Microbiome

Microbiome has co-evolved with insects over millions of years, establishing symbiotic relationships that influence their fitness and shape evolutionary trajectories. These long-term associations are shaped by the hosts ecology, diet, and physiology which determines microbial diversity. In turn, external stresses such as pathogenic infections can adversely affect the host microbiome, further compounding the effects of pathogenic infections on the host. Using a computational pipeline and publicly available transcriptomics datasets, we analyzed the effect of viral infections on transcript derived microbiome diversity and composition in various insects including Drosophila melanogaster, Acyrthosiphon pisum, Antheraea pernyi, and Culex pipiens. The meta-transcriptomic reanalysis provides an indirect but comprehensive comparison of infection-associated transcriptional microbiome changes across hosts with distinct life histories. Our study reveals that viral infections influence the host-associated microbial transcripts, leading to shifts in transcript-derived microbial profiles and offering insights into host-microbe dynamics. In Cx. pipiens, microbial transcript profiles remained relatively stable following RVFV exposure and were dominated by the bacterial endosymbiont Wolbachia. Our study highlights viral infections as key drivers of infection-associated shifts in microbial profiles and suggests a potential association between Wolbachia dominance and transcriptional microbiome stability in insects, with age as a contributing factor in shaping microbial transcript profiles in Culex. This cross-species framework uncovers conserved and host-specific patterns of virus-microbiome interactions, expanding our understanding of microbial resilience mechanisms in insects.

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.

Functional profiling of anaerobic digestion microbiomes is routinely performed against KEGG or MetaCyc, whose paid commercial licences block fee-for-service analysis for biogas operators, the setting where the results have the most immediate operational value. We present digestome, a curated marker-gene panel for anaerobic digestion built exclusively on sources that are free for commercial use (NCBIfam, public domain; Pfam, CC0; Rhea, CC BY), together with a scorer that reports pathway completeness, branch capability and, for hydrolysis, whether the enzyme is built for export. Benchmarked against 1,401 metagenome-assembled genomes from 134 anaerobic digesters, the panel assigned no methanogenesis route to any of 1,361 non-methanogens, and every acetoclastic call fell within Methanosarcina or Methanothrix. The result held for 3,043 species representatives from the Genome Taxonomy Database, spanning 203 phyla, which are built differently from binned metagenomes: the only background genomes given a route were two archaea carrying mcrA, and none of the 90 anaerobic methane and alkane oxidisers was given one. Specificity against non-methanogens does not show that a methanogen gets the right route: with a family-level check, 59 of 156 acetoclastic calls in that sample fell on methylotrophic genera that do not use acetate, and a lineage policy removed them, with 14 more left unconfirmed in unnamed genera. Testing whether a catalytic domain shares a polypeptide with an export module reduced the genomes credited with cellulolytic capacity from 597 to 50. A causal graph of the measurement shows that a profile reports the community together with three measurement choices (sampling, DNA extraction and sequencing depth, and reference genomes), which fixes what one profile can attribute and what a prospective validation would have to record.

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.

Many termites digest lignocellulosic wood using diverse gut microbiota, yet functions are rarely linked to specific symbionts because complete genomes are scarce. We used PacBio HiFi long reads to reconstruct circular single-contig bacterial metagenome-assembled genomes (MAGs) from the gut microbiota of the wood-feeding termite Nasutitermes takasagoensis. From 13.9 Gb of HiFi data, we obtained highly contiguous assemblies, including 11 Breznakiellaceae (Spirochaetota), two Candidatus Fibrimonas (Fibrobacterota), and one Chitinispirillaceae genome, three dominant lineages in this gut ecosystem. The reconstructed genomes encoded extensive carbohydrate-active enzyme repertoires and anaerobic fermentation pathways consistent with lignocellulose degradation. They also carried genes for essential amino acid biosynthesis, while nitrogen-fixation capacity was present in representatives of all three lineages but varied among closely related strains. One Breznakiellaceae genome possessed a complete Wood-Ljungdahl pathway potentially supporting reductive acetogenesis in the presence of external formate, whereas related strains carried incomplete variants likely involved in anabolic C1 metabolism. Differences in CAZymes, C1 metabolism, and nitrogen fixation among closely related strains reveal functional diversification that fragmented MAGs would not resolve. The mapping of metatranscriptomic reads from three biological replicates of the fiber-associated community to the reconstructed genomes provided independent evidence that key predicted metabolic functions are transcriptionally active. Our results demonstrate the power of HiFi metagenomics to recover highly contiguous genomes from complex insect microbiomes and uncover lineage-specific metabolic roles of dominant termite gut symbionts.

Schistosoma mansoni infection is associated with alterations of the host gut microbial community structure, yet its impact on microbial genome dynamics and horizontal gene transfer remains poorly understood. In this study, we investigated the activity of Mobile Genetic Elements (MGEs), and of Insertion Sequences (IS) in particular, in the gut microbiome of wild-type (WT) and human-microbiota-associated (HMA) mice. Taxonomic profiling revealed significant differences in bacterial diversity between WT and HMA mice, with reduced richness and alpha diversity observed in HMA gut microbiomes regardless of the infection status. Principal Coordinates Analysis demonstrated clear separation between microbial communities, reflecting differences in host background and bacterial composition. In contrast, differences were observed between plasmid and viral community profiles and between these and bacterial diversity, suggesting that different MGEs may follow distinct ecological dynamics within the gut microbiome. IS elements within bacterial genomes were widespread across all microbiome communities analysed in this study, with higher insertion counts observed not only in HMA compared to WT mice, but also in infected compared to uninfected mice. IS elements were predominantly associated with dominant gut bacterial taxa, particularly members of Bacteroidia and Clostridia. At the genomic level, insertion events occurred more frequently in intergenic regions than in coding sequences, suggesting a preferential selective pressure towards alteration of gene expression rather than gene disruption. Furthermore, IS activity was dominated by a subset of IS families, with consistent patterns observed across experimental groups. Notably, infection was associated with increased insertion frequencies across multiple taxa and IS families, suggesting enhanced mobilisation under conditions of host-associated stress (the physiological and molecular pressure experienced by microorganisms, pathogens, or the host itself during their interactions within a symbiotic or infected environment) and microbiome perturbation. Together, these findings demonstrate that S. mansoni infection may promote IS mobilisation in the mouse gut microbiome, potentially contributing to microbial genome plasticity and facilitating horizontal gene transfer within host-associated microbial communities.

The genomic and epigenomic complexity of microbiomes with low species richness remains poorly characterized. Here, we integrated 111 non-redundant isolate genomes with deep long-read metagenomics to investigate an undefined mesophilic dairy starter. Closely related isolates harboured distinct combinations of features associated with fermentation traits and phage defence, including surface polysaccharide genotypes, plasmids, prophages, defence systems, and methylation profiles. Metagenomics revealed additional elements and inferred host associations. However, the limited overlap between approaches across core-genome lineages and accessory repertoires underscored complementary but incomplete recovery, to the extent that the dominant metagenomic cell wall polysaccharide genotype (29.6% abundance) was absent from isolates. Metagenomic methylation signatures supported 36 species-level plasmid-host assignments, corroborated by isolate genomes in nine of ten testable cases. Our findings revealed extensive combinatorial diversity hidden from either approach alone, providing a plausible molecular basis for phage resilience, while likely representing only a lower bound on population complexity.

As circular agricultural inputs are increasingly sought, feather hydrolysate (FH), derived from keratin-rich poultry waste, has emerged as a promising fertilizer and biostimulant. However, how FH shapes soil microbiome succession over time, and what functional potential this restructuring carries, remains poorly understood. Here, we used multi-marker high-throughput sequencing of 16S, 18S, and ITS rDNA to track soil microbial communities over 30 days following FH amendment, with sampling at days 0, 1, 4, 14, and 30. FH induced a rapid and reproducible microbial succession characterized by an early decline in alpha diversity and a pronounced day-1 bloom of copiotrophic taxa, as inferred from predicted rrn operon copy-number profiles. This initial response was accompanied by enrichment of bacterial genera previously associated with plant growth-promoting traits, including pathogen suppression, nutrient solubilization, siderophore production, phytohormone synthesis, and ACC deaminase activity. Although these groups gradually declined after the initial peak, several remained at significantly elevated abundances throughout the 30-day experimental period. In parallel, FH-treated soils showed a sustained reduction in the cumulative relative abundance of genera annotated as putative phytopathogens. By integrating temporal community profiling, rrn-based life-strategy inference, and literature-derived functional annotation, we show that FH acts as a strong ecological filter, shifting the soil microbiome toward copiotrophic and putatively plant-beneficial taxa. These findings provide a temporal framework for understanding FH-mediated microbiome restructuring and highlight the need for future studies linking inferred microbial functional potential to measured disease incidence, nutrient fluxes, and plant performance across diverse soil contexts.

The advent of sequencing-based omics has transformed our understanding of the gut microbiome, enabling microbial communities to be characterised at unprecedented scale and resolution. However, these approaches provide limited information about cellular traits such as size and morphology. Giant bacteria remain largely overlooked in gut microbiome research, despite their extraordinary cell size, extreme polyploidy and unusual reproductive strategies. Epulopiscium, one of the largest known heterotrophic bacteria, has been studied primarily as an intestinal symbiont of surgeonfish, leaving its broader host range unresolved. Here we show, by a large-scale analysis of publicly available 16S ribosomal RNA amplicon samples, alongside locally collected human samples, that Epulopiscium spp. occur across diverse hosts, including humans and a broad diversity of animals. Full-length 16S rRNA gene phylogenies resolved multiple Epulopiscium-related lineages with distinct host compositions. Among human samples, Epulopiscium sequences were most abundant during the first year of life. In adults, abundance varied across disease states, increasing in inflammatory bowel disease and colorectal cancer while decreasing in neurological conditions compared with a healthy adult baseline. Similar patterns were also observed in mouse models. Our study broadens the known host range of Epulopiscium and establishes giant bacteria as an overlooked component of gut microbiome diversity. These findings highlight the need to combine sequence-based studies with classical microbiological techniques to identify organisms with unique traits, otherwise hidden by taxonomy or phylogeny.

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.

Hospital sink drains act as persistent reservoirs for nosocomial pathogens, yet the structure and long-term stability of their associated microbial communities remain poorly known. Here, we present an extensive characterization integrating multi-kingdom meta-taxonomic profiling (16S rRNA, ITS, and 18S rRNA, n=93 samples) with culturomics (n>4000 isolates) and phenotypic resistance testing across 14 sinks from a former intensive care unit (ICU). Over a 17-month study period, we tracked community assembly and stability through the renovation and transition from an unpopulated baseline to active clinical use, encompassing both structural remodeling and functional conversion. We showed that former-ICU drains harbor a highly conserved multi-kingdom core that remains resilient despite significant structural changes and periods of inactivity. While a persistent core baseline exists, community composition changes along a continuous temporal gradient driven by structural and anthropogenic shifts. Furthermore, individual sinks assemble as unique microecosystems shaped by local environmental factors. Topological network modeling shows these communities are not random assemblages but integrated ecological units, with clinically relevant, multidrug-resistant WHO-priority pathogens deeply embedded in the network alongside often-overlooked fungi and protozoa. Together, these findings identify hospital sink drains as persistent and structured multi-kingdom microbial reservoirs that harbor clinically relevant multidrug-resistant organisms (MDROs). Their temporal stability and strong sink-specific signatures highlight the need to consider the broader microbial community when monitoring hospital environmental reservoirs and developing infection-control strategies.

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.

Current environmental biosurveillance infrastructure excels at tracking known biological threats but is limited in its ability to proactively identify potential pre-emergent pathogens (PEPs). To address this gap, we developed a microfluidics-based Capture-Enrichment pipeline that isolates individual environmental cells via microdroplet encapsulation, preserving community biodiversity while enabling iterative selection against human sera. When applied to urban wastewater, this approach successfully recovered pathogens, commensals, and PEPs that fell below the detection limits of conventional amplicon sequencing. Genomic analysis revealed that wastewater-derived ESKAPE pathogens cluster closely with clinical isolates, showing that the pipeline captures clinically relevant threats. Furthermore, isolated PEPs exhibited known and emerging phenotypes associated with early-stage pathogenesis and antimicrobial resistance. These findings demonstrate that targeting PEPs can fundamentally expand the scope and quality of biosurveillance by providing a scalable strategy to identify, characterize, and close important gaps in our understanding of emerging microbial pathogens.

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.

Abstract: Background Despite the ecological importance of viral anti-defense systems (ADS), yet how viral ADS repertoires vary across microbial ecosystems remains poorly understood. Here, we integrated global virus datasets with metagenomes from heavy metal-contaminated soils and chromium-stress microcosms to investigate ecological determinants of viral ADS. Results We found that ADS repertoires were constrained by viral genome capacity, with most ADS-carrying viruses encoded a single system, whereas multi-ADS occurred mainly in larger genomes. Viral lifestyle partitioned ADS, with temperate viruses enriched in ADS targeting early-stage defenses and virulent viruses preferentially encoding ADS against mid- and late-stage defense. ADS composition also varied by host lineage and across habitats, linking viral ADS repertoires to host immune landscapes and ecosystem-specific viral strategies. Moreover, along the heavy metal contamination gradient, changes in the prevalence of ADS-carrying viruses occurred alongside shifts in prokaryotic defense profiles. In addition, expanded ADS repertoires may further increase dissemination potential of MGE-borne adaptive genes, highlighting viral ADS as a potential contributor to the adaptive evolution of microbial community. Conclusions These findings reveal that viral ADS are shaped by genomic traits, host immunity and environmental stress, with implications for virus-host interaction and microbial adaptive evolution. More broadly, this study extends anti-defense research beyond individual virus-host systems, offering an integrative, community-level perspective to guide future studies testing whether and how virus-prokaryote immune interactions contribute to microbial community dynamics and evolution in changing environments.

Pulmonary tuberculosis (TB) is associated with systemic alterations extending beyond the lungs, yet the longitudinal impact of anti-tuberculosis therapy (ATT) on gut ecosystem recovery remains poorly understood. Here, we performed a longitudinal multi-omics analysis of gut microbial and metabolic dynamics in pulmonary TB patients (n=8) followed at baseline, 1, 6, and 8 months of ATT, compared with healthy household contacts (HHCs; n=8). Shotgun metagenomics, functional profiling, untargeted fecal metabolomics, resistome, plasmidome, virome, and microbiome-metabolome integration analyses were performed to characterize disease-associated alterations and treatment-associated recovery. Active, treatment-naive TB was characterized by reduced microbial diversity, altered community structure, depletion of beneficial commensals, and disruption of microbial functional pathways and metabolic profiles. During ATT, microbial diversity and functional/metabolic features progressively recovered; however, post-treatment profiles remained distinct from HHCs, indicating incomplete ecological restoration after microbiological cure. Microbiome-metabolome analysis revealed associations between altered bacterial taxa and metabolites involved in amino acid metabolism, bile acid transformation, oxidative stress, and host-microbial metabolic interactions. In contrast, resistome and mobile genetic element profiles demonstrated greater stability throughout treatment. HHCs exposed to index TB cases maintained relative gut ecosystem stability over time. Bacterial-AMR correlation analysis identified taxon-specific resistance associations among gut bacteria. Together, these findings provide a longitudinal systems-level view of gut ecosystem remodeling during TB and highlight that restoration of microbial homeostasis extends beyond pathogen clearance.

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.

Gut microbes metabolize the ellagitannin castalagin to ellagic acid and urolithins, a process associated with health effects for the host. Despite the use of the castalagin-rich camu camu extracts in clinical trials, the genes and taxa responsible for castalagin hydrolysis remain elusive. We used multi-omics to discover a castalagin associated hydrolase (cah) operon sparsely distributed in Lachnospiraceae. Analysis of fecal samples from a camu camu supplementation clinical trial demonstrates functional redundancy with prevalence of cah and other ellagic acid metabolism genes in different taxa explaining the prevalence of metabolites. Employing genome mining strategies, we discovered and validated ellagitannin metabolism genes in uncharacterized and uncultured species. We further identified flavogallonic acid as a prevalent metabolite of castalagin in human fecal samples. Collectively, this work provides a genetic basis for castalagin metabolism to urolithins and flavogallonic acid end-metabolites and sets the stage to study the role of metabolism in clinical trials.

Water kefir is a traditional fermented beverage and a non-dairy alternative to milk kefir. Water kefir grains are cauliflower-like aggregates, ranging in size from 1-20 mm. They are composed of an exopolysaccharide matrix derived from the fermentation substrate sucrose. The microbial community on and within the grains is a complex, highly stable multicellular aggregate typically consisting of 10-15 core species that primarily belong to three major groups: lactic acid bacteria, acetic acid bacteria, and yeasts. Remarkably, the structural organisation, stability, and growth of kefir grains cannot be readily explained by their constituent species alone, and attempts to reconstitute them from the isolated species have so far been unsuccessful. Here, we combined a comprehensive structural investigation of the macro- and micromorphology of water kefir grains with culture-dependent and -independent analyses of its microbial biodiversity. The dynamics and interactions within this complex microbial community were studied by combining flow cytometry, fluorescence-activated cell sorting, and amplicon sequencing of selected subpopulations. Furthermore, the growth of water kefir grains was spatiotemporally resolved and quantitatively characterised, thereby unravelling an emergent multicellular growth function, documented for the first time. Our combined evidence provides an unprecedented understanding of a highly structured and metabolically synergistic multicellular microbial aggregate and lets us propose water kefir as a model system for the aggregative development of microbial multicellularity from multiple species. It should lay the foundation for future mechanistic studies aimed at understanding how multispecies microbial communities self-organise into stable, spatially structured multicellular aggregates.

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.