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In mice on a low microbiota-accessible carbohydrate (MAC) diet, the diversity of the gut microbiota is depleted, and the effect is transferred and compounded over generations; this phenotype is only reversed after supplementation of the missing taxa via faecal microbiota transplantation, suggesting dietary intervention alone may by insufficient at managing diseases characterized by a dysbiotic microbiota. Effect of diet on gut microbiota Over our history, humans have experienced major dietary changes, including a shift to low-fibre intake in Westernized populations, which is paralleled by a general loss in the diversity of the gut microbiota. Microbiota-accessible carbohydrates (MACs), which are abundant in dietary fibre, are the primary source of carbon and energy for the distal gut microbiota. This study shows that in mice on a low-MAC diet, the diversity of the microbiota is depleted and that this effect is transferred and compounded over generations, such that the low abundant taxa are progressively lost from one generation to the next, particularly those of the order Bacteroidales, which are proficient in the consumption of dietary fibre. This phenotype is not reversible simply by reintroducing dietary MACs, but requires supplementation of the missing taxa via faecal microbiota transplantation. These findings suggest that a change in diet alone may be insufficient to restore a healthy microbiota in individuals with dysbiosis. The gut is home to trillions of microorganisms that have fundamental roles in many aspects of human biology, including immune function and metabolism 1 , 2 . The reduced diversity of the gut microbiota in Western populations compared to that in populations living traditional lifestyles presents the question of which factors have driven microbiota change during modernization. Microbiota-accessible carbohydrates (MACs) found in dietary fibre have a crucial involvement in shaping this microbial ecosystem, and are notably reduced in the Western diet (high in fat and simple carbohydrates, low in fibre) compared with a more traditional diet 3 . Here we show that changes in the microbiota of mice consuming a low-MAC diet and harbouring a human microbiota are largely reversible within a single generation. However, over several generations, a low-MAC diet results in a progressive loss of diversity, which is not recoverable after the reintroduction of dietary MACs. To restore the microbiota to its original state requires the administration of missing taxa in combination with dietary MAC consumption. Our data illustrate that taxa driven to low abundance when dietary MACs are scarce are inefficiently transferred to the next generation, and are at increased risk of becoming extinct within an isolated population. As more diseases are linked to the Western microbiota and the microbiota is targeted therapeutically, microbiota reprogramming may need to involve strategies that incorporate dietary MACs as well as taxa not currently present in the Western gut.

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 molecules 1 – 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.

Antibiotic treatment disturbs the commensal microbiota and is often followed by infection with enteric pathogens such as Salmonella typhimurium and Clostridium difficile; pathogen expansion is fuelled by antibiotic-driven accumulation of commensal-liberated host mucosal carbohydrates. Gut microbes support pathogen proliferation Intestinal microbiota can provide protection against invading pathogens through competition for resources and production of specific antimicrobial products. But disruption of the microbiota with antibiotics can contribute to the emergence of several enteric pathogens. Justin Sonnenburg and colleagues show here that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium and Clostridium difficile , catabolize microbiota-liberated host sugars to fuel their growth in the mouse gut. In particular, the ability to use sialic acid cleaved from host polysaccharides by Bacteroides thetaiotaomicron is important for pathogen expansion. These findings identify a role for the gut microbiota in facilitating enteric pathogen infection and provide new options for developing therapeutics. The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens 1 , 2 , 3 , 4 . How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium ( S. typhimurium ) and Clostridium difficile , use a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo . Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo . Colonization of gnotobiotic mice with a sialidase-deficient mutant of Bacteroides thetaiotaomicron , a model gut symbiont, reduces free sialic acid levels resulting in C. difficile downregulating its sialic acid catabolic pathway and exhibiting impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

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. Here, using a mouse model, the authors report a previously undescribed role for medium-chain acyl-CoA dehydrogenase in host metabolism of gut microbiota metabolites, and show that circulating compounds, including the abundant organic acid hippurate, depend on host-microbe co-metabolism of phenylalanine by Clostridium sporogenes .

We investigate how host mucus glycan composition interacts with dietary carbohydrate content to influence the composition and expressed functions of a human gut community. The humanized gnotobiotic mice mimic humans with a nonsecretor phenotype due to knockout of their α1–2 fucosyltransferase (Fut2) gene. The fecal microbiota of Fut2 ⁻ mice that lack fucosylated host glycans show decreased alpha diversity relative to Fut2 ⁺ mice and exhibit significant differences in community composition. A glucose-rich plant polysaccharide-deficient (PD) diet exerted a strong effect on the microbiota membership but eliminated the effect of Fut2 genotype. Additionally fecal metabolites predicted host genotype in mice on a polysaccharide-rich standard diet but not on a PD diet. A more detailed mechanistic analysis of these interactions involved colonization of gnotobiotic Fut2 ⁺ and Fut2 ⁻ mice with Bacteroides thetaiotaomicron , a prominent member of the human gut microbiota known to adaptively forage host mucosal glycans when dietary polysaccharides are absent. Within Fut2 ⁻ mice, the B. thetaiotaomicron fucose catabolic pathway was markedly down-regulated, whereas BT4241–4247, an operon responsive to terminal β-galactose, the precursor that accumulates in the Fut2 ⁻ mice, was significantly up-regulated. These changes in B. thetaiotaomicron gene expression were only evident in mice fed a PD diet, wherein B. thetaiotaomicron relies on host mucus consumption. Furthermore, up-regulation of the BT4241–4247 operon was also seen in humanized Fut2 ⁻ mice. Together, these data demonstrate that differences in host genotype that affect the carbohydrate landscape of the distal gut interact with diet to alter the composition and function of resident microbes in a diet-dependent manner.

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.

A pathway for the production of aromatic amino acid metabolites in Clostridium sporogenes is described; modulation of serum levels of these metabolites in gnotobiotic mice affects intestinal permeability and systemic immunity. Gut bacterial pharmacy The human microbiome has a substantial effect on our health. Our gut microbes produce a range of small molecules, many of which can reach relevant concentrations, yet we know surprisingly little about microbial metabolic pathways and how they affect the host. Here, Justin Sonnenburg, Michael Fischbach and colleagues use genetics and metabolic profiling to identify the gene cluster of Clostridium sporogenes that metabolizes aromatic amino acids, several of the products of which are produced exclusively by the microbiota. For example, the neuroprotective agent indolepropionic acid (IPA) was also produced by several other gut bacteria. In mice with controlled bacterial colonies, the serum levels of IPA and host physiology can be modulated by genetic modification of C. sporogenes . The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream 1 , 2 , where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes , we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.

Dietary polysaccharides play a dominant role in shaping the composition and functionality of our gut microbiota. Dietary interventions using these m icrobiota- a ccessible c arbohydrates (MACs) serve as a promising tool for manipulating the gut microbial community. However, our current gap in knowledge regarding microbial metabolic pathways that are involved in the degradation of these MACs has made the design of rational interventions difficult. The issue is further complicated by the diversity of pathways observed for the utilization of similar MACs, even in closely related microbial strains. Our current work focuses on divergent fructan utilization pathways in two closely related B. thetaiotaomicron strains and provides an integrated approach to characterize the molecular basis for strain-level functional differences. Genomic differences between gut-resident bacterial strains likely underlie significant interindividual variation in microbiome function. Traditional methods of determining community composition, such as 16S rRNA gene amplicon sequencing, fail to capture this functional diversity. Metagenomic approaches are a significant step forward in identifying strain-level sequence variants; however, given the current paucity of biochemical information, they too are limited to mainly low-resolution and incomplete functional predictions. Using genomic, biochemical, and molecular approaches, we identified differences in the fructan utilization profiles of two closely related Bacteroides thetaiotaomicron strains. B. thetaiotaomicron 8736 ( Bt-8736 ) contains a fructan polysaccharide utilization locus (PUL) with a divergent susC / susD homolog gene pair that enables it to utilize inulin, differentiating this strain from other characterized Bt strains. Transfer of the distinct pair of susC / susD genes from Bt-8736 into the noninulin using type strain B. thetaiotaomicron VPI-5482 resulted in inulin use by the recipient strain, Bt ( 8736-2 ). The presence of the divergent susC / susD gene pair alone enabled the hybrid Bt ( 8736-2 ) strain to outcompete the wild-type strain in vivo in mice fed an inulin diet. Further, we discovered that the susC / susD homolog gene pair facilitated import of inulin into the periplasm without surface predigestion by an endo-acting enzyme, possibly due to the short average chain length of inulin compared to many other polysaccharides. Our data builds upon recent reports of dietary polysaccharide utilization mechanisms found in members of the Bacteroides genus and demonstrates how the acquisition of two genes can alter the functionality and success of a strain within the gut. IMPORTANCE Dietary polysaccharides play a dominant role in shaping the composition and functionality of our gut microbiota. Dietary interventions using these m icrobiota- a ccessible c arbohydrates (MACs) serve as a promising tool for manipulating the gut microbial community. However, our current gap in knowledge regarding microbial metabolic pathways that are involved in the degradation of these MACs has made the design of rational interventions difficult. The issue is further complicated by the diversity of pathways observed for the utilization of similar MACs, even in closely related microbial strains. Our current work focuses on divergent fructan utilization pathways in two closely related B. thetaiotaomicron strains and provides an integrated approach to characterize the molecular basis for strain-level functional differences.

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 year 1 . 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 infection 2 , 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.

Gut bacteria face a key problem in how they capture enough energy to sustain their growth and physiology. The gut bacterium Clostridium sporogenes obtains its energy by utilizing amino acids in pairs, coupling the oxidation of one to the reduction of another—the Stickland reaction. Oxidative pathways produce ATP via substrate-level phosphorylation, whereas reductive pathways are thought to balance redox. In the present study, we investigated whether these reductive pathways are also linked to energy generation and the production of microbial metabolites that may circulate and impact host physiology. Using metabolomics, we find that, during growth in vitro, C. sporogenes produces 15 metabolites, 13 of which are present in the gut of C. sporogenes -colonized mice. Four of these compounds are reductive Stickland metabolites that circulate in the blood of gnotobiotic mice and are also detected in plasma from healthy humans. Gene clusters for reductive Stickland pathways suggest involvement of electron transfer proteins, and experiments in vitro demonstrate that reductive metabolism is coupled to ATP formation and not just redox balance. Genetic analysis points to the broadly conserved Rnf complex as a key coupling site for energy transduction. Rnf complex mutants show aberrant amino acid metabolism in a defined medium and are attenuated for growth in the mouse gut, demonstrating a role of the Rnf complex in Stickland metabolism and gut colonization. Our findings reveal that the production of circulating metabolites by a commensal bacterium within the host gut is linked to an ATP-yielding redox process. The gut bacterium Clostridium sporogenes uses reductive Stickland reactions for energy and consequently produces metabolites that circulate in the host.