in disease would benefit from a system in which patient-derived bacterial communities can be studied at scale. We addressed this by validating a strategy to propagate phylogenetically complex, diverse, stable, and highly reproducible stool-derived communities in vitro. We generated hundreds of in vitro communities cultured from diverse stool samples in various media; certain media generally preserved inoculum composition, and inocula from different subjects yielded source-specific community compositions. Upon colonization of germ-free mice, community composition was maintained, and the host proteome resembled the host from which the community was derived. Treatment with ciprofloxacin in vivo increased susceptibility to Salmonella invasion in vitro, and the in vitro response to ciprofloxacin was predictive of compositional changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. These findings demonstrate that stool-derived in vitro communities can serve as a powerful system for microbiota research.

Appreciation is growing of how our gut microbes shape health and disease. Now, a study of ancient human faeces sheds light on how microbial populations in the gut have changed during the past 2,000 years. Insights into gut-microbe changes during the past 2,000 years.

The development of experimental techniques capable of probing the viscoelasticity of soft materials over a broad range of time scales is essential to uncovering the physics that governs their behavior. In this work, we develop a microrheology technique that requires only 12 μL of sample and is capable of resolving dynamic behavior ranging in time scales from 10–6 to 10 s. Our approach, based on dynamic light scattering in the single-scattering limit, enables the study of polymer gels and other soft materials over a vastly larger hierarchy of time scales than macrorheology measurements. Our technique captures the viscoelastic modulus of polymer hydrogels with a broad range of stiffnesses from 10 to 104 Pa. We harness these capabilities to capture hierarchical molecular relaxations in DNA and to study the rheology of precious biological materials that are impractical for macrorheology measurements, including decellularized extracellular matrices and intestinal mucus. The use of a commercially available benchtop setup that is already available to a variety of soft matter researchers renders microrheology measurements accessible to a broader range of users than existing techniques, with the potential to reveal the physics that underlies complex polymer hydrogels and biological materials., A broadly accessible microrheology technique is developed that reveals viscoelasticity across broad time scales for a variety of soft and biological materials.

The first rudimentary evidence that the human body harbors a microbiota hinted at the complexity of host-associated microbial ecosystems. Now, almost 400 years later, a renaissance in the study of microbiota spatial organization, driven by coincident revolutions in imaging and sequencing technologies, is revealing functional relationships between biogeography and health, particularly in the vertebrate gut. In this review, we present our current understanding of principles governing the localization of intestinal bacteria, and spatial relationships between bacteria and their hosts. We further discuss important emerging directions that will enable progressing from the inherently descriptive nature of localization and –omics technologies to provide functional, quantitative, and mechanistic insight into this complex ecosystem.

While the gut microbiota's malleability makes it highly responsive to our environment, it also renders it susceptible to rapid selection by factors associated with an industrialized lifestyle. Recently, in Cell, Vangay et al. (2018) have tracked and revealed rapid and profound changes in the gut community of immigrants to one that resembles long-term residents of the U.S.

In this prospective cohort study, we examine the feasibility of a protocol to optimize microbiota for fecal microbiota transplantation (FMT). Donor stool metrics generally accepted as markers of gut health were used to select a stool donor based on superior microbial diversity, balanced constitution of Bacteroidetes versus Firmicutes and high concentration of fecal butyrate. Selected donor microbiota was then administered via FMT. A total of 10 patients with median age of 12 years with recurrent Clostridium difficile infection received the intervention. The rate of recurrence-free resolution with 1-2 FMTs was 100% at Week 10. With a single FMT, 80% of patients cleared Clostridium difficile infection without recurrence, whereas 20% of patients required a single re-treatment. No serious adverse events occurred. Microbiota sequencing revealed that recipients' gut microbiota phylogenic diversity increased by 72-hours post-transplantation, with sustainment over 10-week follow-up. This study highlights the feasibility of purposefully selecting the most ideal microbiota for transplantation.

Clostridial metabolite production The clostridia are Firmicute bacterial commensals commonly found in the mammalian gut. Clostridia produce a range of metabolites that diffuse into the host's circulation and have been difficult to manipulate genetically, but Guo et al. successfully developed a CRISPR-Cas9 deletion system in Clostridium sporogenes (see the Perspective by Henke and Clardy). The authors used deletion mutants and mass spectrometry to elucidate clostridial synthesis of several different branched short-chain fatty acids (SCFAs), including isobutyrate, 2-methylbutyrate, and isovalerate. Germ-free mice colonized with mutants incapable of synthesizing SCFAs showed altered immunoglobulin A production. This finding potentially links bacterial SCFA production and host responses to the presence of the clostridia. Science , this issue p. eaav1282 ; see also p. 1309

It has long been known that proteolytic Clostridia obtain their energy by coupling oxidative and reductive pathways for amino acid metabolism – the Stickland reaction1. The oxidation of one amino acid is coupled with reduction of another, yielding energy in the former step and re-achieving redox balance with the latter. Here, we find that the gut bacterium, Clostridium sporogenes metabolizes amino acids through reductive pathways to produce metabolites that circulate within the host. Measurements in vitro indicate that reductive Stickland pathways are coupled to ATP formation, revealing their role in energy capture by gut bacteria. By probing the genetics of C. sporogenes, we find that the Rnf complex is involved in reductive amino acid metabolism. Rnf complex mutants are attenuated for growth in the mouse gut, demonstrating the importance of energy capture during reductive metabolism for gut colonization. Our findings reveal that the production of high-abundance molecules by a commensal bacterium within the host gut is linked to an energy yielding redox process.

SummaryClostridium difficile infection (CDI) is an enteric bacterial disease that is increasing in prevalence worldwide. C. difficile capitalizes on gut inflammation and microbiome dysbiosis to establish infection, with symptoms ranging from watery diarrhea to toxic megacolon. We recently reported that the safe in human clinical drug candidate ebselen (NCT03013400, NCT01452607, NCT00762671, NCT02603081) has biochemical, cell-based and in vivo efficacy against the bacterial toxins of C. difficile. Here, we show that ebselen treatment reduces recurrence rates and decreases colitis in a hamster relapse model of CDI. Furthermore, ebselen treatment does not alter microbiome diversity but promotes its recovery back to that of healthy controls after antibiotic-induced dysbiosis in both healthy and C. difficile-infected mice. This increased microbiome recovery upon ebselen treatment correlates with a decrease in host-derived inflammatory markers suggesting that the anti-inflammatory properties of ebselen, combined with its anti-toxin function, help to mitigate the major clinical challenges of CDI, including recurrence, microbial dysbiosis, and colitis.

BACKGROUND The collection of trillions of microbes inhabiting the human gut, called the microbiome or microbiota, has captivated the biomedical research community for the past decade. Intimate connections exist between the microbiota and the immune system, central nervous system, and metabolism. The growing realization of the fundamental role that the microbiota plays in human health has been accompanied by the challenge of trying to understand which features define a healthy gut community and how these may differ depending upon context. Such insight will lead to new routes of disease treatment and prevention and may illuminate how lifestyle-driven changes to the microbiota can impact health across populations. Individuals living traditional lifestyles around the world share a strikingly similar microbiota composition that is distinct from that found in industrialized populations. Indeed, lineages of gut microbes have cospeciated with humans over millions of years, passing through hundreds of thousands of generations, and lend credence to the possibility that our microbial residents have shaped our biology throughout evolution. Relative to the “traditional” microbiota, the “industrial” microbiota appears to have lower microbial diversity, with major shifts in membership and functions. Individuals immigrating from nonindustrialized to industrialized settings or living at different intermediate states between foraging and industrialization have microbiota composition alterations that correspond to time and severity of lifestyle change. Industrial advances including antibiotics, processed food diets, and a highly sanitized environment have been shown to influence microbiota composition and transmission and were developed and widely implemented in the absence of understanding their effects on the microbiota. ADVANCES Here, we argue that the microbiota harbored by individuals living in the industrialized world is of a configuration never before experienced by human populations. This “new,” industrial microbiota has been shaped by recent progress in medicine, food, and sanitation. As technology and medicine have limited our exposure to pathogenic microbes, enabled feeding large populations inexpensively, and otherwise reduced acute medical incidents, many of these advances have been implemented in the absence of understanding the collateral damage inflicted on our resident microbes or the importance of these microbes in our health. More connections are being drawn between the composition and function of the gut microbiota and alteration in the immune status of the host. These relationships connect the industrial microbiota to the litany of chronic diseases that are driven by inflammation. Notably, these diseases spread along with the lifestyle factors that are known to alter the microbiota. While researchers have been uncovering the basic tenets of how the microbiota influences human health, there has been a growing realization that as the industrial lifestyle spreads globally, changes to the human microbiota may be central to the coincident spread of non-communicable, chronic diseases and may not be easily reversed. OUTLOOK We suggest that viewing microbiota biodiversity with an emphasis on sustainability and conservation may be an important approach to safeguarding human health. Understanding the services provided by the microbiota to humans, analogous to how ecosystem services are used to place value on aspects of macroecosystems, could aid in assessing the cost versus benefit of specific microbiota dysfunctions that are induced by different aspects of lifestyle. A key hurdle is to establish the impact of industrialization-induced changes to the microbiota on human health. The severity of this impact might depend on the specifics of numerous factors, including health status, diet, human genotype, and lifestyle. Isolating and archiving bacterial strains that are sensitive to industrialization may be required to enable detailed study of these organisms and to preserve ecosystem services that are unique to those strains and potentially beneficial to human health. Determining a path forward for sustainable medical practices, diet, and sanitation that is mindful of the importance and fragility of the microbiota is needed if we are to maintain a sustainable relationship with our internal microbial world.

Intestinal microbiotas contain beneficial microorganisms that protect against pathogen colonization; treatment with antibiotics disrupts the microbiota and compromises colonization resistance. Here, we determine the impact of exchanging microorganisms between hosts on resilience to the colonization of invaders after antibiotic-induced dysbiosis. We assess the functional consequences of dysbiosis using a mouse model of colonization resistance against Escherichia coli. Antibiotics caused stochastic loss of members of the microbiota, but the microbiotas of co-housed mice remained more similar to each other compared with the microbiotas among singly housed animals. Strikingly, co-housed mice maintained colonization resistance after treatment with antibiotics, whereas most singly housed mice were susceptible to E. coli. The ability to retain or share the commensal Klebsiella michiganensis, a member of the Enterobacteriaceae family, was sufficient for colonization resistance after treatment with antibiotics. K. michiganensis generally outcompeted E. coli in vitro, but in vivo administration of galactitol-a nutrient that supports the growth of only E. coli-to bi-colonized gnotobiotic mice abolished the colonization-resistance capacity of K. michiganensis against E. coli, supporting the idea that nutrient competition is the primary interaction mechanism. K. michiganensis also hampered colonization of the pathogen Salmonella, prolonging host survival. Our results address functional consequences of the stochastic effects of microbiota perturbations, whereby microbial transmission through host interactions can facilitate reacquisition of beneficial commensals, minimizing the negative impact of antibiotics.

The gut microbiota is a complex network of diverse organisms that exhibits plasticity and is capable of impacting host immunity. The malleability of the microbiota presents microbial alteration as an avenue for tuning the immune system to different set points, an approach that has the potential to enable a wide range of therapeutic applications, and ultimately enable disease prevention. Despite the tremendous potential in harnessing the microbiome-immune axis to benefit human health, key barriers must be addressed to hasten translation. Studies using mouse models have identified numerous specific interactions between the gut microbiota and both local and systemic immunity, in several cases using gnotobiotics and other highly controlled approaches to establish causal relationships. Recent advances in our ability to perform expansive profiling of both the microbiota and the immune system now enable exploring human-gut microbiota connections more thoroughly than ever before. An important next step in realizing the power of microbiome reprogramming is to elucidate a human-relevant “map” of microbial-immune wiring, while focusing on animal studies to probe the subset of interactions likely to be relevant to human biology. Such efforts have the potential for revealing new paradigms in immune function as it relates to the microbiome, and for harnessing this connection to improve human health. Here we provide an overview of this field’s current status and discuss two approaches for establishing priorities for detailed investigation: (i) longitudinal intervention studies in humans probing the dynamics of both the microbiota and the immune system, and (ii) the study of traditional populations to assess lost features of human microbial identity whose absence may be contributing to the rise of immunological disorders. These human centered approaches offer a judicious path forward to capitalize on the potent power of the microbiota as a driver in immune health.

Diet is the largest source of plant-derived metabolites that influence human health. The gut microbiota can metabolize these molecules, altering their biological function. However, little is known about the gut bacterial pathways that process plant-derived molecules. Glucosinolates are well-known metabolites in brassica vegetables and metabolic precursors to cancer-preventive isothiocyanates. Here, we identify a genetic and biochemical basis for isothiocyanate formation byBacteroides thetaiotaomicron,a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism inB. thetaiotaomicron.Expression of BT2159-BT2156 in a non-metabolizing relative,Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157in vitro. Monocolonization of mice with mutantBtΔ2157showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.

Small intestinal bacterial overgrowth (SIBO) has been implicated in symptoms associated with functional gastrointestinal disorders (FGIDs), though mechanisms remain poorly defined and treatment involves non-specific antibiotics. Here we show that SIBO based on duodenal aspirate culture reflects an overgrowth of anaerobes, does not correspond with patient symptoms, and may be a result of dietary preferences. Small intestinal microbial composition, on the other hand, is significantly altered in symptomatic patients and does not correspond with aspirate culture results. In a pilot interventional study we found that switching from a high fiber diet to a low fiber, high simple sugar diet triggered FGID-related symptoms and decreased small intestinal microbial diversity while increasing small intestinal permeability. Our findings demonstrate that characterizing small intestinal microbiomes in patients with gastrointestinal symptoms may allow a more targeted antibacterial or a diet-based approach to treatment.

The production and handling of serotonin (5-HT) is an important determinant of colonic motility and has been reported to be altered in gastrointestinal (GI) disorders such as irritable bowel syndrome (IBS). Recent studies suggest that the intestinal microbiota and sex of the host can influence expression of genes involved in 5-HT biosynthesis and signaling. While expression of genes in serotonergic pathways has been shown to be variable, it remains unclear whether genes within this pathway are coregulated. As a first step in that direction, we investigated potential correlations in relative mRNA expression of serotonergic genes, in the proximal colon isolated from male and female mice in different states of microbial association: germ-free (GF), humanized (ex-germ-free colonized with human gut microbiota, HM), and conventionally raised (CR) mice. Among the 10 pairwise comparisons conducted between five serotonergic transcripts, Tph1, Chga, Maoa, Slc6a4, and Htr4, we found a strong, positive correlation between colonic expression of Slc6a4 and Htr4 across different colonization states and sexes. We also identified a positive correlation between the expression of Tph1 and Chga; however, there were no correlations observed between any other tested pair of 5-HT-related transcripts. These data suggest that correlated expression of Slc6a4 and Htr4 likely involves coregulation of genes located on different chromosomes which modulate serotonergic activity in the gut. Further work will need to be done to understand the pathways and cell types responsible for this correlated expression, given the important role of 5-HT in gastrointestinal physiology.

The study of traditional populations provides a view of human-associated microbes unperturbed by industrialization, as well as a window into the microbiota that co-evolved with humans. Here we discuss our recent work characterizing the microbiota from the Hadza hunter-gatherers of Tanzania. We found seasonal shifts in bacterial taxa, diversity, and carbohydrate utilization by the microbiota. When compared to the microbiota composition from other populations around the world, the Hadza microbiota shares bacterial families with other traditional societies that are rare or absent from microbiotas of industrialized nations. We present additional observations from the Hadza microbiota and their lifestyle and environment, including microbes detected on hands, water, and animal sources, how the microbiota varies with sex and age, and the shortterm effects of introducing agricultural products into the diet. In the context of our previously published findings and of these additional observations, we discuss a path forward for future work.

Secondary bile acids (SBAs) are derived from primary bile acids (PBAs) in a process reliant on biosynthetic capabilities possessed by few microbes. To evaluate the role of BAs in intestinal inflammation, we performed metabolomic, microbiome, metagenomic, and transcriptomic profiling of stool from ileal pouches (surgically created resevoirs) in colectomy-treated patients with ulcerative colitis (UC) versus controls (familial adenomatous polyposis [FAP]). We show that relative to FAP, UC pouches have reduced levels of lithocholic acid and deoxycholic acid (normally the most abundant gut SBAs), genes required to convert PBAs to SBAs, and Ruminococcaceae (one of few taxa known to include SBA-producing bacteria). In three murine colitis models, SBA supplementation reduces intestinal inflammation. This anti-inflammatory effect is in part dependent on the TGR5 bile acid receptor. These data suggest that dysbiosis induces SBA deficiency in inflammatory-prone UC patients, which promotes a pro-inflammatory state within the intestine that may be treated by SBA restoration.