High fat intake sustains sorbitol intolerance after antibiotic-mediated Clostridia depletion from the gut microbiota.

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.

Effects of long-term high dose aspartame on body mass, bone strength, femoral geometry, and microbiota composition in a young and aged cohort of male and female mice.

Background: Recent reassessment of the safety of aspartame has prompted increased evaluation of its effect on the health of a range of tissues. The gut microbiome is altered by oral aspartame. One prior study suggested that changes in the microbiome caused by aspartame could influence the strength of bone in young skeletally developing mice. Here we ask how aspartame influences bone in mice of different age and sex. Objective: The objective of this study was to determine the effect of aspartame on the bone strength and gut microbiota of young and aged mice. Methods: Male and female C57Bl/6J mice were untreated or treated with a high dose of aspartame in their drinking water from 1 month of age until 4 (young cohort; n = 80) or 22 months (aged cohort; n = 52). Results: In aged males, mice treated with aspartame had greater body mass, whole bone strength, and femoral geometry relative to untreated. Specifically, in aged males, aspartame led to 9% increase in body mass (p < 0.001), 22% increase in whole bone strength (p = 0.006), and 17% increase in section modulus (p < 0.001) relative to untreated mice. Aged males and females receiving aspartame had a different microbiota than untreated mice and a decreased abundance of Odoribacter. No differences in body mass, whole bone strength, or femoral geometry were associated with aspartame dosing in young males or young or aged females. Conclusions: Aspartame treated aged males had greater whole bone strength and the effect appeared to be explained by greater body mass. Aspartame treatment did not alter whole bone strength in young males or young or aged females despite the aspartame having a similar effect on the microbiota of both aged males and females.

A diet-dependent host metabolite shapes the gut microbiota to protect from autoimmunity.

Diet can protect from autoimmune disease; however, whether diet acts via the host and/or microbiome remains unclear. Here, we use a ketogenic diet (KD) as a model to dissect these complex interactions. A KD rescued the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis in a microbiota-dependent fashion. Dietary supplementation with a single KD-dependent host metabolite (ß-hydroxybutyrate, ßHB) rescued EAE whereas transgenic mice unable to produce ßHB in the intestine developed more severe disease. Transplantation of the ßHB-shaped gut microbiota was protective. Lactobacillus sequence variants were associated with decreased T helper 17 (Th17) cell activation in vitro . Finally, we isolated a L. murinus strain that protected from EAE, which was phenocopied by the Lactobacillus metabolite indole lactic acid. Thus, diet alters the immunomodulatory potential of the gut microbiota by shifting host metabolism, emphasizing the utility of taking a more integrative approach to study diet-host-microbiome interactions.

Rationale and protocol for a safety, tolerability and feasibility randomized, parallel group, double-blind, placebo-controlled, pilot study of a novel ketone ester targeting frailty via immunometabolic geroscience mechanisms.

Background: Frailty is a geriatric syndrome characterized by chronic inflammation and metabolic insufficiency that creates vulnerability to poor outcomes with aging. We hypothesize that geroscience interventions, which target mechanisms of aging, could ameliorate frailty. Metabolites such as ketone bodies are candidate geroscience interventions, having pleiotropic effects on inflammo-metabolic aging mechanisms. Ketone esters (KEs) 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 geroscience 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 generalizable 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 geroscience-related endpoints. This data will inform design of larger trials to rigorously test KE effects on geroscience mechanisms and clinical outcomes relevant to frailty.

Simvastatin induces human gut bacterial cell surface genes.

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

Associations between the Gut Microbiota, Race, and Ethnicity of Patients with Colorectal Cancer: A Pilot and Feasibility Study.

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.

Microbiome single cell atlases generated with a commercial instrument.

Single cell sequencing is useful for resolving complex systems into their composite cell types and computationally mining them for unique features that are masked in pooled sequencing. However, while commercial instruments have made single cell analysis widespread for mammalian cells, analogous tools for microbes are limited. Here, we present EASi-seq ( E asily A ccessible S ingle microbe seq uencing). By adapting the single cell workflow of the commercial Mission Bio Tapestri instrument, this method allows for efficient sequencing of individual microbes' genomes. EASi-seq allows thousands of microbes to be sequenced per run and, as we show, can generate detailed atlases of human and environmental microbiomes. The ability to capture large shotgun genome datasets from thousands of single microbes provides new opportunities in discovering and analyzing species subpopulations. To facilitate this, we develop a companion bioinformatic pipeline that clusters microbes by similarity, improving whole genome assembly, strain identification, taxonomic classification, and gene annotation. In addition, we demonstrate integration of metagenomic contigs with the EASi-seq datasets to reduce capture bias and increase coverage. Overall, EASi-seq enables high quality single cell genomic data for microbiome samples using an accessible workflow that can be run on a commercially available platform.

The global anaerobic metabolism regulator fnr is necessary for the degradation of food dyes and drugs by Escherichia coli.

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.

Variety of Fruit and Vegetables and Alcohol Intake are Associated with Gut Microbial Species and Gene Abundance in Colorectal Cancer Survivors.

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.

SIMMER employs similarity algorithms to accurately identify human gut microbiome species and enzymes capable of known chemical transformations.

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 Similarity algorithms that Identify MicrobioMe Enzymatic Reactions (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.

Mild SARS-CoV-2 infection results in long-lasting microbiota instability.

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.

Systems biology elucidates the distinctive metabolic niche filled by the human gut microbe Eggerthella lenta.

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.

Forging the microbiome to help us live long and prosper.

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.

Mild SARS-CoV-2 infection results in long-lasting microbiota instability.

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 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, as long as 154 days after their positive test. These results were confirmed and extended in the K18-hACE2 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 United States), 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.

Genetic manipulation of the human gut bacterium Eggerthella lenta reveals a widespread family of transcriptional regulators.

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.

Host and gut bacteria share metabolic pathways for anti-cancer drug metabolism.

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.

Effects of caloric restriction on the gut microbiome are linked with immune senescence.

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.