Microbiome research has focused on bacteria for long, largely overlooking other microbial members, incl. archaea. Methanogenic archaea are stable members of the human microbiome. By consuming hydrogen, they support fermentation, stabilize networks, and are responsible for methane production. Recent studies link methanogens to colorectal cancer and intestinal methanogen overgrowth, highlighting their relevance in disease and their potential as targets for novel therapies.

Ammonia-oxidizing archaea (AOA) have repeatedly been detected with molecular methods on human skin, yet their persistence, physiological traits, and adaptations remain poorly understood. This is mostly owed to a lack of cultured representatives of AOA taxa from healthy human skin. Using a customized enrichment scheme, we cultivated two autotrophic strains, Candidatus Nitrosocosmicus epidermidis and Ca. Nitrosocosmicus unguis, from human skin samples. Genomic analyses revealed specific adaptations for skin colonization, including genomic islands, and expanded gene families linked to interactions with host proteins, and signaling pathways, distinguishing these AOA from their soil-dwelling relatives. Profiling of >700 samples from 8 body sites in cross-sectional and longitudinal cohorts consistently validated the detection of Ca. Nitrosocosmicus species with up to 100% prevalence in a longitudinal cohort, particularly in sebaceous areas. Co-occurrence patterns with specific bacterial taxa reinforce their role as stable components of the skin microbiome. Our results establish Ca. Nitrosocosmicus species as common skin commensals that are evolutionarily capable of transitioning from soil to human skin. They likely play a critical role in the skin ecosystem by recovering nitrogen from the sebum through utilization of urea and ammonia. This sheds new light on the role of archaeal species in maintaining the nitrogen balance in the human skin microbiome, which might be of importance in maintaining healthy skin.

This study performs patient-to-mouse fecal microbiota transplantation (FMT) as an experimental platform to investigate gut-brain axis alterations with potential relevance to psychiatric disorders, integrating metabolic modeling with measured metabolites and multi-layer molecular profiling. Microbial communities of a stool sample associated with bipolar disorder (BD) displayed a reduced ecological diversity and diminished metabolic potential, particularly within glutamate, aspartate, and GABA biosynthetic pathways. Upon transplantation with BD patient microbiota, recipient mice displayed a markedly altered microbiome characterized by loss of Akkermansia and expansion of Alloprevotella, alongside disruptions in amino acid and carbohydrate metabolism not evident in mice colonized by a healthy donor microbiota. Metabolic microbiome alterations in BD-recipient mice were also correlated to reduced glutathione levels in gut tissue, likely indicating increased oxidative stress, and decreased mRNA expression of key enteroendocrine hormones, including peptide YY and glucagon. Brain metabolomic profiling of BD-recipient mice revealed significant depletion of glycine, choline, and methionine levels connected to anxiety-like phenotypes in elevated plus-maze and light-dark box behavioral tests. Akkermansia abundance positively correlated with physical activity and exploratory behavior, highlighting an important role of this taxon in gut-brain signaling. Collectively, these findings identify distinct microbial, metabolic, and neurobehavioral signatures transmittable from humans to mice via FMT and demonstrate that differences in donor microbiome diversity and metabolic capacity shape engraftment dynamics in recipient mice, which contribute to differences in gut-brain signaling.

Current planetary protection approaches rely heavily on spore-based tests developed for Mars missions and may not adequately assess contamination risks for icy ocean worlds such as Europa. We developed a genome-based framework combining deep shotgun metagenomics and supervised machine learning to predict survival-relevant microbial traits in ESA JUICE launch-site cleanrooms. From 183 genome bins, 25 representative genomes were analyzed for traits including cryotolerance, desiccation tolerance, salt resilience, anaerobic metabolism, autotrophy, and sporulation. Several skin-associated microbes carried multiple relevant traits, and some appeared actively replicating. A broader meta-analysis of 1,868 genomes showed that trait profiles vary strongly within taxa, demonstrating that taxonomy alone is insufficient for risk assessment. This framework complements current planetary protection assays, helps to predict how microbes would survive in a new biotope, and supports functional, risk-informed contamination monitoring for future space missions.

The human gut is colonized by trillions of microbes that influence the health of their human host. Whereas many bacterial species have now been linked to a variety of different diseases, the involvement of Archaea, an evolutionarily distinct group of microbes, in human disease remains elusive. By analyzing 19 independent clinical studies, we demonstrate that associations between Archaea and human diseases are widespread yet highly heterogeneous, with a pronounced and consistent enrichment of Methanobrevibacter smithii in colorectal cancer (CRC) patients. Metabolic modelling and in vitro co-culture identified distinct mutualistic interactions of M. smithii with CRC-causing bacteria such as Fusobacterium nucleatum, including metabolic enhancement. Metabolomics further reveal archaeal-derived compounds with tumor-modulating properties. Together, our results provide mechanistic insights into how the human gut archaeome may participate in CRC-associated microbial networks through metabolic cooperation with bacteria.

Two mesophilic, hydrogenotrophic methanogens, WWM1085 and M. smithii GRAZ-2, were isolated from human faecal samples. WWM1085 was isolated from an individual in the United States and represents a novel species within the genus Methanobrevibacter. M. smithii GRAZ-2 (=DSM 116045) was retrieved from a faecal sample of a European, healthy woman and represents a novel strain within this species. Both Methanobrevibacter representatives form non-flagellated, short rods with variable morphologies and the capacity to form filaments. Both isolates showed the typical fluorescence of F 420 and methane production. Compared to M. smithii GRAZ-2, WWM1085 did not accumulate formate when grown with H 2 and CO 2 . The optimal growth conditions were at 35–39 °C and pH 6.5–7.5. Full genome sequencing revealed a genomic difference of WWM1085 to the type strain of M. smithii DSM 861 (=PS T ), with 93.55% average nucleotide identity (ANI) and major differences in the sequence of its mcrA gene (3.3% difference in nucleotide sequence). Differences in the 16S rRNA gene sequence were very minor, and thus distinction based on this gene marker might not be possible. M. smithii GRAZ-2 was identified as a novel strain within the species Methanobrevibacter smithii (ANI 99.04% to M. smithii DSM 861 [=PS T ]). Due to the major differences between WWM1085 and M. smithii type strain M. smithii DSM 861 (=PS T ) in phenotypic, genomic and metabolic features, we propose Methanobrevibacter intestini sp. nov. as a novel species with WWM1085 as the type strain (DSM 116060 T = CECT 30992 T ).

When profiling the human gut microbiome, technical biases introduced by analytical approaches impede translational research, reducing data reliability and study comparability. Here, through a global study involving 23 labs, we analyzed a wide range of sequencing and bioinformatic approaches for the taxonomic profiling of two well-defined DNA reference reagents (RRs) comprised of 20 common gut bacteria. Through both shotgun and 16S rRNA gene amplicon sequencing, we aimed to isolate sources of bias and understand their impact on microbiome profiling accuracy. Importantly, minimum quality criteria (MQC) were established and are used to evaluate profiling performance. We found that the variability of shotgun sequencing data sets was greater than that of 16S rRNA gene amplicon sequencing and isolated sources of bias in wet and dry lab steps, such as sequencing depth, primer and database choices, rarefaction, and 16S copy number adjustment. This study presents well-defined RRs and MQC to combat technical bias, paving the way for reliable and comparable microbiome research.IMPORTANCEThis benchmark paper highlights the true level of variability in microbiome data across the world and across sectors, underscoring the critical need for the use of WHO International DNA Gut Reference Reagents (RRs) to elevate the quality of data in microbiome research. This global study is the first of its kind, revealing the reality of the bias in the field, comprehensively testing methodologies used by leading laboratories across the world, but also providing avenues for workflow optimization, to accelerate innovation and translational research and move the field forward.

Emerging evidence highlights the role of gut microbiome in mental health disorders, including depression, raising the question whether the action of antidepressants could be mediated, at least in part, via the microbiome-gut-brain axis. To explore this, we subjected a St. John's wort extract (STW 3-VI), clinically proven to be effective in mild to moderate depression, to a model of the upper and lower intestinal tract, including static in vitro predigestion followed by ex vivo incubation with human microbiota samples. To cover the interindividual diversity of gut microbiome composition, fecal samples from ten healthy volunteers were used. Although unchanged levels of most annotated compounds were observed during simulated upper intestinal tract digestion, incubation with fecal microbiota led to a significant change of the chemical profile of the extract. While hyperforins remained stable, flavonoids and hypericins were rapidly biotransformed, suggesting that they may act as prodrugs. Several metabolites were formed, many of which are known to be involved in gut-brain communication. Differential abundance analysis revealed significant changes in microbiome composition, particularly for taxa known to be potentially associated with depression. Among others, the Firmicutes/Bacteroidetes ratio, known to be lowered in depressive patients, was increased. Functional profiling revealed modulation of pathways involved in gut-brain communication, such as tyrosine and tryptophan metabolism. These bidirectional interactions suggest for the first time the gut microbiome as a potential mediator of the pharmacological effects of St. John's wort extracts via the microbiome-gut-brain axis.

The reciprocal relationship between aging and alterations in the gut microbiota is a subject of ongoing research. While the role of bacteria in the gut microbiome is well-documented, specific changes in the composition of methanogens during extreme aging and the impact of high methane production in general on health remain unclear. This study was designed to explore the association of predominant methanogenic archaea within the human gut and aging. Shotgun metagenomic data from the stool samples of young adults (n = 127, Age: 19–59 y), older adults (n = 86, Age: 60–99 y), and centenarians (n = 34, age: 100–109 years) were analyzed. Our findings reveal a compelling link between age and the prevalence of high methanogen phenotype, while overall archaeal diversity diminishes. Surprisingly, the archaeal composition of methanogens in the microbiome of centenarians appears more akin to that of younger adults, showing an increase in Methanobrevibacter smithii, rather than Candidatus Methanobrevibacter intestini. Remarkably, Ca. M. intestini emerged as a central player in the stability of the archaea-bacteria network in adults, paving the way for M. smithii in older adults and centenarians. Notably, centenarians exhibit a highly complex and stable network of these two methanogens with other bacteria. The mutual exclusion between Lachnospiraceae and these methanogens throughout all age groups suggests that these archaeal communities may compensate for the age-related drop in Lachnospiraceae by co-occurring with Oscillospiraceae. This study underscores the dynamics of archaeal microbiome in human physiology and aging. It highlights age-related shifts in methanogen composition, emphasizing the significance of both M. smithii and Ca. M. intestini and their partnership with butyrate-producing bacteria for potential enhanced health.

Gastrointestinal bacteria interact with the host and each other through various mechanisms, including the production of extracellular vesicles (EVs). However, the composition and potential roles of EVs released by gut archaea are poorly understood. Here, we study EVs produced by four strains of human gut-derived methanogenic archaea: Methanobrevibacter smithii ALI, M. smithii GRAZ-2, M. intestini, and Methanosphaera stadtmanae. The size (~130 nm) and morphology of these EVs are comparable to those of bacterial EVs. Proteomic and metabolomic analyses reveal that the archaeal EVs are enriched in putative adhesins or adhesin-like proteins, free glutamic and aspartic acid, and choline glycerophosphate. The archaeal EVs are taken up by macrophages in vitro and elicit species-specific responses in immune and epithelial cell lines, including production of chemokines such as CXCL9, CXCL11, and CX3CL1. The EVs produced by M. intestini strongly induce pro-inflammatory cytokine IL-8 in epithelial cells. Future work should examine whether archaeal EVs play roles in the interactions of archaea with other gut microbes and with the host.

ABSTRACT The role of the gut archaeal microbiome (archaeome) in health and disease remains poorly understood. Methanogenic archaea have been linked to multiple sclerosis (MS), but prior studies were limited by small cohorts and inconsistent methodologies. To address this, we re-evaluated the association between methanogenic archaea and MS using metagenomic data from the International Multiple Sclerosis Microbiome Study. We analyzed gut microbiome profiles from 115 MS patients and 115 healthy household controls across Buenos Aires (27.8%), Edinburgh (33.9%), New York (10.4%), and San Francisco (27.8%). Metagenomic sequences were taxonomically classified using kraken2/bracken and a curated profiling database to detect archaea, specifically Methanobrevibacter species. Most MS patients were female (80/115), aged 25–72 years (median: 44.5), and 70% were undergoing treatment, including dimethyl fumarate (n = 21), fingolimod (n = 20), glatiramer acetate (n = 14), interferon (n = 18), natalizumab (n = 6), or ocrelizumab/rituximab (n = 1). We found no significant differences in overall archaeome profiles between MS patients and controls. However, treated MS patients exhibited higher abundances of Methanobrevibacter smithii and M. sp900766745 compared to untreated patients. Notably, M. sp900766745 abundance correlated with lower disease severity scores in treated patients. Our results suggest that gut methanogens are not directly associated with MS onset or progression but may reflect microbiome health during treatment. These findings highlight potential roles for M. smithii and M. sp900766745 in modulating treatment outcomes, warranting further investigation into their relevance to gut microbiome function and MS management.IMPORTANCEMultiple sclerosis (MS) is a chronic neuroinflammatory disease affecting the central nervous system, with approximately 2.8 million people diagnosed worldwide, mainly young adults aged 20–30 years. While recent studies have focused on bacterial changes in the MS microbiome, the role of gut archaea has been less explored. Previous research suggested a potential link between methanogenic archaea and MS disease status, but these findings remained inconclusive. Our study addresses this gap by investigating the gut archaeal composition in MS patients and examining how it changes in response to treatment. By focusing on methanogens, we aim to uncover novel insights into their role in MS, potentially revealing new biomarkers or therapeutic targets. This research is crucial for enhancing our understanding of the gut microbiome’s impact on MS and improving patient management.

The human gut is colonized by trillions of microbes that influence the health of their human host. Whereas many bacterial species have now been linked to a variety of different diseases, the involvement of Archaea in human disease remains elusive. Here we searched for gut archaeal signatures of disease by screening 19 cross-sectional clinical studies comprising more than 1,800 individuals. We found that associations between Archaea and medical disorders are common but highly variable and are dominated by a significant increase of Methanobrevibacter smithii in colorectal cancer (CRC) patients. Metabolic modelling and in vitro co-culture identified distinct mutualistic interactions of M. smithii with CRC-causing bacteria such as Fusobacterium nucleatum , including metabolic enhancement. Metabolomics further revealed archaeal-derived compounds with tumor-modulating properties. This provides the first mechanistic link between human gut archaeome and CRC and highlights its role in modulating health in humans through trophic control of the resident bacteriome. ### Competing Interest Statement The authors have declared no competing interest. Austrian Science Fund (FWF) [10.55776/P32697 (given to CME), 10.55776/CoE7 (CME and CD), 10.55776/F8300 (CME)]. C.T. reports a research grant by Bruker Switzerland AG.

The development of hospital-associated microbial communities over time remains poorly characterized, particularly in terms of how microbial populations dynamically respond to changes in building function, the integration of molecular and cultivation-based data, and the early identification of intervention points for flexible, adaptive microbial control strategies. In this longitudinal study, we investigated microbiome dynamics across five newly built departments at the University Hospital of Graz, Austria, over one year. Surface samples were collected at seven time points: before and after hospital operation started. Alpha and beta diversity analyses revealed a distinct two-phase microbial transition, marked by an initial disruption followed by a gradual homogenization of microbial communities. The strongest driver of community change was the arrival of patients, which led to a significant shift in both diversity and taxonomic composition. While early time points were dominated by environmental taxa such as Acinetobacter and Pseudomonas, human-associated genera like Staphylococcus and Corynebacterium became more prevalent over time, particularly on frequently touched surfaces. Department-specific and surface-specific microbial signatures were observed, with outpatient and transplant departments showing more variability than surgical and intensive care units (ICUs). Propidium monoazide treatment indicated that Pseudomonas and Acinetobacter may persist as viable community members, whereas Staphylococcus and Corynebacterium likely reflect frequent human deposition. Cultivation data supported these findings, showing episodic contamination primarily linked to human contact. Phenotypic predictions revealed a decline in aerobic, Gram-negative, and potentially pathogenic bacteria over time, although these trends were less pronounced in the ICU. Together, our findings reveal a longitudinal homogenization of hospital microbiomes driven by human activity and highlight key taxa and surfaces that warrant targeted monitoring to improve hygiene protocols and infection control strategies.IMPORTANCEThis study provides crucial insights into how hospital environments transform microbially after new departments open, a process poorly understood until now. We reveal a two-phase microbial shift, starting with environmental bacteria like Acinetobacter and Pseudomonas before the hospital opens, then rapidly transitioning to human-associated microbes such as Staphylococcus and Corynebacterium once patients and staff arrive. Our findings highlight that human activity is the strongest driver of these changes, especially on frequently touched surfaces. This work is vital for developing targeted and adaptive hygiene concepts, improving infection control, and ultimately making hospital environments safer for patients and staff by focusing on specific surfaces and microbial groups that warrant continuous monitoring.

ABSTRACTThe partial or complete loss of the sense of smell, which affects about 20% of the population, impairs the quality of life in many ways. Dysosmia and anosmia are mainly caused by aging, trauma, infections, or even neurodegenerative disease. Recently, the olfactory area—a site containing the olfactory receptor cells responsible for odor perception—was shown to harbor a complex microbiome that reflects the state of olfactory function. This initially observed correlation between microbiome composition and olfactory performance needed to be confirmed using a larger study cohort and additional analyses. A total of 120 participants (middle-aged, no neurodegenerative disease) were enrolled in the study to further analyze the microbial role in human olfactory function. Olfactory performance was assessed using the Sniffin’ Stick battery, and participants were grouped accordingly (normosmia: n = 93, dysosmia: n = 27). The olfactory microbiome was analyzed by 16S rRNA gene amplicon sequencing and supplemented by metatranscriptomics in a subset (Nose 2.0). Propidium monoazide (PMA) treatment was performed to distinguish between intact and non-intact microbiome components. The gastrointestinal microbiome of these participants was also characterized by amplicon sequencing and metabolomics and then correlated with food intake. Our results confirm that normosmics and dysosmics indeed possess a distinguishable olfactory microbiome. Alpha diversity (i.e., richness) was significantly increased in dysosmics, reflected by an increase in the number of specific taxa (e.g., Rickettsia, Spiroplasma, and Brachybacterium). Lower olfactory performance was associated with microbial signatures from the oral cavity and periodontitis (Fusobacterium, Porphyromonas, and Selenomonas). However, PMA treatment revealed a higher accumulation of dead microbial material in dysosmic subjects. The gastrointestinal microbiome partially overlapped with the nasal microbiome but did not show substantial variation with respect to olfactory performance, although the diet of dysosmic individuals was shifted toward a higher meat intake. Dysosmia is associated with a higher burden of dead microbial material in the olfactory area, indicating an impaired clearance mechanism. As the microbial community of dysosmics (hyposmics and anosmics) appears to be influenced by the oral microbiome, further studies should investigate the microbial oral-nasal interplay in individuals with partial or complete olfactory loss.IMPORTANCEThe loss of the sense of smell is an incisive event that is becoming increasingly common in today’s world due to infections such as COVID-19. Although this loss usually recovers a few weeks after infection, in some cases, it becomes permanent—why is yet to be answered. Since this condition often represents a psychological burden in the long term, there is a need for therapeutic approaches. However, treatment options are limited or even not existing. Understanding the role of the microbiome in the impairment of olfaction may enable the prediction of olfactory disorders and/or could serve as a possible target for therapeutic interventions.

The reciprocal relationship between aging and alterations in the gut microbiota is a subject of ongoing research. While the role of bacteria in the gut microbiome is well-documented, specific changes in the composition of methanogens during extreme aging and the impact of high methane production in general on health remain unclear. To address these questions, we analyzed metagenomic data from the stool samples of young adults (n=127, Age: 19-59 y), older adults (n=86), and centenarians (n=34, age: 100-109 years).Our findings reveal a compelling link between age and the prevalence of high methanogen phenotype, while overall archaeal diversity diminishes. Surprisingly, the archaeal composition of methanogens in the microbiome of centenarians appears more akin to that of younger adults, showing an increase in Methanobrevibacter smithii , rather than Ca. M. intestini. Remarkably, Ca. M. intestini emerged as a central player in the network stability of adults, paving the way for M. smithii in older adults and centenarians. Notably, centenarians exhibit a highly complex and stable network of these two methanogens with other bacteria. Furthermore, the mutual exclusion between Lachnospiraceae and these methanogens throughout all age groups suggests that these archaeal communities may compensate for the age-related drop in Lachnospiraceae by co-occurring with butyrate-producing Oscillospiraceae.This study underscores the crucial role of the archaeal microbiome in human physiology and aging. It highlights age-related shifts in methanogen composition, emphasizing the significance of Ca. M. intestini and the partnership between methanogens and specific butyrate-producing bacteria for enhanced health and potential longevity.### Competing Interest StatementThe authors have declared no competing interest.

Recent research provides new insights into the early establishment of the infant gut microbiome, emphasizing the influence of breastfeeding on the development of gastrointestinal microbiomes. In our study, we longitudinally examined the taxonomic and functional dynamics of the oral and gastrointestinal tract (GIT) microbiomes of healthy infants (n = 30) in their first year, focusing on the often-over-looked aspects, the development of archaeal and anaerobic microbiomes. Breastfed (BF) infants exhibit a more defined transitional phase in their oral microbiome compared to non-breastfed (NBF) infants, marked by a decrease in Streptococcus and the emergence of anaerobic genera such as Granulicatella. This phase, characterized by increased alpha-diversity and significant changes in beta-diversity, occurs earlier in NBF infants (months 1-3) than in BF infants (months 4-6), suggesting that breastfeeding supports later, more defined microbiome maturation. We demonstrated the presence of archaea in the infant oral cavity and GIT microbiome from early infancy, with Methanobrevibacter being the predominant genus. Still, transient patterns show that no stable archaeome is formed. The GIT microbiome exhibited gradual development, with BF infants showing increased diversity and complexity between the third and eighth months, marked by anaerobic microbial networks. NBF infants showed complex microbial co-occurrence patterns from the start. These strong differences between BF and NBF infants' GIT microbiomes are less pronounced on functional levels than on taxonomic levels. Overall, the infant microbiome differentiates and stabilizes over the first year, with breastfeeding playing a crucial role in shaping anaerobic microbial networks and overall microbiome maturation. IMPORTANCE:The first year of life is a crucial period for establishing a healthy human microbiome. Our study analyses the role of archaea and obligate anaerobes in the development of the human oral and gut microbiome, with a specific focus on the impact of breastfeeding in this process. Our findings demonstrated that the oral and gut microbiomes of breastfed infants undergo distinct phases of increased dynamics within the first year of life. In contrast, the microbiomes of non-breastfed infants are more mature from the first month, leading to a steadier development without distinct transitional phases in the first year. Additionally, we found that archaeal signatures are present in infants under 1 year of age, but they do not form a stable archaeome. In contrast to this, we could track specific bacterial strains transitioning from oral to gut or persisting in the gut over time.

This Review aims to coalesce existing knowledge on the human archaeome, a less‐studied yet critical non‐bacterial component of the human microbiome, with a focus on its interaction with the immune system. Despite a largely bacteria‐centric focus in microbiome research, archaea present unique challenges and opportunities for understanding human health. We examine the archaeal distribution across different human body sites, such as the lower gastrointestinal tract (LGT), upper aerodigestive tract (UAT), urogenital tract (UGT), and skin. Variability in archaeal composition exists between sites; methanogens dominate the LGT, while Nitrososphaeria are prevalent on the skin and UAT. Archaea have yet to be classified as pathogens but show associations with conditions such as refractory sinusitis and vaginosis. In the LGT, methanogenic archaea play critical metabolic roles by converting bacterial end‐products into methane, correlating with various health conditions, including obesity and certain cancers. Finally, this work looks at the complex interactions between archaea and the human immune system at the molecular level. Recent research has illuminated the roles of specific archaeal molecules, such as RNA and glycerolipids, in stimulating immune responses via innate immune receptors like Toll‐like receptor 8 (TLR8) and ‘C‐type lectin domain family 4 member E’ (CLEC4E; also known as MINCLE). Additionally, metabolic by‐products of archaea, specifically methane, have demonstrated immunomodulatory effects through anti‐inflammatory and anti‐oxidative pathways. Despite these advancements, the mechanistic underpinnings of how archaea influence immune activity remain a fertile area for further investigation.

Cancer cachexia is a multifactorial syndrome characterized by involuntary weight loss, muscle wasting, systemic inflammation, and metabolic alterations, affecting up to 87% of pancreatic and gastric cancer patients. Unlike simple starvation, cachexia is driven by metabolic disruption involving both host physiology and the gut microbiome. While microbiome changes in cachexia have been documented, a coherent understanding of how these changes translate into functional metabolic shifts remains elusive. In this study, we combined in vivo fecal and plasma metabolomic analyses with a novel computational microbiome simulation pipeline to identify cachexia-associated microbial metabolites. Using the murine MCA207 tumor line and its cachectic derivative CHX207, we differentiated microbiome changes driven by cachexia from those induced by tumor growth. Our computational tool, McMurGut, a murine-tailored extension of MICOM, enabled simulation of microbial metabolic interactions specific to the mouse microbiome, covering 91% of identified genera. We identified significant abundance changes in 35 microbial genera and corresponding shifts in metabolite production, including reductions in short-chain fatty acids (SCFAs) like acetate and butyrate, alongside increased production of galactose, formate, and propionate. Notably, decreases in SCFA production, particularly by genera such as Faecalibaculum and Dubosiella , correlated with exacerbated cachectic symptoms. Additionally, the elevated production of formate and galactose, primarily by Bacteroides and Lactobacillus , suggested altered fermentation pathways in cachexia, potentially linked to increased mucus degradation. Validation of our computational predictions via NMR metabolomics highlighted key congruencies between predicted and experimentally observed metabolites, supporting the role of microbiome-driven metabolic shifts in cachexia pathology. These findings provide crucial insights into the microbiome’s involvement in cachexia and suggest future avenues for therapeutic interventions aimed at modulating microbial taxa and their metabolic outputs to improve patient outcomes. ### Competing Interest Statement The authors have declared no competing interest.

Microorganisms thriving in the human gastrointestinal microbiome have developed numerous mechanisms for microbiome-host and microbe-microbe interaction, including the formation of extracellular vesicles (EVs). However, the interactive mechanisms of members of the human archaeome remained largely unexplored. Here, we report the capability of two Methanobrevibacter strains M. smithii, Cand. M. intestini and Methanosphaera stadtmanae, to produce EVs. While the size (~ 130 nm) and morphology of these archaeal EVs were comparable to bacterial EVs, proteomic and metabolomic analyses revealed unique traits. The proteins found in both the EVs of M. smithii and Cand. M. intestini (n = 229) predominantly included adhesin and adhesin-like proteins, involved in archaea-surface and archaea-bacteria interaction. Additionally, the archaeal EVs contained glutamic acid and choline glycerophosphate, which could be involved in gut-brain signalling. Vesicles from all studied species were efficiently taken up by human monocytes and induced IL-8 production in epithelial HT-29 cells. The identification of archaeal EVs and their components provides novel insights into the complex interactions between the archaeal and bacterial microbiome and the host, potentially offering new paths to modulate vesicle-mediated impacts on host health.

The gut microbiota has been implicated as a driver of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Recently we described, mucosal biofilms, signifying alterations in microbiota composition and bile acid (BA) metabolism in IBS and ulcerative colitis (UC). Luminal oxygen concentration is a key factor in the gastrointestinal (GI) ecosystem and might be increased in IBS and UC. Here we analyzed the role of archaea as a marker for hypoxia in mucosal biofilms and GI homeostasis. The effects of archaea on microbiome composition and metabolites were analyzed via amplicon sequencing and untargeted metabolomics in 154 stool samples of IBS-, UC-patients and controls. Mucosal biofilms were collected in a subset of patients and examined for their bacterial, fungal and archaeal composition. Absence of archaea, specifically Methanobrevibacter, correlated with disrupted GI homeostasis including decreased microbial diversity, overgrowth of facultative anaerobes and conjugated secondary BA. IBS-D/-M was associated with absence of archaea. Presence of Methanobrevibacter correlated with Oscillospiraceae and epithelial short chain fatty acid metabolism and decreased levels of Ruminococcus gnavus. Absence of fecal Methanobrevibacter may indicate a less hypoxic GI environment, reduced fatty acid oxidation, overgrowth of facultative anaerobes and disrupted BA deconjugation. Archaea and Ruminococcus gnavus could distinguish distinct subtypes of mucosal biofilms. Further research on the connection between archaea, mucosal biofilms and small intestinal bacterial overgrowth should be performed.