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Calories from any food have the potential to increase risk for obesity and cardiometabolic disease because all calories can directly contribute to positive energy balance and fat gain. However, various dietary components or patterns may promote obesity and cardiometabolic disease by additional mechanisms that are not mediated solely by caloric content. Researchers explored this topic at the 2017 CrossFit Foundation Academic Conference 'Diet and Cardiometabolic Health - Beyond Calories', and this paper summarizes the presentations and follow-up discussions. Regarding the health effects of dietary fat, sugar and non-nutritive sweeteners, it is concluded that food-specific saturated fatty acids and sugar-sweetened beverages promote cardiometabolic diseases by mechanisms that are additional to their contribution of calories to positive energy balance and that aspartame does not promote weight gain. The challenges involved in conducting and interpreting clinical nutritional research, which preclude more extensive conclusions, are detailed. Emerging research is presented exploring the possibility that responses to certain dietary components/patterns are influenced by the metabolic status, developmental period or genotype of the individual; by the responsiveness of brain regions associated with reward to food cues; or by the microbiome. More research regarding these potential 'beyond calories' mechanisms may lead to new strategies for attenuating the obesity crisis.

The human gut microbiota plays a key role in pharmacology, yet the mechanisms responsible remain unclear, impeding efforts toward personalized medicine. We recently identified a cytochrome-encoding operon in the common gut Actinobacterium Eggerthella lenta

The emergence of massively parallel sequencing technology has revolutionized microbial profiling, allowing the unprecedented comparison of microbial diversity across time and space in a wide range of host-associated and environmental ecosystems. Although t

Background: Frailty is a geriatric syndrome characterized by chronic inflammation and metabolic insufficiency that creates vulnerability to poor outcomes with aging. We hypothesize that interventions which target common underlying mechanism of aging could ameliorate frailty. Ketone bodies are metabolites produced during fasting or on a ketogenic diet that have pleiotropic effects on inflammatory and metabolic aging pathways in laboratory animal models. Ketone esters (KEs) are compounds that induce ketosis without dietary changes, but KEs have not been studied in an older adult population. Our long-term goal is to examine if KEs modulate aging biology mechanisms and clinical outcomes relevant to frailty in older adults., Objectives: The primary objective of this randomized, placebo-controlled, double-blinded, parallel-group, pilot trial is to determine tolerability of 12-weeks of KE ingestion in a broad population of older adults (≥ 65 years). Secondary outcomes include safety and acute blood ketone kinetics. Exploratory outcomes include physical function, cognitive function, quality of life, aging biomarkers and inflammatory measures., Methods: Community-dwelling adults who are independent in activities of daily living, with no unstable acute medical conditions (n = 30) will be recruited. The study intervention is a KE or a taste, appearance, and calorie matched placebo beverage. Initially, acute 4-hour ketone kinetics after 12.5g or 25g of KE consumption will be assessed. After collection of baseline safety, functional, and biological measurements, subjects will randomly be allocated to consume KE 25g or placebo once daily for 12-weeks. Questionnaires will assess tolerability daily for 2-weeks, and then via phone interview at bi-monthly intervals. Safety assessments will be repeated at week 4. All measures will be repeated at week 12., Conclusion: This study will evaluate feasibility, tolerability, and safety of KE consumption in older adults and provide exploratory data across a range of aging-related endpoints. This data will inform design of larger trials to rigorously test KE effects on aging mechanisms and clinical outcomes relevant to frailty., Competing Interests: The principal investigator (Dr. Newman), Dr. Brianna Stubbs, and the Buck Institute hold shares in BHB Therapeutics. Drs. Newman and Stubbs are inventors on patents relating to the use of ketone bodies that are assigned to The Buck Institute. All other authors have no conflicts to declare. Individual and institutional extensive conflict management plans were developed and approved by the Buck Institute and the IRB. Actions and decisions important to subject safety and study integrity are carried out by parties with no potential financial conflict. Participant consent is obtained by licensed registered nurses who have no financial conflict. Decisions on subject enrollment, continuation, and discontinuation are made by independent medical officers unaffiliated with Buck Institute and with no financial conflict. Data analysis for the primary outcome is carried out by an independent statistician with no financial conflict. Study staff, including the principal investigator, will maintain blinding through study completion unless unblinding is required for safety concerns. The principal investigator (Dr. Newman), Dr. Brianna Stubbs, and the Buck Institute hold shares in BHB Therapeutics. Drs. Newman (US 11,773,051 B2, US 11,608,306 B2) and Stubbs (US 11,645,228 B2) are inventors on patents relating to the use of ketone bodies that are assigned to The Buck Institute. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have no conflicts to declare., (Copyright: © 2024 Stubbs et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Drugs represent our first, and sometimes last, line of defense for many diseases, yet despite decades of research we still do not fully understand why a given drug works in one patient and fails in the next. The human gut microbiome is one of the missing puzzle pieces, due to its ability to parallel and extend host pathways for drug metabolism, along with more complex host-microbiome interactions. Herein, we focus on the well-established links between the gut microbiome and drugs for heart disease and cancer, plus emerging data on neurological disease. We highlight the interdisciplinary methods that are available and how they can be used to address major remaining knowledge gaps, including the consequences of microbial drug metabolism for treatment outcomes. Continued progress in this area promises fundamental biological insights into humans and their associated microbial communities and strategies for leveraging the microbiome to improve the practice of medicine.

Background: Exposure to persistent organic pollutants (POPs) and disruptions in the gastrointestinal microbiota have been positively correlated with a predisposition to factors such as obesity, metabolic syndrome, and type 2 diabetes; however, it is unclear how the microbiome contributes to this relationship., Objective: This study aimed to explore the association between early life exposure to a potent aryl hydrocarbon receptor (AHR) agonist and persistent disruptions in the microbiota, leading to impaired metabolic homeostasis later in life., Methods: This study used metagenomics, nuclear magnetic resonance (NMR)- and mass spectrometry (MS)-based metabolomics, and biochemical assays to analyze the gut microbiome composition and function, as well as the physiological and metabolic effects of early life exposure to 2,3,7,8-tetrachlorodibenzofuran (TCDF) in conventional, germ-free (GF), and Ahr -null mice. The impact of TCDF on Akkermansia muciniphila ( A. muciniphila ) in vitro was assessed using optical density (OD 600), flow cytometry, transcriptomics, and MS-based metabolomics., Results: TCDF-exposed mice exhibited lower abundances of A. muciniphila , lower levels of cecal short-chain fatty acids (SCFAs) and indole-3-lactic acid (ILA), as well as lower levels of the gut hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), findings suggestive of disruption in the gut microbiome community structure and function. Importantly, microbial and metabolic phenotypes associated with early life POP exposure were transferable to GF recipients in the absence of POP carry-over. In addition, AHR-independent interactions between POPs and the microbiota were observed, and they were significantly associated with growth, physiology, gene expression, and metabolic activity outcomes of A. muciniphila , supporting suppressed activity along the ILA pathway., Conclusions: These data obtained in a mouse model point to the complex effects of POPs on the host and microbiota, providing strong evidence that early life, short-term, and self-limiting POP exposure can adversely impact the microbiome, with effects persisting into later life with associated health implications. https://doi.org/10.1289/EHP13356.

The past 50 years of interdisciplinary research in humans and model organisms has delivered unprecedented insights into the mechanisms through which diet affects energy balance. However, translating these results to prevent and treat obesity and its associated diseases remains challenging. Given the vast scope of this literature, we focus this Review on recent conceptual advances in molecular nutrition targeting the management of energy balance, including emerging dietary and pharmaceutical interventions and their interactions with the human gut microbiome. Notably, multiple current dietary patterns of interest embrace moderate-to-high fat intake or prioritize the timing of eating over macronutrient intake. Furthermore, the rapid expansion of microbiome research findings has complicated multiple longstanding tenets of nutrition while also providing new opportunities for intervention. Continued progress promises more precise and reliable dietary recommendations that leverage our growing knowledge of the microbiome, the changing landscape of clinical interventions, and our molecular understanding of human biology., Competing Interests: Declaration of interests P.J.T. is on the Scientific Advisory Boards for Pendulum, Seed, and SNIPRbiome; there is no direct overlap between the current study and these consulting duties., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The human gut microbiome vastly extends the set of metabolic reactions catalysed by our own cells, with far-reaching consequences for host health and disease. However, our knowledge of gut microbial metabolism relies on a handful of model organisms, limiting our ability to interpret and predict the metabolism of complex microbial communities. In this Perspective, we discuss emerging tools for analysing and modelling the metabolism of gut microorganisms and for linking microorganisms, pathways and metabolites at the ecosystem level, highlighting promising best practices for researchers. Continued progress in this area will also require infrastructure development to facilitate cross-disciplinary synthesis of scientific findings. Collectively, these efforts can enable a broader and deeper understanding of the workings of the gut ecosystem and open new possibilities for microbiome manipulation and therapy., (© 2024. Springer Nature Limited.)

In a recent study examining the effects of manipulating the gut microbiome on bone, a control group of mice in which the microbiome was altered using a non-caloric, aspartame-based sweetener resulted in whole bone strength being 40% greater than expected from geometry alone, implicating enhanced bone tissue strength. However, the study was not designed to detect changes in bone in this control group and was limited to young male mice. Here we report a replication study examining how changes in the gut microbiome caused by aspartame-based sweetener influence bone. Male and female C57Bl/6 J mice were untreated or treated with a high dose of sweetener (10 g/L) in their drinking water from either 1 to 4 mo of age (young cohort; n  = 80) or 1 to 22 mo of age (aged cohort; n  = 52). Sweetener did not replicate the modifications to the gut microbiome observed in the initial study and did not result in an increase in bone tissue strength in either sex at either age. Aged male mice dosed with sweetener had larger bones (+17% femur section modulus, p <.001) and greater whole bone strength (+22%, p =.006) but the increased whole bone strength was explained by the associated increase in body mass (+9%, p <.001). No differences in body mass, whole bone strength, or femoral geometry were associated with sweetener dosing in males from the young cohort or females at either age. As we were unable to replicate the gut microbiota observed in the initial experiment, it remains unclear if changes in the gut microbiome can enhance bone tissue strength. Although prior work studying gut microbiome-induced changes in bone with oral antibiotics has been highly repeatable, the current study highlights the variability of nutritional manipulations of the gut microbiota in mice., Competing Interests: The authors have no conflicts of interest to disclose., (© The Author(s) [2024]. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Dose-limiting toxicities remain a major barrier to drug development and therapy, revealing the limited predictive power of human genetics. Herein, we demonstrate the utility of a more comprehensive approach to studying drug toxicity through longitudinal study of the human gut microbiome during colorectal cancer (CRC) treatment (NCT04054908) coupled to cell culture and mouse experiments. 16S rRNA gene and metagenomic sequencing revealed significant shifts in gut microbial community structure during treatment with oral fluoropyrimidines, which was validated in an independent cohort. Gene abundance was also markedly changed by oral fluoropyrimidines, including an enrichment for the preTA operon, which is sufficient for the inactivation of active metabolite 5-fluorouracil (5-FU). Higher levels of preTA led to increased 5-FU depletion by the gut microbiota grown ex vivo . Germ-free and antibiotic-treated mice had increased fluoropyrimidine toxicity, which was rescued by colonization with the mouse gut microbiota, preTA + E. coli , or CRC patient stool with high preTA levels. preTA abundance was negatively associated with patient toxicities. Together, these data support a causal, clinically relevant interaction between a human gut bacterial operon and the dose-limiting side effects of cancer treatment. Our approach is generalizable to other drugs, including cancer immunotherapies, and provides valuable insights into host-microbiome interactions in the context of disease., Competing Interests: W.A.K. has received research funding (institution) from Pfizer; there is no direct overlap with the current study. P.J.T. is on the scientific advisory boards of Pendulum, Seed and SNIPRbiome; there is no direct overlap between the current study and these consulting duties. C.E.A served on the scientific advisory board of Pionyr Immunotherapeutics and has received research funding (institution) from Bristol Meyer Squibb, Erasca, Gossamer Bio, Guardant Health, Kura Oncology, Merck and Novartis; there is no direct overlap with the current study. E.V.B. is on the medical advisory board for Fight CRC; there is no direct overlap with the current study.All other authors declare no competing interests.

Recent studies suggest that human-associated bacteria interact with host-produced steroids, but the mechanisms and physiological impact of such interactions remain unclear. Here, we show that the human gut bacteria Gordonibacter pamelaeae and Eggerthella lenta convert abundant biliary corticoids into progestins through 21-dehydroxylation, thereby transforming a class of immuno- and metabo-regulatory steroids into a class of sex hormones and neurosteroids. Using comparative genomics, homologous expression, and heterologous expression, we identify a bacterial gene cluster that performs 21-dehydroxylation. We also uncover an unexpected role for hydrogen gas production by gut commensals in promoting 21-dehydroxylation, suggesting that hydrogen modulates secondary metabolism in the gut. Levels of certain bacterial progestins, including allopregnanolone, better known as brexanolone, an FDA-approved drug for postpartum depression, are substantially increased in feces from pregnant humans. Thus, bacterial conversion of corticoids into progestins may affect host physiology, particularly in the context of pregnancy and women's health., Competing Interests: Declaration of interests A.S.D. is an ad hoc consultant for Axial Therapeutics. M.D.M. and A.S.D. are co-inventors on a provisional patent related to this work. J.R.H. serves as a consultant for CJ CheilJedang and is a co-founder and consultant for Interon Laboratories. He received research funding from CJ Bioscience outside the submitted work. A.G.E. serves as a consultant for Mirvie, Inc. outside the submitted work and receives research funding from Merck Pharmaceuticals outside the submitted work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Drug efflux transporters are a major determinant of drug efficacy and toxicity. A canonical example is P-glycoprotein (P-gp), an efflux transporter that controls the intestinal absorption of diverse compounds. Despite a rich literature on the dietary and pharmaceutical compounds that impact P-gp activity, its sensitivity to gut microbial metabolites remains an open question. Surprisingly, we found that the cardiac drug-metabolizing gut Actinobacterium Eggerthella lenta increases drug absorption in mice. Experiments in cell culture revealed that E. lenta produces a soluble factor that post-translationally inhibits P-gp ATPase efflux activity. P-gp inhibition is conserved in the Eggerthellaceae family but absent in other Actinobacteria. Comparative genomics identified genes associated with P-gp inhibition. Finally, activity-guided biochemical fractionation coupled to metabolomics implicated a group of small polar metabolites with P-gp inhibitory activity. These results highlight the importance of considering the broader relevance of the gut microbiome for drug disposition beyond first-pass metabolism., Competing Interests: P.J.T. is on the scientific advisory boards for Pendulum, Seres, and SNIPR Biome; there is no direct overlap between the current study and these consulting duties., (© 2024 The Author(s).)

Carbohydrate intolerance, commonly linked to the consumption of lactose, fructose, or sorbitol, affects up to 30% of the population in high-income countries. Although sorbitol intolerance is attributed to malabsorption, the underlying mechanism remains unresolved. Here, we show that a history of antibiotic exposure combined with high fat intake triggered long-lasting sorbitol intolerance in mice by reducing Clostridia abundance, which impaired microbial sorbitol catabolism. The restoration of sorbitol catabolism by inoculation with probiotic Escherichia coli protected mice against sorbitol intolerance but did not restore Clostridia abundance. Inoculation with the butyrate producer Anaerostipes caccae restored a normal Clostridia abundance, which protected mice against sorbitol-induced diarrhea even when the probiotic was cleared. Butyrate restored Clostridia abundance by stimulating epithelial peroxisome proliferator-activated receptor-gamma (PPAR-γ) signaling to restore epithelial hypoxia in the colon. Collectively, these mechanistic insights identify microbial sorbitol catabolism as a potential target for approaches for the diagnosis, treatment, and prevention of sorbitol intolerance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Importance: This work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and nonenzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen, and L-Cysteine control the ability of E. coli to degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context., Competing Interests: P.J.T. is on the scientific advisory boards for Pendulum, Seed, and SNIPRbiome; there is no direct overlap between the current study and these consulting duties. A.T.W. is on the scientific advisory board for Enzymetrics Biosciences; there is no direct overlap between the current study and this role. The other authors declare no competing interests.

Drugs intended to target mammalian cells can have broad off-target effects on the human gut microbiota with potential downstream consequences for drug efficacy and side effect profiles. Yet, despite a rich literature on antibiotic resistance, we still know very little about the mechanisms through which commensal bacteria evade non-antibiotic drugs. Here, we focus on statins, one of the most prescribed drug types in the world and an essential tool in the prevention and treatment of high circulating cholesterol levels. Prior work in humans, mice, and cell culture support an off-target effect of statins on human gut bacteria; however, the genetic determinants of statin sensitivity remain unknown. We confirmed that simvastatin inhibits the growth of diverse human gut bacterial strains grown in communities and in pure cultures. Drug sensitivity varied between phyla and was dose-dependent. We selected two representative simvastatin-sensitive species for more in-depth analysis: Eggerthella lenta (phylum: Actinobacteriota) and Bacteroides thetaiotaomicron (phylum: Bacteroidota). Transcriptomics revealed that both bacterial species upregulate genes in response to simvastatin that alter the cell membrane, including fatty acid biogenesis (E. lenta) and drug efflux systems (B. thetaiotaomicron). Transposon mutagenesis identified a key efflux system in B. thetaiotaomicron that enables growth in the presence of statins. Taken together, these results emphasize the importance of the bacterial cell membrane in countering the off-target effects of host-targeted drugs. Continued mechanistic dissection of the various mechanisms through which the human gut microbiota evades drugs will be essential to understand and predict the effects of drug administration in human cohorts and the potential downstream consequences for health and disease., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Background: Colorectal cancer (CRC) is more prevalent among some racial and ethnic minority and low socioeconomic status populations. Although the gut microbiota is a risk factor for CRC and varies with race and ethnicity, its role in CRC disparities remains poorly understood., Methods: We examined the feasibility of recruiting sociodemographically diverse CRC patients for a microbiome study involving a home stool collection. We also explored whether race and ethnicity were associated with gut microbiome composition. We recruited Black/African American, Hispanic/Latino, and non-Hispanic White patients who were receiving care for active CRC to complete a comprehensive dietary and lifestyle survey, self-collect a stool sample, and complete an exit interview. Gut microbial diversity and composition were analyzed using 16S rRNA gene sequencing., Results: 30 individuals consented (of 35 who were eligible and contacted) with 5 (17%) Black/African American, 11 (37%) Hispanic/Latino, and 14 (46%) non-Hispanic White. A total of 22 (73%) completed the dietary and lifestyle survey; 18 (63%) returned a stool sample. Even after controlling for socioeconomic, dietary, or treatment-related covariates, microbiome composition was associated with race and ethnicity. Fusobacteriota (a phylum associated with the development and progression of CRC) was significantly higher in the Black/African American group compared to others, and microbial diversity was higher in samples from non-Hispanic White individuals compared to Hispanic/Latino individuals., Conclusion: Our study shows that it is feasible to recruit and collect stool samples from diverse individuals with CRC and found significant associations in gut microbial structure with race and ethnicity.

Background: Adherence to the American Cancer Society (ACS) guidelines of avoiding obesity, maintaining physical activity, and consuming a diet rich in fruits, vegetables, and whole grains is associated with longer survival in colorectal cancer (CRC) survivors. Dietary components of the ACS guidelines may act in part by changing the microbiome, which is implicated in CRC outcomes., Objectives: We conducted a pilot cross-sectional study to explore associations between ACS guidelines and the gut microbiome., Methods: Stool samples and questionnaires were collected from 28 CRC survivors at the University of California, San Francisco from 2019 to 2020. ACS scores were calculated based on validated questionnaires. Gut microbial community structure from 16S amplicons and gene/pathway abundances from metagenomics were tested for associations with the ACS score and its components using ANOVA and general linear models., Results: The overall ACS score was not significantly associated with variations in the fecal microbiota. However, fruit and vegetable intake and alcohol intake accounted for 19% (P = 0.005) and 13% (P = 0.01) of variation in the microbiota, respectively. Fruit/vegetable consumption was associated with increased microbial diversity, increased Firmicutes, decreased Bacteroidota, and changes to multiple genes and metabolic pathways, including enriched pathways for amino acid and short-chain fatty acid biosynthesis and plant-associated sugar degradation. In contrast, alcohol consumption was positively associated with overall microbial diversity, negatively associated with Bacteroidota abundance, and associated with changes to multiple genes and metabolic pathways. The other components of the ACS score were not statistically significantly associated with the fecal microbiota in our sample., Conclusions: These results guide future studies examining the impact of changes in the intake of fruits, vegetables, and alcoholic drinks on the gut microbiome of CRC survivors., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2-positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls. These results were confirmed and extended in the K18-humanized angiotensin-converting enzyme 2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the USA), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila . Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology. IMPORTANCE Taken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2, it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease., Competing Interests: P.J.T. is on the scientific advisory boards for Pendulum, Seed, and SNIPRbiome; there is no direct overlap between the current study and these consulting duties. The other authors declare no competing interests.

Bacteria within the gut microbiota possess the ability to metabolize a wide array of human drugs, foods, and toxins, but the responsible enzymes for these chemical events remain largely uncharacterized due to the time-consuming nature of current experimental approaches. Attempts have been made in the past to computationally predict which bacterial species and enzymes are responsible for chemical transformations in the gut environment, but with low accuracy due to minimal chemical representation and sequence similarity search schemes. Here, we present an in silico approach that employs chemical and protein S imilarity algorithms that I dentify M icrobio M e E nzymatic R eactions (SIMMER). We show that SIMMER accurately predicts the responsible species and enzymes for a queried reaction, unlike previous methods. We demonstrate SIMMER use cases in the context of drug metabolism by predicting previously uncharacterized enzymes for 88 drug transformations known to occur in the human gut. We validate these predictions on external datasets and provide an in vitro validation of SIMMER's predictions for metabolism of methotrexate, an anti-arthritic drug. After demonstrating its utility and accuracy, we made SIMMER available as both a command-line and web tool, with flexible input and output options for determining chemical transformations within the human gut. We present SIMMER as a computational addition to the microbiome researcher's toolbox, enabling them to make informed hypotheses before embarking on the lengthy laboratory experiments required to characterize novel bacterial enzymes that can alter human ingested compounds., Competing Interests: AB, RN, AA No competing interests declared, PT Reviewing editor, eLife, KP is a consultant for Phylagen Inc, (© 2023, Bustion et al.)

Human gut bacteria perform diverse metabolic functions with consequences for host health. The prevalent and disease-linked Actinobacterium Eggerthella lenta performs several unusual chemical transformations, but it does not metabolize sugars and its core growth strategy remains unclear. To obtain a comprehensive view of the metabolic network of E. lenta, we generated several complementary resources: defined culture media, metabolomics profiles of strain isolates, and a curated genome-scale metabolic reconstruction. Stable isotope-resolved metabolomics revealed that E. lenta uses acetate as a key carbon source while catabolizing arginine to generate ATP, traits which could be recapitulated in silico by our updated metabolic model. We compared these in vitro findings with metabolite shifts observed in E. lenta-colonized gnotobiotic mice, identifying shared signatures across environments and highlighting catabolism of the host signaling metabolite agmatine as an alternative energy pathway. Together, our results elucidate a distinctive metabolic niche filled by E. lenta in the gut ecosystem. Our culture media formulations, atlas of metabolomics data, and genome-scale metabolic reconstructions form a freely available collection of resources to support further study of the biology of this prevalent gut bacterium., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: P.J.T. is on the scientific advisory boards for Pendulum, Seed, and SNIPRbiome; there is no direct overlap between the current study and these consulting duties. D.D. is a co-founder of Federation Bio, a company developing microbiome-based therapeutics. All other authors have no relevant declarations., (Copyright: © 2023 Noecker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aging is often accompanied by an increased risk of an array of diseases spanning the cardiovascular, nervous, and immune systems, among others. Despite remarkable progress in understanding the cellular and molecular mechanisms involved in aging, the role of the microbiome remains understudied. In this Essay, we highlight recent progress towards understanding if and how the microbiome contributes to aging and age-associated diseases. Furthermore, we discuss the need to consider sexually dimorphic phenotypes in the context of aging and the microbiome. We also highlight the broad implications for this emerging area of interdisciplinary research to address long-standing questions about host-microbiome interactions across the life span., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Rock, Turnbaugh. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Eggerthella lenta is a prevalent human gut Actinobacterium implicated in drug, dietary phytochemical, and bile acid metabolism and associated with multiple human diseases. No genetic tools are currently available for the direct manipulation of E. lenta. Here, we construct shuttle vectors and develop methods to transform E. lenta and other Coriobacteriia. With these tools, we characterize endogenous E. lenta constitutive and inducible promoters using a reporter system and construct inducible expression systems, enabling tunable gene regulation. We also achieve genome editing by harnessing an endogenous type I-C CRISPR-Cas system. Using these tools to perform genetic knockout and complementation, we dissect the functions of regulatory proteins and enzymes involved in catechol metabolism, revealing a previously unappreciated family of membrane-spanning LuxR-type transcriptional regulators. Finally, we employ our genetic toolbox to study the effects of E. lenta genes on mammalian host biology. By greatly expanding our ability to study and engineer gut Coriobacteriia, these tools will reveal mechanistic details of host-microbe interactions and provide a roadmap for genetic manipulation of other understudied human gut bacteria., (© 2022. The Author(s).)

Pharmaceuticals have extensive reciprocal interactions with the microbiome, but whether bacterial drug sensitivity and metabolism is driven by pathways conserved in host cells remains unclear. Here we show that anti-cancer fluoropyrimidine drugs inhibit the growth of gut bacterial strains from 6 phyla. In both Escherichia coli and mammalian cells, fluoropyrimidines disrupt pyrimidine metabolism. Proteobacteria and Firmicutes metabolized 5-fluorouracil to its inactive metabolite dihydrofluorouracil, mimicking the major host mechanism for drug clearance. The preTA operon was necessary and sufficient for 5-fluorouracil inactivation by E. coli, exhibited high catalytic efficiency for the reductive reaction, decreased the bioavailability and efficacy of oral fluoropyrimidine treatment in mice and was prevalent in the gut microbiomes of colorectal cancer patients. The conservation of both the targets and enzymes for metabolism of therapeutics across domains highlights the need to distinguish the relative contributions of human and microbial cells to drug efficacy and side-effect profiles., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Caloric restriction can delay the development of metabolic diseases ranging from insulin resistance to type 2 diabetes and is linked to both changes in the composition and metabolic function of the gut microbiota and immunological consequences. However, the interaction between dietary intake, the microbiome, and the immune system remains poorly described., Results: We transplanted the gut microbiota from an obese female before (AdLib) and after (CalRes) an 8-week very-low-calorie diet (800 kcal/day) into germ-free mice. We used 16S rRNA sequencing to evaluate taxa with differential abundance between the AdLib- and CalRes-microbiota recipients and single-cell multidimensional mass cytometry to define immune signatures in murine colon, liver, and spleen. Recipients of the CalRes sample exhibited overall higher alpha diversity and restructuring of the gut microbiota with decreased abundance of several microbial taxa (e.g., Clostridium ramosum, Hungatella hathewayi, Alistipi obesi). Transplantation of CalRes-microbiota into mice decreased their body fat accumulation and improved glucose tolerance compared to AdLib-microbiota recipients. Finally, the CalRes-associated microbiota reduced the levels of intestinal effector memory CD8 + T cells, intestinal memory B cells, and hepatic effector memory CD4 + and CD8 + T cells., Conclusion: Caloric restriction shapes the gut microbiome which can improve metabolic health and may induce a shift towards the naïve T and B cell compartment and, thus, delay immune senescence. Understanding the role of the gut microbiome as mediator of beneficial effects of low calorie diets on inflammation and metabolism may enhance the development of new therapeutic treatment options for metabolic diseases., Trial Registration: NCT01105143 , "Effects of negative energy balance on muscle mass regulation," registered 16 April 2010. Video Abstract., (© 2022. The Author(s).)

Relative abundances of bacterial species in the gut microbiome have been linked to many diseases. Species of gut bacteria are ecologically differentiated by their abilities to metabolize different glycans, making glycan delivery a powerful way to alter the microbiome to promote health. Here, we study the properties and therapeutic potential of chemically diverse synthetic glycans (SGs). Fermentation of SGs by gut microbiome cultures results in compound-specific shifts in taxonomic and metabolite profiles not observed with reference glycans, including prebiotics. Model enteric pathogens grow poorly on most SGs, potentially increasing their safety for at-risk populations. SGs increase survival, reduce weight loss, and improve clinical scores in mouse models of colitis. Synthetic glycans are thus a promising modality to improve health through selective changes to the gut microbiome., (© 2022. The Author(s).)

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1-deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.

The microbiota modulates gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17A (T H 17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits T H 17 cell differentiation 1 . Although it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown, and it was unclear whether 3-oxoLCA and other immunomodulatory bile acids are associated with inflammatory pathologies in humans. Here we identify human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Similar to 3-oxoLCA, isoLCA suppressed T H 17 cell differentiation by inhibiting retinoic acid receptor-related orphan nuclear receptor-γt, a key T H 17-cell-promoting transcription factor. The levels of both 3-oxoLCA and isoLCA and the 3α-hydroxysteroid dehydrogenase genes that are required for their biosynthesis were significantly reduced in patients with inflammatory bowel disease. Moreover, the levels of these bile acids were inversely correlated with the expression of T H 17-cell-associated genes. Overall, our data suggest that bacterially produced bile acids inhibit T H 17 cell function, an activity that may be relevant to the pathophysiology of inflammatory disorders such as inflammatory bowel disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmunity, but how human-associated bacteria impact Th17-driven disease remains elusive. We show that human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by lifting inhibition of the Th17 transcription factor Rorγt through cell- and antigen-independent mechanisms. E. lenta is enriched in inflammatory bowel disease (IBD) patients and worsens colitis in a Rorc-dependent manner in mice. Th17 activation varies across E. lenta strains, which is attributable to the cardiac glycoside reductase 2 (Cgr2) enzyme. Cgr2 is sufficient to induce interleukin (IL)-17a, a major Th17 cytokine. cgr2+ E. lenta deplete putative steroidal glycosides in pure culture; related compounds are negatively associated with human IBD severity. Finally, leveraging the sensitivity of Cgr2 to dietary arginine, we prevented E. lenta-induced intestinal inflammation in mice. Together, these results support a role for human gut bacterial metabolism in driving Th17-dependent autoimmunity., Competing Interests: Declaration of interests P.J.T. is on the scientific advisory board for Kaleido, Pendulum, and SNIPRbiome. K.S.P. is on the scientific advisory board of Phylagen. S.V.L. is a co-founder, board member, and consultant for Siolta Therapeutics Inc. This work was partially supported by a MedImmune research grant., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest., Competing Interests: Declaration of interests K.N.L., P.S., and P.J.T. are listed inventors on a US provisional patent application related to this work (33167/55262P1). P.J.T. is on the scientific advisory boards for Kaleido, Pendulum, and SNIPRbiome. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The particularly interdisciplinary nature of human microbiome research makes the organization and reporting of results spanning epidemiology, biology, bioinformatics, translational medicine and statistics a challenge. Commonly used reporting guidelines for observational or genetic epidemiology studies lack key features specific to microbiome studies. Therefore, a multidisciplinary group of microbiome epidemiology researchers adapted guidelines for observational and genetic studies to culture-independent human microbiome studies, and also developed new reporting elements for laboratory, bioinformatics and statistical analyses tailored to microbiome studies. The resulting tool, called 'Strengthening The Organization and Reporting of Microbiome Studies' (STORMS), is composed of a 17-item checklist organized into six sections that correspond to the typical sections of a scientific publication, presented as an editable table for inclusion in supplementary materials. The STORMS checklist provides guidance for concise and complete reporting of microbiome studies that will facilitate manuscript preparation, peer review, and reader comprehension of publications and comparative analysis of published results., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

East Asians (EAs) experience worse metabolic health outcomes compared to other ethnic groups at lower body mass indices; however, the potential role of the gut microbiota in contributing to these health disparities remains unknown. We conducted a multi-omic study of 46 lean and obese East Asian and White participants living in the San Francisco Bay Area, revealing marked differences between ethnic groups in bacterial richness and community structure. White individuals were enriched for the mucin-degrading Akkermansia muciniphila . East Asian subjects had increased levels of multiple bacterial phyla, fermentative pathways detected by metagenomics, and the short-chain fatty acid end-products acetate, propionate, and isobutyrate. Differences in the gut microbiota between the East Asian and White subjects could not be explained by dietary intake, were more pronounced in lean individuals, and were associated with current geographical location. Microbiome transplantations into germ-free mice demonstrated stable diet- and host genotype-independent differences between the gut microbiotas of East Asian and White individuals that differentially impact host body composition. Taken together, our findings add to the growing body of literature describing microbiome variations between ethnicities and provide a starting point for defining the mechanisms through which the microbiome may shape disparate health outcomes in East Asians., Competing Interests: QA, DA, JB, JC, HL, EB, GW, CN, AP, SK No competing interests declared, PT is on the scientific advisory board for Kaleido, Pendulum, Seres, and SNIPRbiome; there is no direct overlap between the current study and these consulting duties. Reviewing editor, eLife, (© 2021, Ang et al.)

Diet is a major factor that shapes the gut microbiome 1 , but the consequences of diet-induced changes in the microbiome for host pathophysiology remain poorly understood. We conducted a randomized human intervention study using a very-low-calorie diet (NCT01105143). Although metabolic health was improved, severe calorie restriction led to a decrease in bacterial abundance and restructuring of the gut microbiome. Transplantation of post-diet microbiota to mice decreased their body weight and adiposity relative to mice that received pre-diet microbiota. Weight loss was associated with impaired nutrient absorption and enrichment in Clostridioides difficile, which was consistent with a decrease in bile acids and was sufficient to replicate metabolic phenotypes in mice in a toxin-dependent manner. These results emphasize the importance of diet-microbiome interactions in modulating host energy balance and the need to understand the role of diet in the interplay between pathogenic and beneficial symbionts.

Objective: Although oral methotrexate (MTX) remains the anchor drug for rheumatoid arthritis (RA), up to 50% of patients do not achieve a clinically adequate outcome. In addition, there is a lack of prognostic tools for treatment response prior to drug initiation. This study was undertaken to investigate whether interindividual differences in the human gut microbiome can aid in the prediction of MTX efficacy in new-onset RA., Methods: We performed 16S ribosomal RNA gene and shotgun metagenomic sequencing on the baseline gut microbiomes of drug-naive patients with new-onset RA (n = 26). Results were validated in an additional independent cohort (n = 21). To gain insight into potential microbial mechanisms, we conducted ex vivo experiments coupled with metabolomics analysis to evaluate the association between microbiome-driven MTX depletion and clinical response., Results: Our analysis revealed significant associations of the abundance of gut bacterial taxa and their genes with future clinical response (q < 0.05), including orthologs related to purine and MTX metabolism. Machine learning techniques were applied to the metagenomic data, resulting in a microbiome-based model that predicted lack of response to MTX in an independent group of patients. Finally, MTX levels remaining after ex vivo incubation with distal gut samples from pretreatment RA patients significantly correlated with the magnitude of future clinical response, suggesting a possible direct effect of the gut microbiome on MTX metabolism and treatment outcomes., Conclusion: Taken together, these findings are the first step toward predicting lack of response to oral MTX in patients with new-onset RA and support the value of the gut microbiome as a possible prognostic tool and as a potential target in RA therapeutics., (© 2020, American College of Rheumatology.)

The core symptoms of many neurological disorders have traditionally been thought to be caused by genetic variants affecting brain development and function. However, the gut microbiome, another important source of variation, can also influence specific behaviors. Thus, it is critical to unravel the contributions of host genetic variation, the microbiome, and their interactions to complex behaviors. Unexpectedly, we discovered that different maladaptive behaviors are interdependently regulated by the microbiome and host genes in the Cntnap2 -/- model for neurodevelopmental disorders. The hyperactivity phenotype of Cntnap2 -/- mice is caused by host genetics, whereas the social-behavior phenotype is mediated by the gut microbiome. Interestingly, specific microbial intervention selectively rescued the social deficits in Cntnap2 -/- mice through upregulation of metabolites in the tetrahydrobiopterin synthesis pathway. Our findings that behavioral abnormalities could have distinct origins (host genetic versus microbial) may change the way we think about neurological disorders and how to treat them., Competing Interests: Declaration of interests A patent application related to the role of L. reuteri on social behavior has been filed by BCM. Findings regarding the manipulation of endogenous biotperin levels by the microbiome are the subject of a provisional patent application owned by BCM. M.C.-M. is a scientific co-founder of Mirkrovia. The authors declare no other competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Harnessing the microbiota for beneficial outcomes is limited by our poor understanding of the constituent bacteria, as the functions of most of their genes are unknown. Here, we measure the growth of a barcoded transposon mutant library of the gut commensal Bacteroides thetaiotaomicron on 48 carbon sources, in the presence of 56 stress-inducing compounds, and during mono-colonization of gnotobiotic mice. We identify 516 genes with a specific phenotype under only one or a few conditions, enabling informed predictions of gene function. For example, we identify a glycoside hydrolase important for growth on type I rhamnogalacturonan, a DUF4861 protein for glycosaminoglycan utilization, a 3-keto-glucoside hydrolase for disaccharide utilization, and a tripartite multidrug resistance system specifically for bile salt tolerance. Furthermore, we show that B. thetaiotaomicron uses alternative enzymes for synthesizing nitrogen-containing metabolic precursors based on ammonium availability and that these enzymes are used differentially in vivo in a diet-dependent manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Respiratory viral infections remain a scourge, with seasonal influenza infecting millions and killing many thousands annually and viral pandemics, such as COVID-19, recurring every decade. Age, cardiovascular disease, and diabetes mellitus are risk factors for severe disease and death from viral infection. Immunometabolic therapies for these populations hold promise to reduce the risks of death and disability. Such interventions have pleiotropic effects that might not only target the virus itself but also enhance supportive care to reduce cardiopulmonary complications, improve cognitive resilience, and facilitate functional recovery. Ketone bodies are endogenous metabolites that maintain cellular energy but also feature drug-like signaling activities that affect immune activity, metabolism, and epigenetics. Here, we provide an overview of ketone body biology relevant to respiratory viral infection, focusing on influenza A and severe acute respiratory syndrome (SARS)-CoV-2, and discuss the opportunities, risks, and research gaps in the study of exogenous ketone bodies as novel immunometabolic interventions in these diseases., Competing Interests: J.C.N. and E.V. are co-founders of and shareholders in BHB Therapeutics, Ltd., which is developing products relating to ketone bodies. B.J.S. is a shareholder of HVMN, Inc., which markets products relating to ketone bodies and of BHB Therapeutics, Ltd. J.C.N., E.V., B.J.S., and A.P.K. are inventors on patents related to the use of ketone bodies. J.C.N. is on the scientific advisory board of Virta Health, Inc. E.V. is an advisor to Keto Fuel, Inc. P.J.T. is on the scientific advisory boards for Kaleido, Pendulum, Seres, and SNIPRbiome. All other authors declare no competing interests., (© 2020 Elsevier Inc.)

Disease tolerance, the capacity of tissues to withstand damage caused by a stimulus without a decline in host fitness, varies across tissues, environmental conditions, and physiologic states. While disease tolerance is a known strategy of host defense, its role in noninfectious diseases has been understudied. Here, we provide evidence that a thermogenic fat-epithelial cell axis regulates intestinal disease tolerance during experimental colitis. We find that intestinal disease tolerance is a metabolically expensive trait, whose expression is restricted to thermoneutral mice and is not transferable by the microbiota. Instead, disease tolerance is dependent on the adrenergic state of thermogenic adipocytes, which indirectly regulate tolerogenic responses in intestinal epithelial cells. Our work has identified an unexpected mechanism that controls intestinal disease tolerance with implications for colitogenic diseases., Competing Interests: The authors declare no competing interest.