Gut Microbes

Whereas innate pattern recognition receptors bind evolutionary conserved moieties shared by diverse groups of microbes, control over closely related microorganisms is likely based on different principles and can be sensitive to genetic variation of the host. While the influence of host genetics on intestinal microbiome composition has been growing in appreciation, understanding of polymorphic mechanisms that regulate individual bacterial lineages requires complex approaches. Using gnotobiotic mouse monocolonized with Ligilactobacillus murinus (ASF361), we found that C57BL/6J (B6) mice harbored significantly more intestinal ASF361 (or other Lactobacillaceae genera) than did BALB/cJ (BALB/c) mice. The difference in ASF361 abundance was independent of adaptive immunity and bacteriotoxic factors. In B6 mice, ASF361 occupied ileal crypts, showed greater expression of adhesion-related molecules, and enriched expression of carbohydrate-metabolism pathways, whereas in BALB/c mice, ASF361 remained largely luminal and enriched expressed genes associated with starvation and stress. These findings highlight the importance of host genetic variation in shaping microbial colonization and suggest that such variation should be considered when evaluating probiotic strains and microbiota transplantation.

Background and Aims: As a microbiome/diet-derived vitamin, queuine (q) is a micronutrient that eukaryotes acquire from microbiota or diet. Vitamin q, the free base form of queuosine (Q), is used for Q-modification of transfer RNA (tRNA). Although tRNA-Q-modification in regulating eukaryotic cell physiology is recognized, the relationship between Q-tRNA-modification and the human microbiome in health and inflammatory bowel diseases (IBD) remains unknown. Which bacteria produce q and how the q-producing bacteria contributing to human health and IBD are not explored. Here, we hypothesize that disrupted microbial Q-biosynthesis contributes to the dysbiosis of human IBD. We performed comprehensive multi-omics integrative analyses using publicly available datasets. Specifically, we re-analyzed publicly available shotgun metagenomic, single-cell RNA sequencing (scRNA-seq), metabolic datasets, and completed meta-analysis. Methods: To systematically investigate microbial Q-biosynthesis in healthy individuals and IBD patients, we performed a systematic secondary analysis by integrating 9 publicly available metagenomic datasets, 15 scRNA-seq datasets and one metabolic dataset published prior to 2026. We present a novel secondary bioinformatic reanalysis of sequence outputs mapped to the latest microbial taxonomy, functional pathways and microbial Q-related genes. Longitudinal study of shotgun metagenomics across the human lifespan (3146 samples) enabled taxonomic and functional profiling. Results: We first identified key q-producing bacteria (e.g., Bacteroides and Alistipes) in healthy populations. We then found that patients with IBD had significantly reducation of q-producing bacteria and a coordinated downregulation of the Q-related pathways, including Q, preQ0, de novo guanosine ribonucleotide biosynthesis and de novo guanosine deoxyribonucleotide biosynthesis pathways. This functional impairment was primarily driven by reduced contributions from Bacteroides, Alistipes and Akkermansia. The longitudinal stability of the q-producing bacteria community across the human lifespan (1638 samples). We observed a significant negative correlation between host age and the abundance of q-producing bacteria in patients with UC. Moreover, Queuine tRNA-ribosyltransferase 1 (QTRT1) expression in human intestinal epithelial cells was significantly reduced in patients with IBD compared to healthy controls. Conclusions: Our study used multi-omics datasets to define a novel functional dysbiosis characterized by a reduction of microbial Q-biosynthesis in patients with IBD. Vitamin q and its tRNA modification is lesser-recognized in human IBD. Understanding the cellular and organismal mechanisms of this microbiome-dependent micronutrient will advance the prevention of IBD and improve the quality of life of patients with IBD.

Adherent Invasive Escherichia coli (AIEC) colonize the ileal mucosa of Crohn's disease patients. Among bacterial virulence factors potentially implicated, type 6 secretion systems (T6SS) are molecular machineries sending effectors in prokaryotic or eukaryotic target cells. Two T6SS have been reported in AIEC LF82 reference strain. Here, we show that T6SS genes are more prevalent in Crohn's disease patients' microbiota compared to healthy controls, and that T6SS are more frequent in AIEC pathovar compared to E. coli in general and associated with FimH pathoadaptive polymorphism. The use of an in vitro gastrointestinal system coupled with T6SS promoter reporter assay highlighted expression of LF82 T6SS under gastrointestinal conditions, with a major role for bile salts. In mice infected with AIEC LF82 versus T6SS-deleted mutants, T6SS showed a functional impact in modulating the composition of mucosa-associated gut microbiota. Thus, AIEC may use their T6SS to compete with endogenous microbiota to colonize host mucosa.

Cholera toxin (CTx) is a potent enterotoxin and widely used mucosal adjuvant, yet the acute host response to CTx itself, in the absence of a co-administered antigen, remains vague. In this study, we integrated physiological and metabolic phenotyping with intestinal transcriptomics, epithelial imaging, immune profiling, hepatic gene-expression analysis, and paired microbiome profiling to characterize the response to a single oral CTx exposure in mice. Within 24 h, CTx remodeled immune-cell composition in both epithelial and lamina propria compartments, with responses shaped by sex and genetic background. Recurrent changes included increased epithelial group 3 innate lymphoid cells, reduced lamina propria ROR{gamma}t+ regulatory T cells, and decreased epithelial ILC1/NK cells in males of both strains. CTx also increased villus DCLK1+ tuft cells and reduced MUC2 immunoreactivity. Jejunal transcriptomics revealed induction of inflammatory defense, glycolytic, autophagy, and lipid-utilization programs together with suppression of proliferative and mitochondrial programs, consistent with coordinated stress adaptation. These local changes were accompanied by rapid restructuring of the fecal microbial community and by systemic hypometabolism, despite markedly different hypothermic responses across strains. CTx also elicited a selective hepatic stress and metabolic program, including conserved suppression of Cyp7a1 across sexes and strains. Together, these findings show that CTx alone rapidly reorganizes the intestinal immune, epithelial, metabolic, and microbial environment while suppressing whole-body metabolism. This work provides an integrated framework for understanding the acute host response to CTx and for interpreting its widespread use as a mucosal adjuvant.

Background: Dietary fiber intake is broadly associated with reduced colorectal cancer (CRC) risk, yet processed fiber supplements exert inconsistent and occasionally opposing effects on colon tumorigenesis. How structurally distinct dietary fibers differentially shape the gut microbiome-metabolome axis to influence carcinogenic susceptibility remains poorly understood. Objective: To determine whether structurally distinct dietary fibers differentially modulate colon tumor development and characterize the underlying gut microbiome and metabolic mechanisms. Design: Four-week-old male mice were maintained on low-fiber control or fiber-supplemented diets (7.5%w/w: cellulose, agar, pectin, or inulin) for 28 weeks and received eight intraperitoneal azoxymethane injections (AOM; 7.5 mg/kg). Temporal fecal microbiomes were profiled by 16S-rRNA sequencing, cecal metabolites quantified by H-NMR spectroscopy, and microbiome-metabolome interactions assessed by co-occurrence networks. Results: Refined inulin supplementation markedly elevated colon tumor incidence (~70%) compared with all other groups (~10-20%), and cecal extracts from inulin-fed mice promoted HT29 cancer-cell proliferation. Longitudinal microbiome profiling identified 36 inulin-specific microbial signatures converging on two axes: depletion of SCFAs and fermentation cross-feeding taxa and reprogramming of amino acid and nitrogen metabolism. Metabolomics confirmed succinate, fumarate, and lactate accumulation alongside propionate and amino acid depletion exclusively under AOM conditions, with no differences in carcinogen-naive animals. Two stable co-occurrence hubs were identified exclusively in AOM-treated animals, structurally integrating both functional axes. Conclusion: Inulin supplementation preconditions the gut microbiome toward functional vulnerability, characterized by disrupted fermentation throughput and reprogrammed amino acid metabolism, that is amplified by carcinogen exposure into a tumor-promoting ecosystem, providing a mechanistic basis for the disproportionately higher tumor prevalence in inulin-fed animals.

Fermented foods are recognized for their rich microbial diversity and bioactive metabolites, which have recently been linked to anti-inflammatory effects and increased gut microbiome diversity. Despite extensive research on fermented foods including large-scale metagenomic surveys and metabolite characterization, a comprehensive mechanistic understanding of how diverse fermented foods, their microbes, and resulting metabolites interact with human biological pathways remains limited. Here, we systematically profiled over 100 commercially available fermented foods for their potential to prevent inflammation using a human cell-based model. We then generated bulk RNA sequencing of the human cells under these treatment conditions, along with metagenomic sequencing and metabolomics of the fermented foods used in the assays. By generating sample-matched multi-omics data, our work aims to lay the scientific groundwork that will enable scientists to generate predictive models and testable hypotheses about the molecular mechanisms underlying the anti-inflammatory effects of fermented foods. This open-source resource comprising all raw data and parsed intermediary files allows the community to begin elucidating the molecular mechanisms between fermented foods and human biology, paving the way for new, scientifically informed strategies for the user of fermented foods as functional foods.

Disruption of the intestinal microbiota increases susceptibility to Clostridioides difficile infection (CDI), creating an opportunity for prophylactic interventions before symptomatic disease develops. We investigated whether Escherichia coli Nissle 1917 (EcN) engineered to express the bacteriophage-derived endolysin CD27L could enhance pathogen inhibition and attenuate CDI. Using a plasmid-cured EcN chassis and pMUT2-derived vectors, we generated strains expressing CD27L under constitutive or anaerobically regulated promoter control. The constitutive strain, BioEcN1, reduced viable C. difficile recovery relative to parental EcN and was selected for animal studies. BioEcN1 remained recoverable from monoassociated mice without in vivo antibiotic selection, with similar recovery on selective and nonselective media supporting construct retention. Prophylactic administration reduced clinical disease, body-weight loss, and intestinal histopathological injury in germ-free mice. Observed survival was 100% in BioEcN1-treated challenged mice versus 50% in untreated challenged mice during the monitored period. Protection was accompanied by lower fecal and cecal pathogen burdens. In mice harboring a human-derived dysbiotic microbiota, prophylactic BioEcN1 also reduced clinical and stool-consistency scores, with lower early fecal pathogen recovery and less colon shortening. These findings establish preclinical proof of concept for prophylactic administration of an endolysin-expressing probiotic to attenuate CDI.

Acute respiratory distress syndrome (ARDS) is a severe lung disorder influenced by the interplay between the intestinal microbiome, host metabolism, and immune function. We previously found that Bacteroides acidifaciens (BA) has opposing effects on staphylococcal enterotoxin B (SEB)-induced ARDS, worsening the condition in female mice while alleviating it in males. This study aimed to examine whether these outcomes are linked to sex-dependent changes in the intestinal microbiome and gut-lung metabolome. C3H/HeJ mice of both sexes were orally colonized with Bacteroides acidifaciens before SEB exposure. Stool and bronchoalveolar lavage fluid (BALF) were collected for analysis using 16S rRNA sequencing and untargeted liquid chromatography mass spectrometry metabolomics. Our findings revealed distinct sex-dependent metabolic and microbial responses. In females, disease exacerbation correlated with elevated lipid mediators (leukotriene B4, 13-oxoODE, and 13S-HODE) alongside shifts in bile acid and redox metabolism. The intestinal microbiome showed changes in Bacteroides, Akkermansia, and Lachnospiraceae. Conversely, males showed a protective phenotype characterized by increased butyrate and alterations in sphingolipid, amino acid, redox, and nucleotide pathways. Integrated analyses identified sex-associated microbial communities and metabolomic profiles, highlighting distinct patterns of lipid and redox associations in females versus sphingolipid and short-chain fatty acid patterns in males. These results suggest that host sex influences how a commensal bacterium affects inflammatory lung injury and point to pathways for future research.

Epidemiological studies associate replacing animal-sourced with plant-sourced protein with lower colorectal cancer (CRC) risk, but controlled feeding studies have rarely tested how this substitution alters the gut microbiota and CRC-related biomarkers. In a 12-week randomized controlled feeding study, 136 healthy adults consumed diets providing 30 energy percent (E%), 50 E%, or 70 E% of protein from plant sources. Change in gut microbiota composition was the primary outcome; stool characteristics, fecal N-nitroso compounds (NOCs), and fecal and plasma bile acids (BAs) were secondary outcomes. The 70% plant-protein diet significantly restructured the gut microbiota, increasing fiber-associated Lachnospiraceae and Roseburia while reducing Methanobrevibacter and several asaccharolytic taxa. Microbial functional potential shifted toward plant-carbohydrate degradation, with lower amino acid degradation and methanogenesis. Fecal pH decreased, stool characteristics suggested faster colonic transit, and potentially carcinogenic total and haem-derived NOCs declined progressively with plant-protein replacement. The 70% plant-protein diet also reduced fecal secondary and taurine-conjugated BAs, including the CRC-associated BAs DCA and LCA. Plasma glucuronidated BAs increased, expanding the circulating pool of readily excreted BA conjugates. Higher insoluble fiber intake was associated with greater microbiota restructuring and lower fecal NOCs. Methanogen carriers had higher baseline fecal NOCs and features of slower colonic transit, and showed weaker microbiota and less favorable BA responses to the 70% plant-protein diet. Baseline methanogen carriage thus could be exploited for participant stratification in future dietary interventions.

Vietnam's rapid socioeconomic and dietary transition offers a rare window into how industrialization is reflected in the gut microbiome, yet Vietnamese populations remain underrepresented in global reference datasets. We investigated whether healthy Vietnamese adults in Hanoi harbor microbial community configurations associated with different stages of industrialization. Metagenomic data from stool samples of 183 clinically screened participants in Hanoi were analyzed alongside 629 publicly available metagenomic datasets from populations in low to middle income (LMI) and high income (HI) countries. Species-level taxonomic composition and gene-content-based metabolic pathways were characterized using community clustering and multivariable association analyses. Rather than forming a single population profile, the Vietnamese cohort separated into two enterotypes that mirrored the global industrialization gradient. Enterotype 1 more closely resembled populations from LMICs and showed greater microbial richness and diversity, whereas Enterotype 2 aligned with taxonomic and functional profiles observed in HICs. Both taxonomic and functional potential shows similar trajectories. Diet and lifestyle patterns describe some of the variance between enterotypes, supporting an association between contemporary exposures and microbiome structure without establishing causation. Antibiotic resistance profiles also varied between enterotypes, suggesting that the transition may encompass functions relevant to antimicrobial exposure. Together, these findings position urban Vietnam at a microbiome transition, where community states associated with different levels of industrialization coexist within a single population. This Vietnamese reference cohort expands representation in global metagenomic research and provides a baseline for tracking how rapid socioeconomic change may reshape microbial ecology, metabolic capacity, and antimicrobial resistance.

The gut microbiota plays a key role in piglet health, and maternal microbial transmission may represent a promising lever to shape early-life microbiota and prevent post-weaning digestive disorders. This study aimed to better characterize sow-to-piglet microbiota transmission and persistence using a long-read metabarcoding approach targeting the 16S-ITS-23S region. Fecal samples (n = 204) were collected from 17 families, a family being as sow and three of her piglets, at multiple stages: late gestation (G110), early (L6) and late lactation (L28) for sows; early lactation (L6), late lactation (L28), and 5 days post-weaning for piglets. To approximate strain-level resolution, a putative strain (PS) approach was developed by clustering ASVs (n = 6064) affiliated with the same species based on abundance covariance (r > 0.9), resulting in 4857 PS. Piglet microbiota progressively diversified during lactation and converged toward that of sow. In sows, 27 {+/-} 6% of PS were persistent from late gestation to late lactation. In piglets, only 4.2 {+/-} 2.5% of PS persisted from d6 to 5 days post-weaning. Persistent PS in piglets were mainly affiliated with Limosilactobacillus reuteri and Lactobacillus amylovorus followed with Holdemanella porci and H. biformis, Lentihominibacter hominis and Dorea formicigenerans. Shared PS were significantly higher within families than between unrelated pairs (p < 0.05). Maternal transmission peaked at the end of lactation (35 {+/-} 7% at L28). Persistent transmitted PS represented 2.7 {+/-} 1.6% (d6-post-weaning) and 15.4 {+/-} 5.6% (d28-post-weaning). Early-transmitted persistent PS were mainly affiliated with Limosilactobacillus reuteri, Lactobacillus amylovorus, and Paraeggerthella hominis, whereas late-transmitted persistent PS were associated with Prevotella spp., Sphaerochaeta globosa, and Bariatricus comes. These findings highlight the significance of maternal transmission in shaping the post-weaning microbiota and identify late lactation as a critical window for microbiota transfer.

The intestinal mucus layer forms the primary physical barrier separating the host epithelium from the gut microbiota. Antibiotic-induced dysbiosis compromises this barrier, yet the microbial metabolites and host mechanisms responsible for its rapid restoration remain poorly understood. Here, we identify pantothenic acid (vitamin B5) as a microbial metabolite that rapidly restores colonic mucus integrity following antibiotic administration. Integrated 16S rRNA gene sequencing and metabolomic profiling of murine feces revealed a positive association between Muribaculaceae abundance, fecal pantothenic acid level, and colonic mucus thickness. Among four isolated Muribaculaceae species, only Sangeribacter muris produced pantothenic acid. An exogenous pantothenic acid supplement quickly restored the thin mucus layer. This rapid response occurred without detectable transcriptional activation. This response did not recur in isolated colonic epithelium or colonic organoids, indicating that pantothenic acid acts through a non-epithelial, non-autonomous mechanism. Together, these findings identify a previously unrecognized microbe-metabolite-host signaling pathway that rapidly regulates colonic mucus barrier homeostasis and reveal a local barrier-protective function of microbiota-derived pantothenic acid.

Lifestyle changes associated with industrialization, including shifts in diet, are linked to extensive remodeling of the human gut microbiome. Investigating the gut microbiomes of traditional populations living non-industrialized lifestyles, such as the Hadza hunter-gatherers of Tanzania, therefore offers insight into bacterial taxa and functions depleted in industrialized microbiomes. Despite pronounced compositional differences that correspond to lifestyle, many commensal gut species are shared between industrialized and non-industrialized populations, raising a fundamental question of whether and how their functional repertoires diverge across contrasting gut environments. Here, by integrating strain-resolved metagenomics with culturomics and in vivo models, we identify phylogenetic and functional divergence within prevalent gut species shared between Hadza and industrialized populations, particularly among members of the Clostridia class. Dorea longicatena, prevalent across diverse human populations independent of lifestyle, is a poorly characterized member of Clostridia. Using strains of this D. longicatena isolated from either Hadza or industrialized human stool samples we uncover metabolic pathways selectively enriched in Hadza strains, including machinery for utilizing plant-derived pectic polysaccharides. We further demonstrate that this Hadza-enriched polysaccharide-utilization machinery can be leveraged with its cognate dietary substrate to promote D. longicatena colonization within a model industrialized microbiota, reshaping intestinal and portal-vein metabolite profiles. Together, our findings reveal substantial functional divergence within gut bacterial species shared across lifestyles, providing a framework for identifying diet-responsive strains and pathways that may complement ecological and metabolic functions underrepresented in industrialized microbiotas.

Formula-fed infants have altered gut microbiota and immune development relative to breastfed infants. Human milk oligosaccharides (HMOs) and microbes able to ferment these, like Bifidobacterium infantis, promote healthy immune-microbiome interactions in breastfed infants. In this randomized trial, 240 healthy term infants in the Philippines received a synbiotic blend formula (SynF; seven HMOs, bovine milk oligosaccharides, and B. infantis LMG11588) or control formula (CtrlF) for 6 months. SynF supported growth non-inferior to CtrlF and near universal B. infantis colonization (vs <10% in CtrlF). Markers of intestinal inflammation were lower in children fed SynF while secretory IgA was higher. SynF fed infants showed lower markers of systemic inflammation in plasma, slower immune cell maturation, and fewer upper respiratory tract infections (URTI) during their first 6 months of life. These findings suggest that synbiotic formula can dampen inflammatory responses to colonizing microbes and mild infections in fully formula-fed, term infants. Trial registration: NCT06073652.

The human gut microbiota has been explored for the last two decades and studies have uncovered its major role in the degradation of complex glycans from the diet as well as the host tissues. Strikingly, until very recently, no similar investigation has been carried out on the human oral cavity microbiota, which is increasingly recognized has an important parameter in gastrointestinal health. Prevotellaceae are an important and under-investigated family of bacteria in both the gut and the oral cavity microbiota. The oral cavity is particularly rich in glycosaminoglycans which could provide an abundant carbon source for the inhabiting microorganisms, and play a significant role in host-pathogen interactions. Hence, we analyzed Polysaccharide Utilization Loci in oral microbiota species and we identified a glycosaminoglycan-degrading Polysaccharide Utilization Locus in Segatella oris. We produced and characterized the four enzymes, confirmed their ability to degrade glycosaminoglycans, and we focused our efforts on the biochemical and structural characterization of the first reported Prevotellaceae endosulfatase.

Fecal-derived in vitro microbial communities represent promising models for investigating gut microbiome function and facilitating translational applications. However, the factors governing community establishment and the underlying mechanisms remain incompletely understood. We employed large-scale quantitative metaproteomics to investigate the functional and community dynamics of human fecal microbiomes cultured in three media over 25 serial passages. Our results demonstrated that both culture medium and donor-specific microbiome characteristics significantly influence community establishment. Taxon-specific functional analyses revealed nutrient availability in the medium and the microbial stress responses as key determinants of in vitro community establishment. Notably, the metabolism of mucin glycans in RapidAIM medium contributed to the maintenance of Bacteroidaceae species that were poorly supported in other media, resulting in an in vitro community that most closely resembled the uncultured fecal inoculum. Overall, these findings provide mechanistic insights into in vitro gut microbial community establishment, supporting further optimization and application of fecal-derived in vitro microbiome models.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects nearly a third of adults worldwide, yet how diet shapes the microbiota's contribution remains unclear. In a diet-induced mouse model varying protein content independently of caloric intake, microbiota depletion ameliorated hepatic steatosis and liver injury under protein- sufficient but not protein-restricted conditions. Caecal and plasma metabolomics implicated microbial phenylalanine catabolism and its host conjugates phenylacetylglutamine (PAGln) and phenylacetylglycine (PAGly). PAGln supplementation exacerbated steatosis and liver injury without altering body weight, adiposity or glucose homeostasis. Individual-antibiotic perturbation with metagenomics linked bacterial phenylalanine catabolic capacity to disease severity, and in gnotobiotic mice, genetic disruption of bacterial phenylalanine-to-phenylacetic acid conversion lowered circulating conjugates and attenuated steatosis and fibrosis. In a human cohort, circulating PAGln was selectively elevated in cardiometabolic MASLD. These findings identify dietary protein availability as a modifier of the contribution of microbial phenylalanine metabolism to MASLD and highlight PAGln as a microbiota-derived metabolite that exacerbates hepatic disease.

Background: The gut-liver axis is involved in Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD), but the host molecular actors involved in such dialogue have not yet been fully characterized. Objective: We investigated the sex-dependent role of the Constitutive Androstane Receptor (CAR) as a hepatic sensor of microbial signals in MASLD pathophysiology. Design: Male and female Car+/+ and Car-/- mice were fed a western diet (WD) for 4, 14 or 35 weeks. We performed plasma biochemistry, liver histology, bile acid and cholesterol quantification, untargeted multi-omics approach combining liver RNA-sequencing and 16S rRNA sequencing, together with co-housing, fecal microbiota transplantation and analysis of publicly available transcriptomics data in human cohorts. Results: Short-term WD revealed CAR-dependent protection against steatosis, together with perturbations in the transcriptional signatures of multiple transcription factors involved in hepatocyte identity in males. After 14 weeks, CAR deletion increased WD-induced liver damage, inflammation and fibrosis, and disrupted bile acid, cholesterol and gut microbiota homeostasis, in males only. Co-housing and fecal microbiota transplantations revealed that a Car+/+ gut microbiota protected against WD-induced liver damage, while a Car-/- microbiota was reshaped by a Car+/+ host. No CAR-dependent phenotype was observed in females. Finally, in human cohorts, hepatic CAR expression decreased in advanced clinical stages of MASLD. Conclusion: CAR is a sex-dependent protective nuclear receptor against MASLD progression, acting both as a transcriptional regulator of hepatocyte identity and as a mediator of the host-microbiota crosstalk.

Metformin is the first-line therapy for type 2 diabetes, yet the mechanisms underlying its glucose-lowering effects remain incompletely understood. Increasing evidence implicates gut microbiota as an important mediator of metformin action, but the bacterial targets of the drug remain poorly defined. Here, we identify bacterial NADH oxidoreductase (NDH-1), the homolog of mitochondrial complex I, as a key target of metformin. Metformin inhibited bacterial growth at millimolar concentrations and, at subinhibitory concentrations, markedly impaired carbohydrate fermentation in Escherichia coli and other NDH-1-containing bacteria. Supplementation with nicotinic acid and Mg2+; restored fermentation, whereas pyruvate bypassed the metabolic defect, implicating impaired NAD+; regeneration and disruption of metabolism upstream of pyruvate formation. Genetic analysis further established NDH-1 dependence: metformin failed to inhibit fermentation in an E. coli delta-nuoF mutant lacking functional NDH-1 but retained activity in an NDH-2-deficient delta-ndh strain. Molecular docking predicted metformin binding within the NDH-1 quinone-binding region, proximal to conserved residues involved in quinone reduction. Consistent with this mechanism, Staphylococcus aureus, which lacks NDH-1, was not affected in carbohydrate fermentation, while several gut-associated probiotic and short-chain fatty acid-producing bacteria, including Lactobacillus, Bifidobacterium, and Akkermansia muciniphila, lacked detectable NDH-1 homologs, suggesting intrinsic resistance to this metabolic effect of metformin. These findings identify bacterial NDH-1 as a metabolic target of metformin and provide a mechanistic framework for its effects on the gut microbiota. Selective inhibition of NDH-1-dependent bacteria could alter microbial substrate utilization and ecological interactions in the intestine, potentially contributing to the microbiome-mediated component of metformin's antidiabetic effects.

Antibiotic disruption of gut microbiota creates opportunities for toxigenic Clostridioides difficile. One strategy is to occupy this niche with a non-toxigenic or weakly virulent strain, but even strains that cause little disease may fail to compete during pathogen challenge. We screened 21 sequence type 1 (ST1) isolates for their ability to protect mice co-colonized with toxigenic VPI10463, separating disease potential from protection. Some strains, such as ST1-75, caused little disease and protected, whereas others, such as ST1-68, caused little disease but failed to protect. Metabolomics showed that ST1-75 depleted more proline than ST1-68, whereas another protective strain, ST1-6, had a different metabolic profile; both protective strains nevertheless responded to many substrates. Substrate-utilization profiling revealed that protection across the 21 isolates correlated with amino-acid overlap with VPI10463, but not overall nutrient overlap or shared genome-encoded protein families. Relative amino-acid access explained variation beyond mono-colonization disease potential, and a consumer-resource model showed how nutrient overlap together with additional resource access can support persistence during competition. Additional experiments support that ST1-mediated protection occurs even in the absence of mature B and T cells. These findings distinguish low disease potential from protection, link protection to strain competition, and identify relative resource access as an informative phenotype for prioritizing strains for biotherapeutic testing.