Microbiome

Microbial eukaryotes are prevalent members of host-associated and free-living microbial communities, but are routinely excluded from studies of these communities. Existing methods for eukaryote detection from whole metagenome sequencing are limited by contamination of eukaryotic reference genomes and incomplete taxonomic coverage. Our previously published tool EukDetect addressed these challenges using a curated database of universal BUSCO marker genes, but lacked validated quantitative abundance metrics and was built from a limited number of genomes. Here we present EukDetect2, incorporating a database containing 6,948 microbial eukaryotic genomes representing 6,594 unique species, 2,339 of which are newly added since EukDetect version 1, alongside quantitative metrics for estimating absolute and relative abundance of microbial eukaryotes. Using simulated data, we demonstrate accurate abundance estimation, no false positives from bacterial or host-derived reads, and equivalent or greater sensitivity and specificity than alternative taxonomic profiling tools across a range of microbial abundances and community compositions. Applying EukDetect2 across globally distributed human gut microbiome cohorts, we find that Blastocystis spp. and Dientamoeba fragilis are the most prevalent gut eukaryotes across cohorts, while host-associated fungi are consistently less prevalent than commensal protists. Blastocystis abundance is positively associated with a gut microbial community enriched for fiber-fermenting microbes and depleted for pro-inflammatory and industrialization-associated taxa. EukDetect2 provides sensitive, accurate, and quantitative metrics for investigating microbial eukaryotes from metagenomic samples.

In vitro gut microbiome-culturing models are essential for studying host-microbe interactions, yet achieving high microbial recovery and long-term stability remains a long-standing challenge. Here we devise X-Gutor, an ecologically designed in vitro platform that recapitulates the monogastric digestive system with precise control of pH, redox potential, and a mucin-based biofilm matrix. Using an integrated assessment framework, microbial source tracking, a curated gut metabolism database (GutDB), and short-chain fatty acid profiling, we benchmarked gut community recovery. Building on this platform, HanGutor and PigGutor were developed to recover 96.27{+/-}4.92% and 90.48{+/-}5.95% of human and swine gut microbiota, respectively. Ecological fine-tuning revealed that redox potential drives the Prevotella-Bacteroides trade-off and that spatial biofilm structure suppresses cheater (Succinivibrio) growth. The X-Gutor provides a scalable, modular platform for gut microbiome engineering, dietary intervention, and microbiota-targeted therapy development.

The human gut microbiome harbours a diverse community of microeukaryotes, predominantly fungi, which may potentially play important roles in gut ecology and homeostasis. Despite their potential, the study of gut microeukaryotes has been hampered by the limited sensitivity of standard sequencing approaches, which struggle to capture DNA from low-abundance microorganisms against the overwhelming background of bacterial biomass. To address this, we developed a method to selectively enrich for microeukaryotic cells in human faecal samples by depleting bacterial cells prior to metagenomic sequencing. Through systematic comparison and optimisation at each processing step, we established a robust standard operating procedure (SOP) for microeukaryotic cell enrichment. By benchmarking this SOP across eight human faecal samples with three technical replicates each, we showed that it consistently increased microeukaryote representation in metagenomic libraries, greater microeukaryotic taxonomic diversity, and a reduced proportion of unclassified taxa. Together, these improvements enabled substantially deeper characterisation of the microeukaryotic fraction of the human gut microbiome.

Background: Early-onset colorectal cancer (EOCRC) is increasing globally, particularly among Hispanic/Latino (H/L) populations, yet the contribution of tumor-colonizing microbiota to age-associated colorectal cancer (CRC) biology remains poorly understood. Most microbiome studies have focused on fecal communities or non-Hispanic populations, leaving the intratumoral microbial landscape of H/L patients largely unexplored. Methods: We performed an exploratory characterization of tumor-colonizing microbiota using whole-exome sequencing (WES) data from four primary colorectal tumors obtained from H/L patients treated at City of Hope, including two EOCRC (<50 years) and two late-onset colorectal cancer (LOCRC; [&ge;]50 years) cases. Following removal of host-derived sequences, microbial taxonomic profiling was conducted at the family, genus, and species levels, and microbial metabolic pathways were inferred. Clinical and pathological data were integrated to evaluate age-associated differences in microbial composition and predicted function. Results: Family-, genus-, and species-level analyses consistently demonstrated greater microbial diversity in LOCRC than EOCRC. LOCRC contained more than twice the number of unique bacterial families, nearly three times as many unique genera, and more than twice as many unique bacterial species. A conserved core microbiota, including Fusobacteriaceae, Prevotellaceae, Fusobacterium, and Prevotella, was identified across both age groups, whereas LOCRC was enriched in CRC-associated taxa including Fusobacterium nucleatum, Bacteroides fragilis, Parvimonas micra, Porphyromonas asaccharolytica, and Dialister pneumosintes. Species-level analyses revealed only a single shared bacterial species between EOCRC and LOCRC, indicating progressive microbial divergence with increasing taxonomic resolution. In contrast, functional profiling identified 11 predicted microbial metabolic pathways, of which nine were shared between age groups, two were unique to EOCRC, and none were exclusive to LOCRC. Core metabolic pathways involved in energy metabolism, amino acid biosynthesis, phospholipid metabolism, and central carbon metabolism exhibited comparable abundance across both groups, demonstrating substantial functional conservation despite pronounced taxonomic differences. Conclusions: Tumor-colonizing microbiota differ markedly between EOCRC and LOCRC in H/L patients, with late-onset tumors exhibiting substantially greater microbial richness and taxonomic complexity. Despite these compositional differences, microbial metabolic functions remain largely conserved, supporting the concept of functional redundancy within the colorectal tumor microenvironment (TME). Although exploratory, this proof-of-concept study provides one of the first characterizations of intratumoral microbiota in H/L EOCRC and establishes a foundation for larger multi-omics investigations aimed at identifying microbiome-based biomarkers and therapeutic targets for precision oncology.

Industrialization has been identified as the single biggest factor driving global microbiome diversity. While many studies examining gut microbiomes attribute these shifts to dietary increases in fat and reductions in protein, oral microbiome responses to industrialization remains debated. The oral microbiome is more resilient due to long-standing coevolution with host tissues and biofilm stability. However, limited geographic and historical representation has constrained our understanding of how these transitions unfolded globally in the oral microbiomes. Here, we investigate oral microbiome variation in Batwa rainforest hunter-gatherers and neighboring Bakiga subsistence farmers from southwestern Uganda, comparing them with publicly available data from Tanzanian, Venezuelan, and industrialized populations from North America, Europe, and Australia. Using 16S rRNA gene sequencing, we characterized salivary microbiota and evaluated differences in local and global diversity, composition, and differential abundance. Ugandan populations contained significant compositional differences but similar levels of diversity, suggesting that shared environments and dietary overlap may shape microbial assemblages despite distinct cultural histories. Globally, strong continental and industrialization effects were observed in the oral microbiome, with all industrial populations clustering separately from people living in other locations. African populations also clustered separately from non-African groups. Oral microbiome diversity was highest in Ugandan individuals and lowest in industrialized populations, mirroring patterns previously observed in the gut microbiome. Together, these findings demonstrate that both geography and subsistence strategy structure global oral microbiome variation. They also clarify the position that oral microbial communities record biocultural transitions and highlight the need to better understand the industrial mechanisms that shape microbial diversity in the oral cavity.

Non-digestible oligosaccharides are widely used as prebiotics, yet structurally related glycans can elicit distinct gut microbiome responses. Here, we combined controlled ex vivo fermentation, deep DIA metaproteomics, and targeted metabolomics to determine how oligosaccharide structure and donor age shape microbiome function. Stool microbiomes from 18 healthy donors across three age groups were cultured with seven structurally related oligosaccharides from two glycan families, fructo-oligosaccharides (FOS) and galactosyl-sucrose derivatives (GSD). We found that oligosaccharide structure organized a functional response landscape rather than simply separating substrates into broad prebiotic classes. Structurally related glycans produced more similar response profiles overall, yet closely related FOS substrates remained functionally distinguishable, indicating that subtle structural differences were resolved by the microbiome as graded functional changes. These structure-responsive functions were further associated with producer-level reorganization relative to baseline, while targeted enzyme-level analyses indicated that substrate-specific CAZyme responses could also reflect altered functional investment within shared producer backgrounds. Despite these substrate-specific entry processes, network analysis revealed convergence onto shared downstream physiological states enriched for translation, amino-acid biosynthesis, secretion/export, and chemotaxis-related pathways. Across treatments, major short-chain fatty acids increased while mucin glycan degradation-associated markers decreased, suggesting coordinated shifts toward saccharolytic metabolism and reduced host-glycan foraging. Tryptophan-associated metabolism was also consistently linked to primary fructan processing, accompanied by higher extracellular tryptophan availability. Donor age modified selected microbial functional axes and enzyme-metabolite coupling relationships rather than the overall direction of core fermentation outputs. In particular, oligosaccharides attenuated an Methanobrevibacter smithii (M. smithii) and M00567 methanogenesis-related signature in microbiomes from older adults and altered age-dependent relationships between butyrate-pathway enzymes and extracellular butyrate levels. Together, these findings show that oligosaccharide structure determines how gut microbial communities organize carbohydrate processing and downstream functional states, while donor age reshapes the taxonomic and metabolic context of these responses. This work provides a mechanistic framework for structure-aware and age-aware precision prebiotic design.

The assembly and maturation of the infant gut microbiome is a critical developmental process. Yet the dynamics of the viral community, particularly in the context of stunting (chronic malnutrition) remain underexplored. Leveraging longitudinal fecal metagenomes from Zimbabwean infants with normal and stunted growth trajectories, we characterized the development of the gut bacterial and temperate phage communities from birth to 18 moths old. We found that infant gut temperate phages target hallmark early-life bacterial taxa, such as Bifidobacteriaceae, and exhibit an age-dependent maturation that parallels bacterial succession. Notably, both bacterial and temperate phage alpha diversity increased with age. This contrasts with previous studies focused on the extracellular viral fraction and highlights a strong coupling between prophage early-life dynamics and during bacterial gut colonization. Using abundance-based maturation models, we identified successional phases of colonization for both bacteria and their associated temperate viral clusters. Importantly, a viral microdiversity maturation model provided a stronger prediction of chronological age than viral abundance-based model, revealing within-phage genomic variation as a key signal of virome assembly, particularly around weaning. Contrary to findings in wasting (or severe acute malnutrition), stunted growth trajectories were not associated with a significant delay in either bacterial or temperate phage maturation. These results demonstrate that viral genomic variation is a new, informative dimension of early-life gut microbial assembly and that stunting may not impair infants gut maturation process. ImportanceThe early-life period represents a critical window for the establishment of the gut microbial communities, a process that is often affected by environmental factors such as diet. While severe acute malnutrition (SAM) is known to delay bacterial maturation, the impact of chronic, moderate undernutrition, such as stunting is poorly understood. Stunting is a highly prevalent global health condition with irreversible consequences on long-term host health, yet its implications on gut microbiome assembly remain unclear. Our study provides novel insights into the maturation of temperate phages, which to prime the infant gut by colonizing alongside their bacterial hosts and acting as drivers of bacterial evolution via lysogeny. By demonstrating that viral strain-level (genomic) variation captures a stronger age-related signal than viral abundance, we identified an underexplored dimension of microbial assembly. The finding that stunting, in contrast to SAM, does not impact microbial maturation provides essential context for public health interventions and future studies addressing this condition.

BackgroundLeafhoppers are among the most important insect vectors of plant pathogens worldwide and depend on microbial symbionts to exploit nutrient-poor phloem diets. However, most studies of leafhopper-associated microbiota have focused on a limited number of taxa or marker-gene surveys, leaving the genomic diversity, ecological organization, and functional potential of these microbial communities poorly understood. Here, we generated the Global Leafhopper Microbiome Catalog by integrating genome-resolved metagenomics from 171 leafhopper species across 11 subfamilies and 13 countries, including the first microbiomes characterized from Arctic leafhoppers. ResultsDe novo assembly and genome reconstruction generated 337 high-quality non-redundant microbial genomes and 18.6 million non-redundant genes, substantially expanding the known microbial diversity associated with Cicadellidae, including several previously undescribed bacterial lineages. Comparative analyses revealed a recurrent modular microbiome architecture composed of: (i) a conserved core of obligate nutritional symbionts, dominated by Candidatus Karelsulcia and Candidatus Nasuia; (ii) a heterogeneous layer of secondary symbionts, including Wolbachia, Arsenophonus, Rickettsia, and Diplorickettsia; and (iii) a dynamic pool of environmentally acquired bacteria. While obligate symbionts remained highly conserved across divergent hosts, secondary and environmental taxa varied substantially among species and regions, suggesting repeated acquisition shaped by ecological filtering rather than host phylogeny alone. Comparative analyses between the specialist corn leafhopper Dalbulus maidis and the more polyphagous aster leafhopper Macrosteles quadrilineatus further showed that closely related vectors can maintain conserved ancestral symbionts while harboring markedly distinct accessory microbiomes. Arctic populations contained unique microbial assemblages enriched in functions associated with cold tolerance, oxidative stress, and reproductive manipulation. In addition, we identified numerous plant-associated bacteria, including phytoplasmas, spiroplasmas, Pantoea, and Erwinia, alongside taxa with predicted nutritional and plant growth-promoting functions. ConclusionsOur findings reveal that leafhopper microbiomes are structured through the interaction of ancient obligate symbioses and flexible environmentally responsive microbial layers. This work establishes a genome-resolved framework for understanding microbiome evolution in insect vectors and highlights the potential role of microbial community structure in host adaptation, pathogen ecology, and sustainable pest management.

The gut microbiota plays a fundamental role in human health, nutrition, immune development, and disease, driving widespread adoption of 16S rRNA gene sequencing for microbial community characterization. Short-read V3-V4 sequencing remains the dominant approach for large-scale microbiome studies; however, interrogation of only a small fraction of the 16S gene limits phylogenetic resolution and frequently restricts biological interpretation at the species level. Although full-length (V1-V9) 16S sequencing has emerged as a promising alternative, comprehensive evaluation of highly multiplexed full-length workflows in complex human gut microbiomes remains limited. Here, we establish and evaluate a full-length 16S framework for species-resolved human gut microbiome profiling. The workflow was assessed using defined microbial communities, technical replicates, and healthy human fecal microbiomes. Full-length sequencing generated highly concordant taxonomic profiles across independent technical workflows and enabled reproducible recovery of complex microbial communities at both genus and species levels. Application to human fecal microbiomes revealed substantial inter-individual heterogeneity together with extensive ASV-level microdiversity, highlighting the ability of full-length sequencing to resolve fine-scale phylogenetic variation within dominant gut-associated taxa. To quantify the analytical gain afforded by full-length sequencing, V3-V4 datasets were computationally reconstructed directly from identical full-length reads, eliminating methodological and biological confounders. While alpha diversity metrics and overall community structure remained highly concordant between approaches, full-length sequencing markedly improved taxonomic resolution, increasing species-level assignment from approximately 20% to 98% and resolving substantial intra-genus diversity within clinically and ecologically relevant genera including Bifidobacterium, Prevotella, Blautia, Enterococcus, and Klebsiella. Collectively, these findings position full-length 16S sequencing as an enabling technology for the next generation of microbiome studies, where species-level resolution can be integrated with large-scale cohort, longitudinal, and population-health investigations.

The human gut microbiome exhibits reproducible configurations, yet the ecological forces connecting them to health remain unclear. Here, using enterosignature-based stratification of 5,170 individuals from the Le French Gut cohort, we identified hydrogen disposal as a key determinant of population-scale microbiome configurations, independently replicated in a meta-cohort (n = 5,107). Microbial configurations followed a continuum of hydrogen recycling capacity and redox-associated functions, aligned with dietary patterns and health indicators. Methanogenesis-dominant partitions were associated with more favorable health profiles, whereas acetogenesis-enriched partitions exhibited features of low-grade inflammation, and increased digestive symptoms, perceived stress and antidepressant use. Experimental characterization of mucin profiles highlighted differences across partitions and alterations in Bacteroides-enriched configurations. Together, our findings support an ecological host-microbiome framework linking hydrogen metabolism, redox ecology, and host health, offering microbiome-informed targets for precision intervention. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/722951v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@e7b8d1org.highwire.dtl.DTLVardef@116ab09org.highwire.dtl.DTLVardef@136f1f8org.highwire.dtl.DTLVardef@480097_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fungi are key members of microbial communities, yet microbiome surveys often lack trait-based information required for ecologically meaningful interpretation of mycobiome data. To demonstrate the value of fungal trait-based phenotyping in microbiome research, we re-analyzed N=3,221 samples across four case studies spanning human, agricultural, and environmental systems. In human cancer and vineyard datasets, trait-based analysis detected fungi producing macroscopic fruiting bodies, likely introduced via airborne spore dispersal, indicating widespread contributions of transient or contaminant fungi that can confound interpretation of sequencing data from tumor biopsies and grape berries. In sourdough fermentations, filamentous fungi were highly abundant alongside traditionally-recognized yeast and occupied distinct ecological niches. In forest soils, increasing habitat disturbance was associated with increased prevalence and abundance of plant pathogens, and a marked decline in ectomycorrhizal and lichenized fungi. These changes were accompanied by a shift toward large-spored taxa in urban soils, consistent with enhanced stress tolerance. To facilitate broader adoption of fungal phenotyping in microbiome studies, we introduce q2-fungal-traits, a QIIME2 plugin for automated integration of fungal taxonomy derived from marker-gene or shotgun metagenome sequencing surveys with ecological and functional trait data. The plugin assigns lifestyle-related traits and spore size estimates through hierarchical taxonomic matching and integrates directly into standard microbiome workflows. Our case studies demonstrate that integrating trait-based ecology with mycobiota datasets can generate novel findings and testable hypotheses, enabling inference of the functional (ir)relevance of community constituents. Our work contributes to bridging the gap between descriptive community profiling and functional ecology in microbiome research.

Microbiome research has expanded globally, yet the Middle East and North Africa (MENA) region remains severely under-represented in international sequencing repositories. Here we present the MENA Microbiome Database, the first systematically harmonized catalog of publicly available metagenomic sequencing data from 24 MENA countries, consolidating 60,126 runs across 51,365 biological samples and 2,373 BioProjects deposited between 2008 and 2026. Records were retrieved from ENA, NCBI SRA, and PubMed, enriched with BioSample and study-level metadata, and classified into microbiome subtypes using a 73-rule keyword-based harmonization framework. Amplicon sequencing accounted for 80.6% of runs, with Illumina platforms dominating at 92.7%. Geographic coverage is highly skewed: Saudi Arabia and Turkey together contribute over half of all records, while five countries (Libya, Syria, Palestine, Yemen, and South Sudan) remain critically under-sampled. Metadata completeness averaged 73.97% under a MIxS-MIMS proxy framework, with geographic coordinates available for fewer than 15% of runs. Ecological analyses revealed that country-level factors significantly structure environmental, animal-associated, and plant-associated microbiomes, but not human-associated microbiomes. Spatial autocorrelation confirmed non-random clustering of sampling effort around Red Sea coastal and eastern Mediterranean hotspots. This open, reproducible resource, comprising harmonized data files, analysis code, and an interactive browsing platform, establishes a foundational infrastructure for regional microbiome science and equitable global comparative studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/722303v1_ufig1.gif" ALT="Figure 1000"> View larger version (69K): org.highwire.dtl.DTLVardef@16ebcd3org.highwire.dtl.DTLVardef@12ed2d1org.highwire.dtl.DTLVardef@112b5b1org.highwire.dtl.DTLVardef@156b8a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Viruses of the phylum Nucleocytoviricota are paradigm-shifting entities due to their exceptionally large genomes and complex gene repertoires, which blur the lines between viral and cellular life. Previous research has leveraged computational approaches to map their extensive diversity, while experimental work has started to elucidate the intricate networks they form with hosts, bacterial and other symbionts, co-infecting virophages and other mobile genetic elements. Here, we analyzed deeply sequenced metagenomes sampled from wastewater treatment plants in Denmark, an environment with rapid abiotic changes and known to be a hotbed of dense microbial communities. We discovered 61 novel nucleocytoviruses, 15 virophages and 14 polinton-like viruses. By integrating them with microbial contigs into a multilayered interaction network, we explore the role of these entities on a mesocosm scale. We demonstrate the centrality of nucleocytoviruses, positioning them as important players shaping microbial community structure and evolution in wastewater treatment plants.

Shotgun metagenomic sequencing enables high-resolution profiling of host-associated microbial communities. However, contaminant DNA can substantially distort biological interpretations, especially in low-biomass samples. Here, we introduce Metacontam, a control-free method for species-level decontamination of shotgun metagenomic data. Metacontam integrates blacklist-guided community detection within a species correlation network with average nucleotide identity (ANI) to identify contaminants arising from shared sources. Across diverse low-biomass and mixed-biomass datasets, Metacontam outperformed existing approaches, improving the detection of low-abundance and low-prevalence contaminants while retaining biologically plausible taxa. It also reduces kit-specific biases in skin metagenomes and improves downstream analyses of tissue microbiome data. Together, these results demonstrate that Metacontam enables accurate identification of contaminant taxa across diverse metagenomic datasets, even in the absence of negative controls.

BackgroundRestrictions on the types of food available on land have repeatedly triggered the convergent evolution of herbivory in terrestrial animals. This pattern also holds true in many terrestrially adapted crabs, which have independently colonized land more than 17 times since the Cretaceous, and many clades are now almost completely herbivorous, standing in contrast to the ancestral pattern of detrivory. While many bacteria possess efficient pathways for degrading lignin and cellulose, the role of gut microbiomes in facilitating these dietary shifts in terrestrial crabs remains poorly understood. To explore the relationship between microbial community structure and the ability of land crabs to digest lignocellulose, we conducted read-based and assembly-based metagenomic analyses on feces collected from the guts of 14 crab species across six genera, representing a gradient of terrestriality from the lower intertidal zone to forested habitats. ResultsWe generated 129 metagenome-assembled genomes (MAGs) that represent key members of these gut microbial communities, establishing a foundational resource for future studies on crab-microbiome interactions. We found that host genus explained most of the variation in bacterial community composition, while degree of terrestrial adaptation (i.e. terrestrial grade) explained a smaller proportion. We also identified multiple bacterial genera that strikingly differed in relative abundance across terrestrial grades, crab genera, and diet type. Broad-scale functional analyses of general carbon metabolism across crabs revealed an absence of complete pathways in crabs from lower terrestrial grades, suggesting a functional divergence in gut communities linked to habitat transition. Fine-scale functional analyses of carbohydrate-active enzyme (CAZyme) domains allowed us to connect specific MAGs to lignocellulose degradation pathway genes, demonstrating that different crab genera harbor distinct microbial taxa that have similar CAZyme profiles in their guts. ConclusionsThis work provides a foundational metagenomic resource for genomic exploration of microbial communities in terrestrial crab guts. These results suggest that the gut microbiomes of terrestrially adapted crabs are structured primarily by host identity and have convergently acquired microbes with similar functions to help perform lignocellulose degradation. Overall, different degrees of adaptation to terrestrial environments, including resulting dietary shifts, may be responsible for functional divergence in crab gut community assembly.

Wastewater surveillance has emerged as a critical tool for global epidemiology, yet the functional diversity of wastewater microbiomes remains poorly characterized at the protein level. Here, we present WasteFams, the first comprehensive database dedicated to the systematic exploration of protein families in wastewater metagenomic and metatranscriptomic studies worldwide. Integrating data from 580 metagenomes, 132 metatranscriptomes, and 1,709 reference genomes, WasteFams catalogs 3,887 non-redundant protein families (containing {succeq}100 members) derived from over 105 million predicted proteins. Each protein family is enriched with multi-layered annotations, including AlphaFold3 structural predictions, taxonomic classifications, and biome-specific metadata. To further expand their functional annotation, we integrated deep genomic context analysis to link protein families to Mobile Genetic Elements (MGEs), Biosynthetic Gene Clusters (BGCs), Antibiotic Resistance Genes (ARGs), and CRISPR elements. Accessible through the EnvoFams portal, WasteFams provides a user-friendly interface featuring advanced search capabilities, sequence and structural similarity tools, and interactive visualization modules. As global initiatives increasingly leverage wastewater for public health and environmental insights, WasteFams can serve as a critical resource for discovering novel microbial functions, monitoring resistance mechanisms, and exploring the biotechnological potential of secondary metabolites within wastewater-engineered ecosystems.

Metagenomic studies of arthropod-associated microbiomes have generated vast amounts of sequence data, yet the functional and structural organization of these proteins remains largely unexplored. Here, we present ArthroVerse, the first comprehensive database of protein families derived from arthropod-associated metagenomes. Non-redundant protein families were generated after rigorous filtering, deduplication, and clustering. The protein families were further annotated with microbial taxonomy, host associations, protein structural information, and Carbohydrate-active enzymes (CAZyme) predictions. The resulting dataset integrates both metagenomic and reference genome-derived proteins, enabling systematic exploration of functional diversity, evolutionary relationships, and host-microbe interactions in insect microbiomes. ArthroVerse provides a valuable resource for the study of microbial ecology and arthropod physiology, offering unprecedented insight into the protein landscape of insect-associated microbial communities.

Background Colonisation resistance provided by the gut microbiota is a critical barrier to pathogen invasion, yet its study in vivo is constrained by the complexity and cost of vertebrate models. Here, we developed a humanised Galleria mellonella infection model by inoculating wax moth larvae with complex human faecal microbiota. 16S rRNA gene sequencing confirmed stable, reproducible establishment of a diverse human associated community across larvae over four days. Results Humanised larvae exhibited colonisation resistance against Salmonella enterica serovar Typhimurium, with mortality reduced to 20% compared to 90% in non colonised controls. To test whether prophages could overcome this barrier, we infected larvae with isogenic S. Tm strains differing in the presence of prophage P22. Infection with the P22 carrying strain resulted in a threefold higher larval mortality (60% vs. 20%), increased pathogen load, and a significant reduction in the abundance of resident E. coli. Free P22 virions were detected early after infection, indicating extensive prophage activity. Notably, P22 can neither adsorb nor lyse resident E. coli, indicating that prophage mediated invasion success did not rely on direct lysis. Instead, using high throughput metabolic profiling paired with whole genome sequencing of three replicate lineages, we found that phage activation intensified resource partitioning, accelerating functional metabolic adaptations in E. coli that significantly reduced the niche overlap between the invading pathogen and the commensal E. coli. Conclusion Our findings establish the first humanised G. mellonella model supporting complex human microbiota and provide a novel non lytic mechanism by which prophages influence species interactions. This scalable, low cost model offers a new platform to dissect pathogen phage microbiota interactions relevant to human gut ecology.

The human duodenum harbours a complex, dynamic microbial community that is challenging to study due to inaccessibility, particularly postprandially when nutrient-rich chyme and fluctuating metabolites create unique microbial niches. We used naso-duodenal intubation to longitudinally sample duodenal luminal contents following pea-based meals of differing food structure, alongside parallel blood collection. Shotgun metagenomic sequencing, comprehensive metabolomic profiling and gut hormone measurements were combined to explore microbe-metabolite-hormone interactions. Food structure significantly affected postprandial bacterial composition, with saccharolytic oral taxa increasing after meals with intact structure. Alpha diversity was influenced by structure type (P = 0.025), with whole pea seeds promoting greater diversity than pea flour. Network analysis revealed complex interactions between the duodenal microbiome, luminal metabolites and gut hormones, with most microbial associations linked to glucose-dependent insulinotropic polypeptide (GIP) rather than glucagon-like peptide-1 (GLP-1). Metabolic profiling showed meal-dependent changes in amino acid metabolism, including shifts in D/L amino acid ratios over time consistent with microbial metabolism. The duodenal microbiome showed close phylogenetic relationships with the oral microbiome, with composition influenced by food structuring and swallowing. These findings reveal dynamic microbe-metabolite interplay in the human duodenum during digestion and its relationship to gut hormone responses.

Bifidobacteria dominate the gut microbiota of breast-fed infants, and are strongly associated with human health, including immune regulation, colonization resistance, and protection against inflammation. However, bifidobacteria persist at high abundance after weaning in only a subset of individuals, and the factors that regulate intestinal persistence of bifidobacteria are poorly understood. Using gnotobiotic mouse models, we identified a common dietary fiber, raffinose, as a critical determinant of bifidobacterial persistence during microbial transitions associated with weaning. Bifidobacterial persistence depends on an intact raffinose utilization operon and is associated with disease resistance and restrained inflammation in adult mice. Specific dietary fiber recommendations commencing at weaning are a potential strategy to maintain bifidobacteria persistence beyond infancy, with potential long-term benefits for host resilience and reduced risk of inflammatory disease.