Gut Microbes

Background and AimsThe rising incidence of Crohns disease (CD) in Westernized countries has been linked to changes in diet and increased consumption of food additives, yet the mechanisms by which these factors fuel intestinal inflammation remain unclear. Adherent-invasive Escherichia coli (AIEC), a pathobiont involved in CD pathogenesis, lacks a clear genetic hallmark but exhibits intestinal colonization and virulence traits, raising questions about the evolutionary forces promoting its emergence among select individuals. Here, we investigated how chronic exposure to two common dietary emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (P80), along with host inflammation, drives AIEC genomic evolution and pathogenic potential. MethodsWild-type and Il10-deficient mice were monocolonized with AIEC and chronically exposed to CMC, P80, or water. Bacterial isolates were collected and analyzed for genomic diversification, mutations, and phenotype both in vitro and in vivo. ResultsEmulsifiers accelerated AIEC genomic diversification and selected for mutations linked to increased motility, invasion, and pro-inflammatory activity. Moreover, dietary emulsifier-evolved strains displayed a marked fitness advantage in vivo, outcompeting their counterparts in murine hosts, with the greatest advantage observed when evolution occurred under inflammatory conditions. Notably, evolutionary pathways and phenotypic outcomes were shaped by both emulsifier and the hosts inflammatory status, highlighting synergy between diet and host genetics in fostering pro-inflammatory pathobionts. ConclusionThese findings provide an evolutionary framework connecting modern dietary habits to the emergence of pathogenic AIEC strains, and underscore the importance of dietary interventions in individuals at risk for inflammatory bowel disease.

Bile acids modulate the intestinal microbiota and serve as key signaling molecules in host physiology. Bile acid dysregulation has been implicated in nutritional and inflammatory diseases; however, data on the pool of bile acids present in stunted children or children suffering of environmental enteric dysfunction (EED) is limited, particularly in the upper intestinal compartment where disease phenotypes are most relevant. In this study, we performed a targeted metabolomics approach on 75 bile acids and their derivatives, including gastric and duodenal aspirates and fecal samples from almost 1000 children from two Sub-Saharan cities. We found that levels of secondary bile acids, especially lithocholic acid, are significantly lower in the feces of stunted and EED children, while ursocholic acid and its derivatives are significantly higher. Levels of primary and sulfated bile acids are also increased in the feces of children with EED. Microbiota sequencing revealed that high lithocholic acid levels are positively associated with butyrate-producing bacteria, while negatively associated with oral taxa like Streptococcus and Veillonella. In vitro tests on a panel of reference strains showed that oral bacteria bioaccumulate and are inhibited by a variety of bile acids, while lithocholic and chenodeoxycholic acids modulate the growth of several butyrate-producing bacteria. This effect was even stronger with tauro- or glycol-conjugated bile acids. Exposing stool-derived in vitro communities from children in Afribiota to these bile acids confirmed their positive impact on butyrate producers and negative effect on overgrowing oral taxa. Our findings suggest that secondary bile acids, reduced in stunting and EED, modulate the growth of butyrate-producing bacteria while suppressing harmful oral taxa, highlighting their potential as tools to modulate microbiota composition.

BackgroundThe enterotype concept proposed that gut microbiomes cluster into discrete types, but subsequent critiques demonstrated that such clustering depends on methodological choices, that the number of clusters is not fixed, and that faecal samples cannot capture spatial heterogeneity along the gastrointestinal tract. The stomach remains particularly understudied, and no systematic classification exists for gastric microbial community types. MethodsWe assembled a multi-cohort dataset of 566 gastric mucosal samples spanning healthy controls to gastric cancer, with both Helicobacter pylori (HP)-negative and HP-positive individuals. Critically, we applied the key methodological lessons of the enterotype debate: we used a variational autoencoder (VAE) for dimensionality reduction to learn a continuous latent representation without forcing discrete structure, determined the optimal number of clusters using the Silhouette index (an absolute validation measure) across K=2 to K=10 rather than arbitrarily selecting a cluster number, and performed transparent evaluation of multiple clustering solutions. This VAE-plus-silhouette workflow directly addresses the critiques leveled against the original enterotype analysis. ResultsFour gastotypes were identified, with K=4 achieving the highest mean silhouette score, indicating good cluster cohesion and separation. Two gastotypes (Variovorax-type and Trabulsiella-type) were significantly enriched in HP-positive samples, while two gastotypes (Bacteroides-type and Streptococcus-type) were significantly enriched in HP-negative samples. Random Forest and Gradient Boosting achieved excellent baseline performance for predicting HP infection (AUC = 0.990 and 0.993). ConclusionsThe VAE-plus-silhouette workflow provides a robust, data-driven approach for identifying gastotypes without forcing discrete structure or arbitrarily fixing cluster numbers. Using this framework, we identified four gastotypes with significantly different HP infection rates. Variovorax-type and Trabulsiella-type showed strong HP-positive enrichment, while Bacteroides-type and Streptococcus-type showed strong HP-negative enrichment. These findings demonstrate that methodological advances from the enterotype controversy can be successfully transferred to the stomach, offering a reproducible taxonomy for stratifying HP infection status with potential clinical utility.

The incidence of colorectal cancer (CRC) has been increasing in Taiwan and is associated with multiple risk factors, including aging, obesity, and dietary habits. Increasing evidence suggests that gut microbiota dysbiosis contributes to CRC development. This study aimed to characterize microbial and metabolic alterations across premalignant and malignant colorectal lesions and to identify potential microbiome-associated biomarkers. Individuals undergoing colonoscopy for screening or surveillance at Taipei Veterans General Hospital were enrolled. Gut microbial composition was analyzed using full-length 16S rRNA gene sequencing to achieve high-resolution taxonomic profiling. Predicted functional pathways were inferred from microbial communities, and targeted metabolomic profiling was performed to evaluate microbial metabolic outputs. A total of 122 individuals were included, comprising 62 healthy controls, 15 adenoma cases, and 45 CRC cases. Progressive shifts in microbial composition and predicted functional pathways were observed along the adenoma-carcinoma sequence. Several bacterial taxa, including Phocaeicola dorei, Anaerotignum faecicola, Negativibacillus massiliensis, and Dysosmobacter segnis, were enriched in CRC. At the functional level, CRC samples showed enrichment of pathways associated with energy metabolism and bacterial stress responses. Metabolomic analysis further revealed increased levels of tauro-ursocholanic acid in CRC samples, whereas short-chain fatty acids (SCFAs) were reduced compared with controls. Integrative analysis combining full-length 16S sequencing, functional pathway prediction, and metabolomic profiling revealed coordinated microbial and metabolic alterations across the adenoma-carcinoma sequence. These findings provide insight into microbiome-associated processes in colorectal tumorigenesis and suggest potential microbial and metabolic biomarkers for CRC. ImportanceColorectal cancer (CRC) develops through a adenoma-carcinoma sequence, yet the microbial and metabolic alterations accompanying this progression remain incompletely understood. In this study, we integrated full-length 16S rRNA gene sequencing with metabolomic profiling to characterize taxonomic, functional, and metabolic changes across healthy controls, adenoma, and CRC. Our results reveal synchronized shifts in specific microbial taxa, predicted metabolic pathways, and fecal metabolites along the adenoma-carcinoma sequence. Several bacterial species, including Phocaeicola dorei, Anaerotignum faecicola, and Dysosmobacter segnis, increased in CRC, whereas short-chain fatty acids decreased progressively from controls to adenoma and CRC. Functional pathway analysis further indicated alterations in microbial fermentation, amino acid metabolism, and energy-related pathways. Together, these findings highlight the potential role of microbiome-associated metabolic changes in colorectal tumorigenesis and suggest candidate microbial and metabolic markers that may aid in understanding disease development and improving risk stratification.

BackgroundEmerging evidence suggests that the oral microbiome may contribute to aberrant gut immune responses in Inflammatory Bowel Disease (IBD). MethodsWe performed a comprehensive, harmonised analysis of aggregated oral microbiome 16S rRNA datasets across multiple cohorts. Data were processed using a unified bioinformatics pipeline including DADA2 for taxonomic assignment, PICRUSt2 for functional prediction, MaAsLin2 for multivariable modelling, and machine learning. ResultsAcross 25 studies (n = 1,136 IBD; n = 759 controls), meta-analysis showed significantly reduced oral microbial Shannon diversity in IBD (standardised mean difference -0.31, p = 0.007). Secondary bioinformatics analysis of six datasets plus in-house data confirmed this reduction (Shannon diversity; Hedges SMD = -0.372, p < 0.001), driven primarily by Crohns disease (CD). Beta diversity demonstrated global compositional shifts, with CD demonstrating greater divergence from controls than ulcerative colitis (UC). Multivariable modelling identified reproducible taxa enriched in IBD, including Corynebacterium, Serratia and Streptoccocus, while Porphyromonas and Ruminococcaceae.G1 were enriched in controls. Functional pathway prediction indicated reduced butyrate metabolism in IBD sub-types and increased aromatic amino acid and related metabolite degradation pathways. Machine learning classifiers achieved modest discrimination (mean AUC [~]0.67), supporting the potential of saliva-based microbiome profiling to study dysbiosis in IBD. ConclusionsThese findings demonstrate that the oral microbiome in IBD is characterised by reduced diversity and reproducible structural community reorganisation. Together, these data support a contributory role for the oral-gut axis in CD pathogenesis and provide a rationale for targeted mechanistic and longitudinal studies to define causal links between oral dysbiosis and intestinal inflammation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/26351936v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@57306corg.highwire.dtl.DTLVardef@2c0ef0org.highwire.dtl.DTLVardef@88b0b3org.highwire.dtl.DTLVardef@8ed62_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microbiome-based machine learning classifiers show increasing promise for disease identification across gastrointestinal, metabolic, and immune-mediated conditions. Inflammatory bowel disease (IBD), a chronic immune-mediated disorder associated with disruption of the gut microbiome, has been a particularly successful application area. However, while many predictive models achieve high performance within individual datasets, their ability to generalize across independent populations and geographic contexts remains unclear. Here, we tested whether model class and training dataset composition influence model generalizability across geographically diverse evaluation studies. We compiled seven publicly available shotgun metagenomic studies spanning five geographic regions, comprising 697 individuals with IBD or healthy controls. We trained 246,986 model configurations across seven model classes and five distinct training dataset combinations and evaluated top-performing models on independent studies from the USA, Ireland, Germany, Israel and China Extreme gradient boosting and random forest models showed the highest and most consistent performance across training datasets, a ranking that was maintained on independent evaluation studies. However, models trained on geographically diverse datasets did not outperform those trained on USA-only datasets. Instead, model performance was strongly dependent on the evaluation study itself, with consistent differences in achievable accuracy across studies. Despite most models achieving similar AUC scores, there was limited overlap in the key microbial species identified. Furthermore, even for the small set of disease predictive microbes shared between models, the direction of enrichment between IBD or healthy subjects often varied in opposing directions across study populations. These findings suggest that study-specific factors constrain generalization and may help explain the lack of consistent microbiome-based biomarkers for IBD. ImportanceMachine learning models based on the human gut microbiome are increasingly proposed as diagnostic tools for inflammatory bowel disease, but our findings suggest that identifying reliable microbiome biomarkers poses a challenge. Models trained on different datasets often selected different species as important predictors, even when diagnostic performance was similar, indicating that disease-associated microbes may depend strongly on the patient populations studied. Even species repeatedly selected across training datasets frequently showed inconsistent associations with disease, helping explain low agreement across microbiome studies. Importantly, models performed well across new patient groups independent of the geographic diversity present in the training datasets. By identifying microbial species repeatedly selected across datasets, model types, and evaluation studies, we identified a smaller group of more consistent biomarkers, including enrichment of Klebsiella pneumoniae and Erysipelatoclostridium ramosum and depletion of Lachnospiraceae and Alistipes species, which may represent stronger candidates for transferable microbiome markers.

The gut microbiome is a key regulator of metabolic homeostasis and contributes to obesity progression through effects on immune signaling, gut barrier integrity, and systemic inflammation. Microbiome-targeted strategies are therefore being explored as complementary approaches to conventional weight-loss therapies. Here, we investigated the probiotic yeast Saccharomyces boulardii in a murine model of diet-induced obesity (DIO) using an integrated multi-omics framework combining metabolic phenotyping, gut microbiome profiling, cecal metabolomics, colonic transcriptomics, and portal cytokine analysis. S. boulardii reduced food intake, attenuated weight gain, and increased energy expenditure without major changes in circulating metabolic hormone levels. Microbial diversity remained largely preserved, but selective enrichment of Bacteroidales lineages, including Muribaculaceae, was observed alongside functional remodeling of microbial pathways. Cecal metabolomics revealed increased B-vitamins, betaine, and GABA, with reduced stress-associated metabolites. Colonic transcriptomics showed attenuation of TNF/NF-{kappa}B signaling and enrichment of interferon and epithelial programs, while portal cytokine profiling indicated reduced inflammatory chemokines with trends toward increased IL-17A and IL-22. Integrated multi-omics analysis identified coordinated host-microbe interactions across metabolic, transcriptional, and immune layers. Collectively, these findings demonstrate that S. boulardii modulates the gut-immune-metabolic axis in obesity, supporting microbiome-based interventions as potential adjunct strategies targeting metabolic inflammation.

Vitamin B2 (riboflavin) is a key redox cofactor that may modulate gut microbial ecology, yet conventional supplements are absorbed proximally and have limited colonic exposure. We evaluated whether colon-targeted riboflavin alters microbiome composition, function and network structure as well as host biomarkers in healthy older adults. In a randomized, double-blind, placebo-controlled, parallel-group clinical trial (N=348; 50-70 years), participants received colon-targeted riboflavin (1.4, 10, or 75 mg/day) or placebo for 12 weeks. The primary endpoint was the change in fecal microbial composition, while secondary endpoints encompassed microbiome function, host health biomarkers, and clinical outcomes. Shotgun metagenomics and fecal/blood biomarkers were assessed at baseline, week 4, and week 12. Although no significant changes were observed between groups in overall community-wide diversity metrics (alpha and beta diversity), colon-delivered riboflavin significantly altered the relative abundance of several microbial taxa compared with placebo. The most pronounced effects on microbiome composition, function, and network structure were observed with the 10 mg dose at week 12, reflected by within-group increases in alpha diversity, the largest rise in total species counts, higher HACK index values indicating greater community resilience, and distinct shifts in KEGG module abundance, including enhanced potential for riboflavin biosynthesis. Supplementation with 75 mg riboflavin led to higher fecal butyrate concentrations at week 4 versus placebo, while the lowest dose (1.4 mg) significantly reduced the dysbiosis index within groups and modestly improved network structure across groups. All three doses (1.4, 10, and 75 mg) influenced keystone species abundance. No between-group differences were observed for gastrointestinal symptoms, quality-of-life measures, fecal pH, high-sensitivity C-reactive protein (hs-CRP), calprotectin, or soluble CD14, except for an increase in plasma riboflavin concentrations at 75 mg after 12 weeks, indicating colonic absorption. The product was safe and well-tolerated across all doses. These findings indicate that colon-targeted riboflavin can act as a functional modulator of the human gut microbiome, with the most consistent effects observed at 10 mg and additional dose-specific effects at 1.4 mg and 75 mg. Future studies are warranted to establish related health benefits, either as a standalone intervention or in combination with classical pre-, pro-, or postbiotics, particularly in target populations such as individuals with IBS, stress, mild cognitive decline, or early metabolic or inflammatory alterations.

Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection and is increasingly explored for other dysbiosis-related disorders. However, its implementation as a regulated therapeutic strategy still requires robust donor screening, biosafety frameworks, and standardized processing workflows. Here, we describe the establishment of the first fecal microbiota biobank in the south of Brazil and evaluate the incorporation of metagenomic sequencing as a complementary layer of donor safety assessment. A structured donor selection pipeline based on international guidelines was implemented, integrating clinical screening, biochemical and serological testing, and microbiological analyses. Of 100 screened candidates, only four donors met all eligibility criteria and were included in the biobank, highlighting the stringency of the selection process. Shotgun metagenomic sequencing revealed a diverse resistome across all donors, including a shared core set of resistance-related genes alongside marked interindividual variability. Dominant antibiotic resistance genes included tetracycline-associated determinants, as well as ermF, CfxA-type {beta}-lactamases, and aminoglycoside-modifying enzymes, each linked to specific gut taxa. Notably, the relatively high abundance of tetW and ermF in Bacteroides fragilis suggests that this dominant commensal species may act as a reservoir for tetracycline and multidrug resistance determinants within the intestinal microbiota. Rather than serving as exclusion criteria, such determinants highlight the importance of integrating functional genomic profiling into donor characterization. Overall, this study provides a framework for microbiota biobank implementation and supports the use of metagenomics as a complementary strategy to improve biosafety and functional assessment in FMT.

Background: Irritable bowel syndrome (IBS) is characterized by heterogeneous symptom trajectories and high treatment discontinuation rates. Traditional analyses examine longitudinal outcomes and time-to-event endpoints separately, potentially missing informative dropout and the association between symptom dynamics and treatment persistence. Objective: To jointly model patient-reported IBS symptom trajectories and time-to-treatment discontinuation using shared random effects, characterizing the association between individual symptom dynamics and treatment persistence in a large Canadian prospective cohort. Methods: We analyzed 2,847 adults with Rome IV diagnosed IBS enrolled in the Canadian Gut Project (2018 to 2024) across 14 gastroenterology centres in Alberta, British Columbia, and Ontario. The longitudinal submodel used linear mixed-effects regression for the IBS Severity Scoring System (IBS-SSS) measured at baseline and months 3, 6, 12, 18, and 24. The survival submodel used a Weibull proportional hazards model for time-to-treatment discontinuation. The joint model linked both processes through shared random effects (random intercept and slope), estimated via maximum likelihood with adaptive Gauss-Hermite quadrature (15 nodes). We conducted sensitivity analyses using Bayesian estimation, alternative association structures (current value, time-dependent slopes), and multiple imputation for intermittent missingness. Results: Mean baseline IBS-SSS was 298.4 (SD 72.1). Over 24 months, 1,042 participants (36.6%) discontinued treatment. The longitudinal submodel revealed a mean IBS-SSS decline of -8.7 points/month (95% CI: -10.2, -7.1) with substantial between-person heterogeneity in both intercepts (STD = 4,218.3) and slopes (STD = 12.4). The association parameter linking the shared random intercept to the hazard of discontinuation was = 0.0034 (95% CI: 0.0021, 0.0047; p < 0.001), indicating that each 10-point increase in individual-specific baseline severity increased the hazard of discontinuation by 3.5%. The shared slope association parameter was 2 = -0.187 (95% CI: -0.264, -0.110; p < 0.001), demonstrating that individuals with steeper symptom improvement had lower discontinuation hazards. IBS-D subtype (HR = 1.41; 95% CI: 1.18, 1.69), concurrent anxiety (HR = 1.28; 95% CI: 1.09, 1.50), and social media health information use (HR = 0.82; 95% CI: 0.71, 0.95) were significant predictors in the survival submodel. Conclusion: Joint longitudinal-survival modelling reveals that IBS symptom trajectories and treatment discontinuation are dynamically linked through individual-level latent processes. Higher baseline severity and slower improvement trajectories significantly predict earlier discontinuation. These findings support personalized treatment monitoring approaches that use real-time symptom trajectory data to identify patients at risk of discontinuation.

Background Recent mouse model data demonstrate that chronic colonization with toxigenic Clostridioides difficile promotes colonic tumorigenesis via intraluminal toxin B (TcdB), its main virulence factor. In a prior multisite hospital cohort, we found that history of positive tcdB stool testing was associated with increased CRC risk in a dose-dependent manner, though limited by small sample size. We aimed to validate this association in a larger cohort with extended follow-up and greater geographic distribution using the Veterans Health Administration (VHA) Corporate Data Warehouse (CDW). Methods We conducted a retrospective cohort study among adults receiving care through the VA from 2000-2025 who underwent C. difficile testing. Data collected from the VHA CDW and National Death Index (NDI) included demographics, comorbidities, medications, CRC risk factors, and cancer incidence and death. The first C. difficile test date defined cohort entry; individuals with prior CRC were excluded. Ever C. difficile positivity was defined by a positive PCR or EIA results. The number of positive tests (episodes) was also determined to define recurrent positivity. Follow-up time ended at the first occurrence of CRC incidence or mortality, death from other causes, or censor date. Follow-up time was split for individuals who converted from negative to positive, with follow-up time updated accordingly. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HRs) for C. difficile exposure and CRC incidence and mortality after adjustment for confounders. Tests for linear trend and tests for interaction were conducted to assess effect modification by sex and IBD status, while time-lag intervals were evaluated for 1, 3, 5, and 10 years before the outcome. Results Among 806,844 veterans with C. difficile testing, those with positive tests were more likely to be older, male, to have diabetes, to use aspirin, and to have a lower BMI than those with negative tests. Race and IBD prevalence were similar between the groups. There was no overall association between ever C. difficile positivity and CRC incidence (HR = 0.99, 95% CI 0.93-1.05). However, recurrent C. difficile positivity was associated with increased risk in a dose-response manner [2-3 episodes HR = 1.30 (95% CI 1.16-1.47), and >3 episodes HR = 1.58 (95% CI 1.17-2.14) compared to negative tests; ptrend< 0.001]. Further, ever C. difficile positivity was associated with increased CRC mortality risk (HR = 1.21, 95% CI 1.13-1.30; p < 0.001). Recurrent C. difficile positivity was associated with increased mortality risk but was particularly strong for those with >3 episodes among individuals with IBD (HR=3.84, 95% CI 1.98-7.45). In sensitivity analyses, the increased risk of CRC incidence and mortality attenuated beyond 10 years. Conclusion Prior positive C. difficile testing was associated with increased CRC incidence and mortality in a dose-dependent manner, particularly among patients with IBD. These findings extend animal model evidence, epidemiologically establishing C. difficile presence as an independent risk factor for subsequent colorectal tumorigenesis and supporting investigation into recurrent CDI, especially among patients with IBD, as a potential modifiable CRC risk factor.

The gut microbiome is a critical part of host homeostasis, yet its resilience following opioid exposure remains poorly understood. While opioid-induced short-term dysbiosis is well documented, the long-term recovery dynamics following oxycodone remain unclear. This study characterized the temporal dynamics of the fecal microbiota in male C57BL/6J mice following a brief 3-day oxycodone regimen (5mg/kg, BID). 16S rRNA gene sequencing was performed at baseline, day 3, 10, 17, and 70. While acute post-treatment phases (day 3 to 10) showed subtle taxonomic shifts in Clostridium_sensu_stricto_1 and Romboutsia, significant community disruption emerged later. By day 17, beta diversity significantly differed from saline controls (P =0.002). At day 70, both alpha diversity (p=0.02) and beta diversity (P=0.007) remained significantly altered, characterized by enriched Akkermansia and Marvinbryantia alongside depleted Eubacterium_xylanophilum. These findings demonstrate that even brief oxycodone exposure triggers persistent, non-recovering dysbiosis that became detectable only after treatment cessation and persisted through day 70. This suggests that the window for microbiome recovery exceeds two months in mice (equivalent to several human years), highlighting a potential long-term risk for patients prescribed short-term opioid courses. ImportanceShort-term opioid exposure is generally assumed to cause only transient disruption of the gut microbiome. However, the duration of microbiome recovery following clinically relevant opioid treatment remains poorly defined. In this study, we show that a brief three-day course of oxycodone in mice resulted in delayed and persistent alterations in gut microbial community structure that remained detectable for at least 70 days after treatment cessation. Notably, significant divergence in microbial composition emerged weeks after exposure rather than immediately following treatment, suggesting that short-term opioid use may initiate longer-lasting remodeling of the gut microbiome than previously appreciated. These findings highlight the importance of considering extended recovery timelines when evaluating the microbiological consequences of opioid exposure.

1.Fermentable fibres such as inulin can support metabolic health but may exacerbate gastrointestinal symptoms in individuals with irritable bowel syndrome (IBS) due to rapid fermentation and gas production. The gel-forming fibre psyllium improves IBS symptoms, although the underlying mechanisms remain unclear. We hypothesised that fibre gelation alters fermentation by modulating microbial access to substrates. To test this, we compared psyllium with methylcellulose, a chemically modified, gel-forming fibre, to determine the effects of gelation on inulin fermentation. Inulin alone or combined with psyllium or methylcellulose was fermented for 48 hrs in a colonic fermentation model inoculated with healthy human faeces. Gas production, metabolite profiles, microbial community composition and microbial localisation within fibre gels were assessed. Bioactivity of fermentation products was evaluated in STC-1 cells. Psyllium co-fermentation significantly accelerated fermentation and enhanced production of metabolites, while methylcellulose had minimal effects. Psyllium maintained higher diversity and enriched polysaccharide-degrading taxa including Bacteroides and Phoecaeicola species, which were strongly associated with metabolic activity. Bacterial penetration into the psyllium matrix was observed but not into methylcellulose. Fermentation products from psyllium but not methylcellulose stimulated GLP-1 and 5-HT secretion in STC-1 cells. These findings demonstrate that delayed-onset fermentable gel-forming fibres enhance microbial access to entrapped substrates, driving metabolic and hormonal responses.

Enteroaggregative Escherichia coli (EAEC) is a leading cause of persistent diarrhea in children in low- and middle-income countries, and the emergence of multidrug-resistant (MDR) strains necessitates non-antibiotic therapeutic strategies. This study evaluates Lactobacillus johnsonii, previously characterized by our group, as a probiotic candidate against a clinically confirmed MDR EAEC isolate resistant to ampicillin, ciprofloxacin, azithromycin, amoxicillin, and gentamicin. L. johnsonii demonstrated robust gastrointestinal resilience, high cell surface hydrophobicity, phenol tolerance, and rapid autoaggregation reaching 80.4 {+/-} 2.3% by 4 hours, collectively supporting mucosal colonization potential. In antimicrobial assays, L. johnsonii produced zones of inhibition against MDR EAEC substantially exceeding those of gentamicin, reduced viable biofilm-associated EAEC by over 80%, and displaced pre-adhered EAEC from HCT-116 intestinal epithelial cells in a time-dependent manner. L. johnsonii also attenuated MDR EAEC-induced gas production and reduced nitric oxide levels by 67.7% in infected RAW 264.7 macrophages, suggesting immunomodulatory activity. Nutrient competition did not appear to contribute to EAEC suppression under tested conditions, indicating inhibition is predominantly secretome-dependent. Fractionation of the L. johnsonii cell-free supernatant by fast protein liquid chromatography yielded five fractions below 75 kDa; fractions S5 and S6 exhibited sustained bactericidal activity at 6 hours. Gram staining confirmed that both fractions reduced viable EAEC cell numbers, with S6 producing a greater reduction than S5, indicating quantitatively distinct bactericidal potencies. These in vitro findings support the potential of L. johnsonii as a biotherapeutic candidate for antibiotic-resistant enteric infections. In vivo validation and chemical characterization of active fractions remain important next steps.

Shigella sonnei is a leading cause of bacterial dysentery and a high priority WHO pathogen because of the spread of multidrug resistant strains. Understanding microbiome-Shigella-host interactions during colonization of the gastrointestinal tract, and the development of vaccines have been hampered by the lack of small animal models of shigellosis. Here, we developed a murine model of intestinal colonization with S. sonnei. Pre-treatment of mice with antibiotics disturbed the intestinal microbiome and rendered mice susceptible to high level, gastrointestinal colonization with S. sonnei for over one week. Infection with S. sonnei CS14 harbouring a stable virulence plasmid induced an initial inflammatory response in wild type mice, with weight loss and elevated levels of fecal lipocalin 2; the S. sonnei Type III Secretion System was responsible for this inflammatory response. Expression of O-antigen and Group IV capsule by S. sonnei promoted sustained intestinal colonization, with infected mice developing mucosal and systemic antibody responses predominantly directed at these glycans. Finally, infection with S. sonnei induced a degree of protection against subsequent re-challenge. Overall, this murine model successfully mimics aspects of S. sonnei colonization and should be helpful in understanding how S. sonnei successfully survives within the gastrointestinal tract and competes with the microbiota as well as the evaluation of vaccine candidates.

The study of human enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) has been limited by the inability of these pathogens to effectively colonize murine models without prior antibiotic treatment. Because it mimics key features of human EPEC and EHEC infection, Citrobacter rodentium, a natural mouse pathogen that colonizes the lower intestine, has become the primary model for investigating these organisms. C57BL/6 mice are most commonly used for C. rodentium research, however, unless they carry specific genetic mutations, they typically develop only mild disease and clear the infection within weeks. As a result, models of severe disease in genetically unmodified hosts are lacking. Here, we describe the development of a non-genetically modified C57BL/6 mouse line with an undisturbed intestinal microbiota, highly susceptible to severe C. rodentium infection. Early infection in these mice was marked by significantly elevated cecal bacterial burdens and tissue pathology. Immune profiling revealed broad reductions in multiple lymphoid subsets, indicating impaired early mucosal activation. Although overall cytokine expression patterns were similar between groups, ceca of susceptible mice exhibited elevated baseline and early post-infection IL-18, as well as increased G-CSF at day 1. Microbiota analyses showed broadly comparable communities with wildtype controls, with some altered groups, such as Lachnospiraceae, Prevotellaceae, Desulfovibrionaceae, and Erysipelotrichaceae. Together, these findings characterize a robust C57BL/6 model that reproducibly develops severe C. rodentium-induced disease. This phenotype is driven by microbiota-associated alterations and impaired early cecal immunity, providing a valuable system for studying host-microbiota interactions in enteric infections.

The role of gut microbiome in regulating vertebrate metabolism has been well-recognized. However, the effects of gut bacteria on growth have been less studied. Bacillus is a prevalent genus in the gut microbiota of human and animals. In this study, the effect of gut-derived Bacillus velezensis T23 on growth was investigated in zebrafish. B. velezensis T23 improved the growth of zebrafish and promoted IGF1 production in the liver and muscle, with a concomitant activation of the AKT/mTOR signaling pathway. The growth-promoting effect of B. velezensis T23 was not dependent on lipopeptides and polyketides. Cell wall peptidoglycan isolated from B. velezensis T23, as well as muramyl dipeptide (MDP), was sufficient to stimulate IGF1 signaling and growth. Further, the effect of B. velezensis T23 on growth and IGF1 production was abrogated in nod2-/- zebrafish, confirming that B. velezensis T23 promoted growth via MDP-NOD2 signaling. Gut transcriptomic analysis indicated that B. velezensis T23 promoted renewal and differentiation of intestinal cells, suggesting an involvement of gut-liver axis in the effect of B. velezensis T23 on systemic IGF1 production. Together, our results revealed an effect of gut Bacillus-derived muropeptide on growth via NOD2-IGF1 signaling, and provided novel mechanistic insights in the beneficial effect of Bacillus spp. as probiotics.

BackgroundHigher-quality diets have been associated with lower levels of ectopic fat deposited in the viscera and liver, which is hypothesized to be mediated in part by the gut microbiota. ObjectivesWe tested this hypothesis in a multi-ethnic imaging study using global (microbiome-wide) testing as well as a high-dimensional multiple-mediators regression framework to identify bacterial genera in the human gut that mediate the association between diet quality and ectopic adiposity. MethodsWe analyzed the cross-sectional data of 1,400 older adults (age 60-77) from five racial/ethnic groups in the Multiethnic Cohort Adiposity Phenotype Study (2013-2016). Overall diet quality was defined by adherence to the MIND diet. The relative abundance of 151 bacterial genera was quantified from 16S rRNA gene sequencing of the stool samples. Visceral fat, liver fat, and the presence of MASLD (metabolic dysfunction-associated steatotic liver disease) were determined based on magnetic resonance imaging (MRI). We used high-dimensional mediation analysis (HDMA) to estimate gut microbial mediation in the linear regression of visceral fat or liver fat, or in logistic regression of MASLD, on the MIND adherence score, adjusted for potential confounders. ResultsHigher diet quality was associated with lower ectopic adiposity: 12% less visceral fat area, 23% less liver fat, and a 49% less likelihood of having MASLD, comparing the highest to the lowest quartile of the MIND score. Using a distance-based global test, we confirmed overall significant microbial mediation of the inverse diet-ectopic fat association. From HDMA, four bacterial genera were identified as mediating the protective association with visceral fat, with the largest mediation conferred by Lachnospiraceae UCG.001 (12.2%). Two genera (Lachnoclostridium, Weissella) were shown to mediate the MIND association with both liver fat and MASLD. In particular, Lachnoclostridium mediated 13.6% of the liver fat association and 10.8% of the MASLD association, and Lachnospiraceae UCG.001 additionally mediated 12.1% of the liver fat association. ConclusionsOur results support the hypothesis that the gut microbiota contributes to conveying the effect of diet quality on preferred body fat distribution, e.g., involving bacteria that are known to produce short-chain fatty acids (Lachnospiraceae) or secondary bile acids (Lachnoclostridium).

T helper 17 (Th17) cells are a critical T lymphocyte subset involved in mucosal immunity and host defense against enteric pathogens. Although ketogenic diets (KD) and the major ketone body {beta}-hydroxybutyrate (BHB) reshape gut microbiota and suppress Th17 responses under defined diet conditions, it remains unclear whether elevation of BHB alone, independent of dietary macronutrient composition and systematic metabolic shift, is sufficient to remodel Th17-inducing commensals and alter host susceptibility to enteric infection. Here, we used 1,3-butanediol (BD), a precursor metabolized to BHB independently of KD, to elevate systemic BHB levels in mice. BD treatment significantly reduced the frequency of ileal Th17 cells, as assessed by flow cytometry for Th17 markers IL-17A and ROR{gamma}t. 16S rRNA gene sequencing revealed that BD altered gut microbial community structure, as indicated by beta-diversity analysis based on Bray-Curtis dissimilarity, and reduced Shannon diversity and evenness. Linear discriminant analysis effect size identified segmented filamentous bacteria (SFB) as significantly decreased in the ileum following BD treatment, and SFB abundance positively correlated with Th17 markers. Microbiota transplantation demonstrated that BD-shaped microbiota was sufficient to suppress Th17 responses in recipient mice, accompanied by reduced SFB abundance. In a Citrobacter rodentium infection model, BD treatment was associated with increased pathogen burden, and fecal C. rodentium levels were negatively correlated with SFB abundance. Together, these results indicate that BD-induced elevation of BHB reshapes commensal microbiota, including decreasing SFB levels, resulting in dampened Th17 responses and increased susceptibility to enteric infection. IMPORTANCEDiet is a key determinant of gut microbial composition and mucosal immune function, yet the microbial mechanisms linking how diet-mediated changes to metabolism regulate immune responses remain incompletely understood. Th17 cells play central roles in both protective mucosal immunity and inflammatory pathology, making them a critical target of immunometabolic regulation. In this study, we show that {beta}-hydroxybutyrate (BHB), generated independently of diet, suppresses intestinal Th17 responses by reshaping the gut microbiota, reducing SFB levels, a potent Th17-inducing murine commensal. We further demonstrate that BHB-associated microbiota changes are linked to increased susceptibility to enteric infection. This work provides a mechanistic framework illustrating how metabolic state can influence host immunity through selective effects on commensal microbes. These findings inform future studies of microbiota-mediated immune regulation.

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.