Position statement of the Microbiota International Clinical Society

PREAMBLE The intestinal microbiota is an extraordinarily complex ecosystem composed of trillions of microorganisms, including bacteria, viruses, fungi, and protozoa, which interact with each other and with the human host. This microbial community plays a crucial role in regulating human health, influencing vital processes such as digestion, immune modulation, inflammation control, and protection against pathogens (1). Scientific and clinical interest in In review the microbiota has grown exponentially in recent years, with implications ranging from metabolic and immune health to mental well-being (2). Microbiota analysis has emerged as a promising diagnostic and therapeutic resource for various clinical conditions. However, the lack of standardized regulations and limited concrete evidence of its clinical utility present significant challenges. Interest in the human microbiome has transformed biomedical research and clinical practice (3). The microbiome is now recognized as a key player in multiple biological processes, associated with conditions such as obesity, inflammatory bowel diseases (IBD), cardiovascular diseases, and neurological disorders (4), thereby offering new opportunities for the development of novel therapeutic strategies as well as for complementing existing ones. Objectives of the Position Statement This document aims to provide a comprehensive, state-of-the-art overview of the analytical, interpretative, and clinical requirements for microbiota testing, while also outlining current challenges, future opportunities, and operational guidelines based on the most recent scientific evidence. The Microbiota International Clinical Society (MICS) aims to promote the development and a conscious, rational, appropriate, and standardized use of microbiota testing that can offer real benefits to clinical practice. THE ROLE OF THE MICROBIOTA IN HEALTH AND DISEASE The relationship between gut microbes and the human body is a mutualistic symbiosis, wherein the host provides a nutrient-rich environment that supports the microbiota, which in turn contributes to host well-being through the production of metabolites and other health-promoting substances, while simultaneously fostering the development of a functional immune system. A growing body of scientific evidence highlights the fundamental role of the gut microbiota in maintaining host homeostasis (5) through the following functions: METABOLIC FUNCTIONS: The microbiota plays a crucial role in digestion, nutrient absorption, and the synthesis of essential vitamins, such as vitamin K and the B-vitamin complex (including biotin, cobalamin, folate, niacin, pantothenic acid, pyridoxine, riboflavin, and thiamine (6)). It also contributes to the production of a broad range of metabolites, primarily derived from the bacterial fermentation of indigestible carbohydrates such as fibers and resistant starch. Among the most relevant metabolites are short-chain fatty acids (SCFAs), including butyrate, propionate, and acetate, which exhibit key bioactive properties. These include the regulation of transepithelial fluid transport, enhancement of oxidative status, and reduction of mucosal inflammation. SCFAs also strengthen the intestinal barrier by increasing mucus production and enhancing the integrity of tight junctions, thereby impeding, for example, the progression of colorectal In review cancer [7]. Butyrate, in particular, acts on the GPR109a receptor to stimulate the production of the cytoprotective cytokine IL-18 and promotes the differentiation of regulatory T cells (Treg) through the induction of IL-10, while inhibiting the formation of pro-inflammatory Th17 cells (7). They stimulate the release of anorexigenic peptides such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) from L-endocrine cells, contributing to satiety (8),(9). STRUCTURAL FUNCTIONS: Certain microbial species play a fundamental role in inducing the expression of proteins essential for tight junction function. They stimulate mucin production, a key component of the intestinal mucus barrier, and promote molecular signaling that supports epithelial cell survival, enhancing the organism's ability to respond to inflammatory or infectious challenges (10). PROTECTIVE FUNCTIONS: In a balanced intestinal ecosystem, specific microbial species can outcompete and suppress potentially pathogenic bacteria by producing antimicrobial compounds, altering intestinal pH, depriving pathogens of nutrients, and preserving the integrity of the mucosal barrier—an essential line of defense. Moreover, the microbiota modulates both innate and adaptive immune responses by influencing the production of inflammatory cytokines (11). NEUROLOGICAL FUNCTIONS: The microbiota plays a key role in the gut-brain axis, a bidirectional communication system linking the gastrointestinal tract and the central nervous system (CNS). SCFAs stimulate enterochromaffin cells to produce and release serotonin, a neurotransmitter involved in mood regulation, memory, and learning (12). EUBIOSIS AND DYSBIOSIS The human body hosts a complex ecosystem of microorganisms—primarily bacteria, but also fungi, viruses, and others. The gut microbiota (GM) plays a critical role in health and disease, as its activity influences digestive, metabolic, and immune functions. Once underestimated, the essential role of GM in physiological processes is now widely recognized (14). The human body consists of approximately 37.2 trillion cells, while the GM includes about 39 trillion microbial cells, highlighting a near 1:1 ratio. This balance emphasizes the symbiotic relationship between human and microbial cells. Found on our skin, in the mouth, respiratory tract, and especially the gut, the microbiota is a fundamental and indispensable ecosystem for human health and survival (1),(15). To describe the complexity of the symbiotic coexistence between a host organism, human, animal, or plant, and its associated biological entities that do not share the same DNA, the term "holobiont" is used. This concept extends to an integrated system that fulfills the biological needs defined by human DNA and the teleonomy of its ecosystem, including the GM. This paradigm shift also underscores the role of the mind in mediating between human needs and the microorganisms we host (1). Maintaining well-being inevitably requires the promotion of the teleonomy of specific bacterial taxa through dietary choices that favor their proliferation over microbes that may induce metabolic disturbances or dysbiosis (16). A eubiotic GM composition includes a balanced variety of bacterial, fungal, and viral taxa, which is crucial for health. Eubiosis refers to a harmonious and functional microbiota that supports the organism's well-being. In contrast, dysbiosis is not merely an imbalance, but rather a microbial configuration that is misaligned with the host's physiological needs, often influenced by genetic, dietary, and environmental factors. This condition may lead to reduced microbial diversity and promote the proliferation of pathogenic microorganisms, contributing to chronic inflammation. In health, host–microbe symbiosis underpins digestive, metabolic and immune homeostasis, whereas dysbiosis - a departure from host-aligned community structure—has been associated with diverse pathologies spanning metabolic, inflammatory, oncologic and neuro-behavioural domains (14, 17, 18, 19). Four mechanistic axes recur across these associations: barrier dysfunction, including tight-junction impairment and mucus thinning; immune signalling driven by microbe-and diet-derived ligands that sustain low-grade inflammation (7, 20); metabolic mediation via short-chain fatty acids and bile-acid derivatives that influence epithelial energetics and entero-endocrine output (6, 7); and neuroactive routes, such as tryptophan/indole metabolism along the gut–brain axis (12, 13, 19). Because these links are largely correlative, we stress that cross-sectional taxonomic snapshots cannot establish causality; longitudinal multi-omics studies are essential to connect microbial function with clinical trajectories. Another key aspect of dysbiosis is its impact on intestinal permeability. Alterations in the microbiota can compromise the integrity of the mucosal barrier, increasing intestinal permeability and promoting systemic inflammatory responses. Moreover, increased permeability may trigger neurogenic inflammation, crossing the blood-brain barrier and contributing to neuroinflammatory processes associated with conditions such as depression and anxiety (19). The gut microbiota and the intestinal mucosa, therefore, engage in a complex network of interactions that influence health under both physiological and pathological conditions. In a state of eubiosis, the mucus layer, rich in mucins, acts as a physical barrier against pathogenic colonization. Commensal bacteria also produce or stimulate the production of antimicrobial peptides and contribute to immune education (20). In summary, the role and impact of the GM on human life are fundamental and indispensable. Understanding and maintaining a balanced microbiota through informed dietary and lifestyle choices can significantly support overall health and well-being (18),(19). Key Factors Influencing Microbiota Composition • Age: The composition of the microbiota evolves with age. In newborns, it is less diverse and gradually develops through exposure to solid foods and environmental factors. The of solid foods the community from a an by and and often with whereas production, and mucosal promotes the of health, while to a diverse but less microbiota, often in and the of solid foods and increased dietary the gut microbial community a significant from a to In review in and a and microbiota In the microbiota a may microbial diversity and overall health • of microbiota the immune system development and a via are to a limited microbiota, by and which may influence immune and health also plays a critical role in microbiota and bioactive such as and that promote the of bacteria and to favor the proliferation of less intestinal bacteria • modulates microbial composition through and and diversity of key with increased diversity in whereas with and in in gastrointestinal mucosal and contribute to community • Certain may influence the immune ability to interact with microorganisms, thereby the composition of the microbiota • A immune system supports symbiosis between the host and the immune dysfunction, such as in diseases, can balance • or The of diseases, especially chronic or inflammatory conditions such as inflammatory bowel (IBD), or cardiovascular diseases, can significantly the microbiota conditions stress and depression also impact gut health • The use of and the gut microbiota by bacterial diversity and promoting the of resistant the same gut microorganisms produce of and • is the most in the rich in as based on and the of bacteria and promote microbial diversity to microbiota composition significantly based on and status, both of which influence dietary and example, in to less diverse microbiota in often to a less and reduced microbial exposure A recent foods and the gut community in fermentation with human microbiota The significant in both composition and as increased the of potentially taxa such as and and to whereas to However, while significantly the the of of the current about the human microbiota from the a that and the microbiome in various body including the gut, skin, mouth, and The also the role in conditions such as obesity, gastrointestinal diseases, and the for new and In the of the the that a microbiome with microbial composition is influenced by such as and environment The the the between the microbiome and chronic diseases, that a of microbial diversity is associated with increased of such conditions the significant in gut microbiota by the The gut microbiota over species across and are the most by and which for to of the microbial in the are approximately including and species are for their health-promoting such as and pathogenic Among the and are the The is less and primarily by for its impact on health. includes pathogens such as and is a central concept in and microbial to the and of species a complex ecosystem such as the Understanding microbial in the gut is a key for the health of the host. analysis potentially to various pathologies such is critical to therapeutic strategies microbiota balance and host health. This on the of a to microorganisms based on an includes from the of the from a that share a widely by the scientific community for taxonomic The is a in microbial to its bacteria and its of and The for while the between bacterial taxa while with and of provide critical on the and composition of the gut microbiota, across and clinical conditions To intestinal microbiota are • the variety of species a gut include the which both species and and the which the of species by for • microbial diversity between or across health in microbial of these such as the are This the relationship between the of and the of in the In recent years, for analysis has been to from to and Among is of the most widely for from microbiome taxonomic and of the of microbial composition across the of a with that of a from a or an the of the of and the of microbial diversity This provides fundamental the and of the gut microbiota under physiological or pathological conditions. microbial that is or to may an microbiota has been associated with diseases such as metabolic disorders inflammatory bowel and has been to conditions such as and neurological disorders associated with diversity of the intestinal microbiome is recognized by the scientific community as a of various research the impact of use with that the of bacterial to immune responses and reduced gut has been to which the immune ability to suppress In review This condition has been associated with significantly survival in with that a and diverse gut microbiome may play a crucial role in immune responses against cancer In the of through such as and diversity and a critical for and clinical various been in the to human and This and A to and gut are not associated with or body but are influenced by dietary on current the human can defined as associated with rich in of in In and of the microbial These are based on the of specific bacterial and are to dietary rather such as or This underscores the significant impact of on gut microbiota composition in the the relationship between gut microbiota functional and microbiota or as defined by or The taxonomic based on the of specific bacterial primarily and or and The refers to functional the metabolic diversity of the microbial community rather taxonomic or The that by or by exhibit functional to that may influence the functional of the gut microbiome refers to metabolic not to taxonomic and In review across taxonomic defined by or diversity from and exhibit functional therefore, a by genetic, dietary, and lifestyle factors. This the gut microbiota a key of health and the to diagnostic and therapeutic The various not in the bacterial taxa but also in their functional and as microbial associated with to diverse and in a and microbial over In contrast, are often by and a of These influence not the composition of the microbiota but also its interactions with the host and its overall impact on health the for and therapeutic based on an microbial Understanding and these provide and with diagnostic a to scientific and clinical interest in gut microbiota is research in in its are such as microbiota a and for These a future in which microbiota analysis and clinical therapeutic strategies for a range of Microbiota testing of the gut microbial a to the and of diseases in a This supports the of by microbial such as which may host health and to specific pathological conditions. to an microbiota has the to health by and therapeutic strategies It is essential to that growing for the clinical role of the gut microbiota, current scientific evidence most clinical are based on studies or specific and is not evidence to support the use of microbiota testing or in clinical practice (3). AND OF MICROBIOTA The of a microbiota requires a and of the clinical its This based on a analysis of the current and overall health to that the is for diagnostic or therapeutic microbiota testing by or other with the to the and the clinical However, the clinical of microbiome testing is • A lack of in • The proliferation of clinical with limited or clinical • in and a lack of education that microbiota analysis is primarily by from gastrointestinal functional in by and These also about their microbiota, as well as by various most or other systemic interest in these is driven by of these are for to that has to that not been as or The of microbiota analysis has under to such testing not the fundamental that clinical the ability to the Clinical scientific evidence linking microbiome to specific health conditions. Clinical the to influence that the can and the Clinical refers to the ability to the of a specific Clinical utility is defined as the ability to therapeutic (3). The of microbiota testing is essential to and This concept refers to the ability to it is to with To and However, in the of microbiota these is to the of microbial composition and the complexity of a microbiota to provide that are as as In review Key requirements to • and such as or other to bacterial DNA integrity and These specific on the is or • is to microbial to specific implications with • to and is or a compromise with an of the by microbiota testing by to and are recognized by the scientific of such microbiota to for standardized and The has been the for taxonomic and of bacterial in microbiota research This the a present in approximately the both and The as for the of the across a range of bacterial while the or for the differentiation and of bacteria various taxonomic including and In gut microbiota of the most widely to its and in complex microbial which can significantly by a less of the microbial by bacterial a overview of microbial of include and the ability to a broad range of bacterial taxa, including that are or to the benefits from and for taxonomic it for broad taxonomic and snapshots of microbial diversity and its is its taxonomic to can bacteria the it often to diverse bacterial Moreover, it not provide functional the microbial as it a rather the The for key • DNA DNA is from or other biological • of the are specific of the are with and can taxa, that in the of bacterial on the and the • such as are to the • The are that include control, of or of and taxonomic such as or the To the of we a on studies that such as and may in or microbial diversity and to the same in but across diverse while a of diversity and across and by the same These the of and to and in microbiome provides a of the microbiome to such as as it DNA present in a including bacterial, fungal, and functional metabolic and the of the microbial This also the of novel and that may not analysis of microbial for the of the of species and The includes critical In review • DNA DNA is from the of • The DNA is to in • The are for by to their These for to the and the • The is a each DNA is in producing • The are by these are and a In are to the of species the • The are to and functional and to microbial In includes taxonomic to community composition and functional analysis to the of various AND is a to both and This limited of DNA rather the it provides less and to or it a overview of microbial multiple such as The is to that of but with significantly However, of these in the of provide about the mucosal microbial or the physiological of the It is to that analysis is on which is The of these are their and However, the key in the of the mucosal which is from the in of and a of the of these to physiological the of the metabolic, or functional activity or intestinal on the or of specific bacterial is as it from is the of to the production of short-chain fatty acids such as acetate, propionate, and butyrate, based on the taxa such functional can that for of microbial the can through microbiota • This refers to the variety and of bacterial species present in a It is such as the of the and the These provide the and of microbial • microorganisms from to species In can to and offering on microbial • These are of microbiota that and may influence gut health. The include and is associated with specific dietary and health • and associations: between microbial can provide These can as for in microbiota composition but with and in with clinical (3). AND To and a of the of and is • such as bacterial with and are to the and of the • are crucial for DNA or These to the integrity of the the use of such as the by and of is a critical to It • • DNA • and microbial However, it is to in the in to the of about the mucosal which plays a and significant role in microbiota A standardized is essential to in microbiota It requires with and through to and The a that includes a to a for and a to bacterial to the with for to and of in with for both and to of the is crucial to microbial DNA The use of and is to analysis of the The of the can significantly influence microbiome The use of the for of and for This an essential for clinical DNA of the DNA is to DNA to the standardized to and and DNA or to a of microbial to that the are and for analysis and The DNA is to of the These are which provide and the of a broad of microbial species in the are that taxonomic of bacterial and analysis to microbial diversity and In review are with to microbial and to operational and to It is also essential to the taxonomic for microbial as the of on the of the which and by the scientific community Clinical Once analysis is the a to the or The may include such as and to in along with and for clinical microbiota and fundamental and including that microbes preserving and DNA and for both and as of microbial DNA in the The most scientific been In to a community used. of the with A in which taxonomic and are A with potentially of to from the analysis This analysis the bacterial network the microbiota and may AND OF MICROBIOTA Clinical on • and diversity as and in microbiome as provide the complexity and of the microbial ecosystem, which may associated with clinical However, studies are to its role in clinical practice the between microbial longitudinal multiple or pathological conditions. clinical needs to defined • to or species and to species or with their across the community or with of can in with a to diversity and composition A in an control, which to the an to • Key bacterial of the is in microbiome testing, as current evidence not support the use of such as diagnostic dysbiosis may to the complexity of microbiome and can lead to In review example, of may a or an ecosystem on the various dysbiosis been dysbiosis an concept for clinical use such are it is essential that are and by analysis is not as a diagnostic for specific diseases, but rather to the influence of the microbiota on an existing pathological In other it can the microbiota plays a role in but it not the is of to has a microbial composition influenced by genetic, and dietary factors. the microbiota composition may a for and chronic diseases, metabolic diseases, and gastrointestinal conditions. It also therapeutic such as the of and dietary strategies to and microbial studies the use of in therapeutic including as an for in with for and in the of respiratory diseases Another is in the of Scientific evidence that physical as or physiological that impact not but also metabolic, and intestinal health to often gastrointestinal such as bowel gut and These are associated with tract inflammation, increased and microbial imbalance, including of and a This dysbiosis can promote bacterial and systemic inflammation, both and gut health studies that and with dietary strategies and specific may and However, studies are to these microbiota analysis may also support colorectal cancer describe in which of in with gastrointestinal to with clinical In review microbiome analysis with such as and strategies in disorders with gastrointestinal the microbiota to play a critical role in clinical that the microbiota may in therapeutic dietary and may studies are to the involved AND its microbiota testing with • Microbiota composition is and influenced by and other factors. It significantly across it to establish a or Microbiota testing as a a clinical and by to or It not to but rather to support to AND Microbiota testing significant but scientific evidence A to is the lack of which across Another critical in intestinal microbiota is often or in and a lack the to microbiota or to both and are on the often to for on the use and of microbiota clinical utility through scientific research and • by Microbiota analysis for but a scientific also that the of and can influence clinical To we of and key and their to the of The Microbiota International Clinical Society (MICS) for increased and for to that microbiota testing can contribute to clinical and well-being.