The human intestinal microbiota

Gastroentérologie Clinique et Biologique Vol. 34/1 (2010) 379-441

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The intestinal microbiotia: Mini-symposium on New Perspectives in Irritable Bowel Equilibrium and disorders Predicting liver failure after major hepatectomy Syndrome d’activation macrophagique

Guest editors: G. Corthier & P. Marteau Cholécystectomie ambulatoire

ELSEVIER MASSON

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THE norMAL MiCroBioTA oF THE GASTroinTESTinAL TrACT

The human intestinal microbiota

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Le microbiote intestinal humain J. Doré*, G. Corthier INRA, MICALIS ex-UEPSD, Domaine de Vilvert, 78350 Jouy-en-Josas, France

Summary The human intestinal microbiota constitutes a complex ecosystem which is now well recognized for its impact on human health and well-being. it contributes to maturation of the immune system and provides a direct barrier against colonization by pathogens. its possible implication in diseases of modern societies, currently increasing in prevalence, has been reported. These include allergies, inflammatory bowel diseases and possibly metabolic and degenerative disorders. The analysis of the molecular composition of the human intestinal microbiota indicates marked inter-individual variations which may seem paradoxical considering the high degree of conservation of major functions of the intestinal microbiota such as anaerobic digestion of alimentary fibres. We have characterized a phylogenetic core within the human intestinal microbiota, in terms of composition, i.e., a set of conserved species that could be responsible for major conserved functions. Based on culture-independent molecular assessments, current knowledge enables a definition of criteria qualifying the normal state of the human intestinal microbiota that we call normobiosis. This further enables the identification of specific deviations from normobiosis, i.e., dysbiosis in immune, metabolic or degenerative diseases. Notably, Crohn’s disease, an inflammatory bowel disease of yet unknown aetiology, is associated with intestinal dysbiosis with a lower representation of the Clostridium leptum group among the Firmicutes phylum. We further showed that the bacterial species Faecalibacterium prausnitzii exerts anti-inflammatory properties in vitro and in animal models; this could explain its ability, when detected in the mucosa-associated microbiota of patients in vivo, to protect patients from post-operative recurrence of endoscopic signs of inflammation 6 months after surgical resection of the ileocecal region of the gut.By confirming the major role of the microbiota in bowelrelated disorders, which are especially associated with a disruption of homeostasis, we are currently applying high throughput functional metagenomic screens in order to identify signal molecules and mechanisms of bacteria-host cross-talk. Together with the high resolution description of the human intestinal metagenome, as well as explorations of environmental proteins and metabolites, these observations will further our understanding of the functional roles bacteria play in the maintenance of health and * Corresponding author. E-mail address: [email protected] (J. doré). ✰ A reprint of the french translation of this article is available on request. © 2010 Elsevier Masson SAS. All rights reserved.

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well-being in humans. It will open new perspectives for the monitoring and design of strategies for modulating the microbiota for health. © 2010 Elsevier Masson SAS. All rights reserved. Résumé  Le microbiote intestinal humain constitue un écosystème complexe, qui est maintenant bien reconnu pour son impact sur la santé et le bien-être de l’homme. Il contribue à la maturation du système immunitaire et assure une barrière directe contre la colonisation par des agents pathogènes. Son implication possible dans les maladies des sociétés modernes, dont la prévalence est en augmentation, a été décrite. Ces maladies sont notamment les allergies, les maladies inflammatoires de l’intestin et, peut-être, des troubles métaboliques et dégénératifs. L’analyse de la composition moléculaire du microbiote intestinal humain indique des variations interindividuelles marquées, qui pourraient sembler paradoxales étant donné le haut degré de conservation des fonctions majeures du microbiote intestinal, telles que la digestion anaérobie des fibres alimentaires. Nous avons caractérisé un noyau phylogénétique au sein du microbiote intestinal humain, en termes de composition, c’est-à-dire un ensemble d’espèces conservées qui pourraient être responsables des fonctions conservées majeures. Sur la base d’évaluations moléculaires indépendantes des cultures, les connaissances actuelles permettent de définir les critères qualifiant l’état normal du microbiote intestinal humain, ou « normobiose ». Cela permet en outre l’identification d’écarts spécifiques par rapport à la normobiose, c’està-dire d’une dysbiose, dans les maladies immunitaires, métaboliques ou dégénératives. La maladie de Crohn notamment, qui est une maladie inflammatoire de l’intestin dont l’étiologie est encore inconnue, est associée à une dysbiose intestinale avec une plus faible représentation du groupe Clostridium leptum parmi le phylum des Firmicutes. Nous avons en outre montré que l’espèce bactérienne Faecalibacterium prausnitzii possède des propriétés anti-inflammatoires in vitro et dans des modèles animaux ; cela pourrait expliquer sa capacité, lorsqu’elle est détectée dans le microbiote associé à la muqueuse de patients in vivo, à protéger ces patients contre une récidive postopératoire des signes endoscopiques d’inflammation 6  mois après la résection chirurgicale de la région iléocæcale de l’intestin. En confirmant le rôle majeur joué par le microbiote dans les troubles en relation avec l’intestin, qui sont en particulier associés à une rupture de l’homéostasie, nous procédons actuellement à des analyses métagénomiques fonctionnelles à haut débit dans le but d’identifier les molécules signal et les mécanismes d’interaction bactérie-hôte. Conjointement avec la description de haute résolution du métagénome intestinal humain, ainsi qu’avec des explorations des protéines et métabolites environnementaux, ces observations nous permettront de mieux comprendre les rôles fonctionnels joués par les bactéries dans l’entretien de la santé et du bien-être chez l’homme. De nouvelles perspectives s’ouvriront alors pour la surveillance et la mise au point de stratégies de modulation du microbiote pour l’optimisation de la santé. © 2010 Elsevier Masson SAS. Tous droits réservés

Introduction From the very moment of birth on, microorganisms colonize the newborn and establish a mutual relationship with their host that lasts the entire life and essentially contributes to health and well-being. Yet microorganisms that surround us are still most often perceived as dangerous potential invaders against which we should be ready to fight. This oddly negative perception contrasts with their acknowledged utility for the environment, agriculture, industry and mankind. Over millions of years, humans, and animals before them, have long co-evolved with microorganisms, leading to the establishment of mutually beneficial adaptation. Although fine mechanisms or temporal dynamics are not yet known, the host develops a tolerance towards non-pathogenic bacteria that colonize mucosal surfaces and skin and in return,

the bacteria control the development and maintenance of numerous essential functions of their host. The intestinal microbiota contributes to the bioconversion of unabsorbed food-born compounds in the upper parts of the digestive tract. Colonization by commensal microorganisms is a key to immune development [1-4]. In addition, the microbiota exerts a direct role of colonization prevention by keeping pathogens at population levels that prevent them from expressing virulence [5-7]. More recently, a potential role of the intestinal microbiota in the regulation of fat storage has been described in mice [8] and its operation in humans has been suggested [9,10]. Our vision of the human intestinal microbiota has recently been thoroughly revisited with the use of molecular tools with a gene marker approach based on the use of ribosomal RNA as a phylogenetic marker. For an overview of the main stages of the development of the complex microbiota

The human intestinal microbiota

associated with humans, the reader is invited to consult the appropriate chapter in this monograph. We herein will give an updated view of the intestinal microbiota composition and stability. We will thereafter outline its beneficial roles, and illustrate the association of microbiota dysbiosis and disease. We will finally provide perspectives on the metagenomic exploration application of the functions of the dominant human intestinal microbiota.

Composition of the human intestinal microbiota It is currently accepted that the human digestive tract, known as the microbiota, is made up of hundreds of species in each individual. Microbial population densities in this complex bacterial consortium reach their maximum values in the colon with 1011 bacteria per gram of content. Yet a thorough description of all intestinal bacteria does not exist for two main reasons: Traditional culture-based characterization [11-13] can take into account no more than 30% or so of the microorganisms that can be seen and enumerated by microscopic observation. Worldwide species diversity of commensal intestinal bacteria would be immense. In that respect, the use of molecular tools has indicated that the majority of the dominant bacterial species observed in the faecal microbiota of an individual (approximately 80%) are specific to this individual [14-16]. If species diversity of the dominant intestinal microbiota gives a faecal print which is essentially specific to the individual, the taxa composition (genera and/or large phylogenetic groups) highlights consistent components found in all individuals. Some of these taxa have been known for a long time and are represented by numerous collection strains; others have been demonstrated only recently via molecular approaches and still have not been cultured. Culturable genera of the dominant faecal microbiota of adults are Bacteroides, Eubacterium, Ruminococcus, Clostridium and Bifidobacterium [13, 17] Accounting for non-culturable microorganisms has refined this vision and put it in a phylogenetic framework. The phylum Firmicutes is always highly represented. It comprises the Eubacterium rectale – Clostridium coccoides, often the most represented (14 to 31% of total bacteria depending on the studies) [18-22]. It is composed of species belonging to the genera Eubacterium, Clostridium, Ruminococcus, Butyrivibrio. The phylum Firmicutes also comprises the Clostridium leptum group with the species Faecalibacterium prausnitzii, Ruminococcus albus and R. flavefaciens; this group is also very often dominant (16 to 22% on average) [20, 23]. Bacteroidetes are represented by genera related to Bacteroides. They are always present and share dominance with the above groups (9 to 42% of total bacteria on average). The phylum Actinobacteria is less consistently detected as dominant but represents a few percentage of the total bacteria. It comprises bifidobacteria (0.7 to 10%) and bacteria of the Collinsella-Atopobium group (0.3 to 3.7% on average) [21,24]. Enterobacteria are more seldom observed in the top two logs of population of the faecal microbiota (0.4 to 1%), similar to lactobacilli and streptococci (2%) [23]. Also occasionally found are species

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related to Clostridium ramosum, Eubacterium cylindroides, Phascolarctobacterium, Verrucomicrobium, Sporomusa, Selenomonas or Veillonella. Highly conserved compositional traits of phylogenetic groups and phyla on the one hand, together with subject specificity of species on the other hand suggest that there exists, on functional grounds, some degree of redundancy between species, and that different levels of resolution provide complementary pieces of information. Finally, the observed bacterial species are strictly associated with the intestinal ecosystem. This is the result of a long co-evolution with the host [9] which has been confirmed by recent studies of cross-association of microbiota from/ with different hosts [25]. At present, phylogenetic reassessment of the human intestinal microbiota has been essentially restricted to the dominant fraction, and our knowledge of subdominant bacteria, i.e., below 108 per gram of stool, may be incomplete and remains restricted to culturable isolates. The ability to isolate and grow microorganisms in vitro remains a key step in knowledge building, especially considering that phylogeny does not provide information on the in situ activity of microbes. On a more focussed standpoint, the assessment of the contribution of archaea or phages, which may be highly significant in terms of populations, has remained anecdotal up to now.

Colonization of the human intestine Any microorganism from the environment, from another human or surrounding animal can in principle lead to colonization. It will nonetheless face a competitive challenge with other microorganisms and, soon after birth, with a complex community that expresses a barrier effect as a main function, i.e., the ability to prevent colonization of non-established species. The foetus of mammals develops in utero in a sterile environment, and it is well accepted that colonization starts during the process of birth. Sterile mucosal surfaces (digestive, urogenital, naso-buccal and respiratory as well as skin) constitute a set of ecological niches which are highly favourable to microbial colonization. In the absence of the sophisticated immunological mechanisms of the adult, the digestive tract of the newborn is an especially permissive environment in which population levels or early colonizers quickly reach levels of 1011 bacteria per gram of stool. Colonization follows a fairly organized scheme under the control of exogenous and endogenous factors. Exogenous factors include exposure to microorganisms of maternal (faecal, vaginal and cutaneous) and environmental origin, but also food intake and occasionally antibiotherapy which may induce significant perturbations. A few recent studies have indicated that maternal milk could also be a vector of microorganisms from mother to child. Even when collected under sterile conditions, maternal milk may contain live and culturable bacteria [26], which may be transferred to the infant via breastfeeding [27,28]. Exogenous factors include several secretions from the digestive tract but also products from the first colonizing microorganisms that globally condition the ecosystem’s physical chemistry. Anaerobic bacteria that dominate the adult intestinal microbiota are among the first microorganisms encountered after vaginal delivery. They will nonetheless develop and become dominant in the

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Major questions still remain concerning the process of microbiota development following birth. For example, the concept of a transient “window of permissivity”, possibly allowing any microorganism encountered to become a dominant component of the adult microbiota, has yet to be challenged and the duration of such a period of permissivity is totally unknown.

intestine, following facultative anaerobes that will prepare the ecosystem by consuming the oxygen as a privileged energy source. Recent work has indicated that dominant anaerobic species do modify their metabolism following weaning and the corresponding change in dietary exposure [29]. This first succession of species occurs in the very first few hours following birth (Fig. 1). Antagonistic and synergistic relationships will thereafter govern the succession of dominant species, leading by about the age of 2 years to a functionally stable microbiota [30]. Eventually anaerobic bacteria will dominate facultative anaerobes one thousandfold. Hygiene at birth and during the first moments of life will markedly influence the dynamics of colonization. It appears clear today that colonization by the usual early colonizing commensal species such as E. coli is delayed in industrialized countries compared to the past (by a few days to six months) and compared to developing countries, owing most likely to the hygiene conditions applied to date [31, 32]. Bacteria commonly associated with the skin microbiota may thus temporarily become part of the dominant intestinal microbiota [33]. Birth by caesarean section compared to vaginal delivery most often leads to retarded acquisition of usual dominant microbial groups and the frequent occurrence of environmental bacteria. The geographical location of the birth place may have an impact on early microbiota development. While comparison of adult microbiota does not indicate much difference from country to country in western Europe, we recently observed a north to south gradient in the faecal microbiota composition of 6 week old infants [34], such that Bifidobacterium is a dominant colonizer in the north (Sweden and Scotland) while Bacteroides is dominant within a microbiota characterized by early diversification in the South (Italy and Spain) (Fig. 2).

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The dynamics and homeostasis of the intestinal microbiota may be considered in time (for any given individual) and space, i.e., between individuals or intestinal compartments. The overall composition of the dominant intestinal microbial community appears to be consistent between individuals and over time. The same major phyla are present, with proportions that vary between individuals but most likely remain within the same log-unit equivalent in terms of population. Dominant species diversity appears remarkably stable over time for a given individual from day-to-day and even across years [22,35,36], while a large fraction of the dominant species appear to be specific to the subject. With regard to strains, stability is much less clear and will depend on the subject [37,38]. Hence the stability observed in groups and species would hide an important rate of renewal at the level of strains. Genomic plasticity may in fact come into play in that respect. It has also been shown that species diversity for subdominant groups, e.g., Lactobacilus, is far less stable over time than that of dominant ones [36] and that stability of communities is greater in the colon than in the ileum. It must also be considered that lactic acid bacteria brought by the ingestion of fermented food may occasionally have a survival rate during transit that leads to their dominant transient passage in the small intestine and colon.

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Homeostasis of the intestinal microbiota

Figure 1 Fecal microbiota of 6 week-old European infants. Infant fecal microbiota differs from that of adults, until the 2nd year of life. Genus Bifidobacterium is most represented; Bacteroides and relatives are early colonizers following enterobacteria. Adult phylogenetic groups C. coccoides and C. leptum are not ‘yet’ dominant [34].

The human intestinal microbiota

By definition, a microorganism that colonizes a given niche will persist and multiply without requiring reinoculation. For an individual, modifications of the intestinal microbiota may derive either from colonization by exogenous microorganisms or by modulation of population levels of commensal bacteria. in most cases it will essentially be the consequence of relays in dominance in response to factors modulating ecological niches. numerous factors may affect the stability of microbial communities - among others transit time, pH, quality and quantity of exogenous substrates and endogenous mucins. Although microbial communities appear ready to deal with changes in ecological settings, it seems difficult to induce durable alterations of established dominant populations, at least in terms of composition. numerous observations hence illustrate the ability of the dominant intestinal microbiota to resist modification. The administration of an allochtonous strain such as a probiotic or an exogenous non-absorbable substrate such as a prebiotic often leads to transient modifications of microbial equilibrium. Even a major stress such as an antibiotic administration can be followed by return of the community to its initial dominant species profile within a month or so [39,40]. This ability to recover its original make-up following a stress, known as resilience, suggests a finely-tuned adaptation of the microbiota to the gut and even to the host that harbours it. This can be linked to the observation that monozygotic twins have faecal microbial communities that have significantly more closely related patterns than these of unrelated individuals, suggesting that genotype may play a role in the development and structuring of the intestinal bacterial populations. The spatial distribution of intestinal microbes according to digestive sites requires the collection of samples within and along the intestine via invasive methods and is hence rather difficult to study. The preservation of topological relations

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between bacteria and epithelium is also a challenge. This most probably explains some remaining controversy on this topic. The luminal microbiota (within the intestinal cavity) has been explored in several ways. The proximal colon luminal microbiota differs from the faecal microbiota, whose composition is only found in the distal parts of the colon [41]. Between the proximal and the distal colon, microbial populations increase overall 100-fold and the increase is essentially due to an increase in strictly anaerobic bacteria. The layer of mucus that covers the intestinal wall constitutes a specific ecological niche. Several studies have shown that the microbial community that colonizes this niche is stable over time and is remarkably similar from the ileum to the rectum for a given individual [42,43]. Conversely, species that dominate in the mucus layer differ from dominant luminal species found in [16,42]. The ability of commensal gut bacteria to adhere in situ to intestinal epithelial cells has thus far not been unequivocally documented. indirect evidence does exist, derived from the presence of genes encoding adhesions in the genome of strains of E. coli able to durably colonize their host. Adhesions could nonetheless contribute to the recognition of mucosal sites and structures or sloughed cells. in ecological terms, a given strain must divide at least as quickly as it is eliminated from its niche in order to be maintained at a stable population level in the ecosystem [44]. Hence adhesion to the epithelium does not appear to be an absolute necessity, but recognition of sites within the mucus or in the contents would provide a selective advantage for slow growing strains [45]. if adhesion to the epithelium does not seem to be a relevant criterion for commensal bacteria, this property has been associated with intestinal bacteria in patients with inflammatory bowel diseases, and of course, many intestinal pathogens.

Figure 2 Fecal microbiota of 6 week-old European infants. Birth location has an effect on the composition of the fecal microbiota. North-South gradient from Stockholm, Sweden to Granada, Spain : more bifids in the north ; early diversification in the south [34].

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It remains clear that not all mechanisms involved in the maintenance of homeostasis of the human intestinal microbiota are understood to date, especially the determinants of resistance to change and resilience. It hence is still fully relevant to question the right level of phylogenetic depth and time period for which stability of the ecosystem should be defined. As far as resilience is concerned, it is also reasonable to speculate that a certain level of stress will disturb the equilibrium of the gut ecosystem, resulting in irreversible perturbation. The threshold above which the human intestinal microbiota loses its ability to return to its original balance is still unknown today and may differ from one bacterial group or niche to another. Finally, the parameters of homeostasis will apply both to functions as well as composition; yet the relevance of these parameters from a functional standpoint is totally unexplored. Functional resistance and resilience of the intestinal microbiota have yet to be determined, as does the link between phylogeny and functions. There are only speculations at present on a potential link between “quantity of diversity” and functional resistance and resilience. Finally it would seem highly relevant to explore the ability of functional foods to modulate these parameters of homeostasis since improving resistance to change or resilience upon stress (especially antibiotherapy) can be associated with reduced risk of infection for example.

Normobiosis and dysbiosis of the human intestinal microbiota We will refer to the normal state of the human intestinal microbiota that we call normobiosis. Based on culture-independent molecular assessments, it appears possible to define criteria qualifying normobiosis. Obviously, emphasis has been put for technical reasons on the phylogenetic evaluation of composition, diversity, core species and the dynamics of these over time and space, and it is obvious that defining normobiosis will benefit from the addition of functional parameters. This context further allows specific deviations from normobiosis to be identified, i.e., dysbiosis, which can be specifically investigated in immune, metabolic or degenerative diseases. We have recently validated the concept in the case of Crohn’s disease. Crohn’s is an inflammatory bowel disease of yet unknown aetiology that has a prevalence of one per 2000 in European countries. We have demonstrated that Crohn’s disease is associated with an intestinal dysbiosis with a lower representation of the Clostridium leptum group among the Firmicutes phylum [22,46-48]. We further showed that the bacterial species Faecalibacterium prausnitzii, when detectable in the mucosa-associated microbiota of the patient’s ileum, is protective against post-operative recurrence of endoscopic signs of inflammation 6 months after surgical resection of the ileocecal region of the intestine [47]. We finally demonstrated that Faecalibacterium prausnitzii could exert anti-inflammatory properties in vitro and in animal models with chemically-induced inflammation. The exploration of dysbiosis may be viewed as a primary step providing key information for the design of strategies aimed at restoring or maintaining homeostasis and normobiosis. Although thus far restricted to microbiota

J. Doré, G. Corthier

composition and/or diversity, dysbiosis has been suspected and in a few cases is already well documented. This is true for irritable bowel syndrome [49], ulcerative colitis [47,50], obesity [51,52], type-1 diabetes [53,54], type-2 diabetes [55], celiac disease [56,57], allergy [58,59], autism [60,61], cardiovascular health [62] and in cases of infections with Clostridium difficile [63] or HIV [64]. These observations, just as that of dysbiosis in Crohn’s disease above, are not indicative of a causal relationship between microbiota imbalance and onset of the disease. Indeed it is quite reasonable to argue that, owing to the disruption that such diseases will cause in the immune system and in the physicochemical properties of the intestinal milieu, dysbiosis could in fact be a consequence rather than a cause. In the case of Faecalibacterium prausnitzii in Crohn’s disease, we nevertheless have a situation in which deprivation in populations of a normal commensal bacterium, which belongs to the most dominant core species of the healthy gut microbiota and is potentially anti-inflammatory in vivo, is associated with a reduced ability of the ecosystem to promote a return to immune homeostasis. It can even be anticipated that a vicious circle is set in place, combining the detrimental effects of higher bacterial densities close to the mucosa [65], increased populations of gram-negative, pro-inflammatory, endotoxin-producing bacteria which are usually subdominant in healthy subjects [66,67], reduced proportions of anti-inflammatory commensal bacteria [47] and even increased occurrence of protein biomarkers, potentially promoting auto-immune reactivity [Juste and Doré, personal communication]. The current strengthening of the concept of normobiosis/ dysbiosis confirms the major role of the microbiota in bowelrelated disorders, especially for immune mediated diseases associated with a disruption of homeostasis. It stresses the need to apply the emerging tools of microbiomics to provide diagnostic models and also to identify signal molecules and describe bacteria-host crosstalk mechanisms at play.

Human intestinal microbiomics Beyond the phylogenetic level, which has been extensively explored thus far, lie the combined gene pool, transcripts, proteins and even metabolites of the members of the intestinal microbial community. These are known today as the metagenome, metatranscriptome, metaproteome, and all together as the human intestinal microbiome. Since the vast majority of dominant intestinal bacteria have not yet been cultured to date, the genomic content of this microbial community and its derived components at the various levels of omic integration are essentially unknown. Metagenomics is emerging today as this most powerful approach to characterize the repertoire of genes of any complex microbial setting, independent of whether its components can be cultured. The development of very high throughput sequencing technologies is further contributing to this development. In practical terms, the microbial community may be extracted from its environment and its DNA purified and/or cloned in order to determine its sequence. The complete gene repertoire of culturable and non-culturable dominant microbes can hence be obtained. This further offers the possibility of designing high

The human intestinal microbiota

throughput profiling tools which can be informative with regard to functional potentials of the complete community. In addition, cloning of genome fragments of intestinal bacteria in large insert metagenomic libraries allows the use of functional screens in order to seek functions of non-culturable bacteria after heterologous expression in E. coli, giving access to yet totally unexplored biological resources. Beyond its innovative character, the potentials of metagenomics have already largely been documented [68,69]. The first developments of the metagenomics approach applied to the human intestinal microbiota focussed on the diversity of the microbial community [70,71] and the gene repertoire for a few subjects [72,73]. Functional metagenomics applications have remained confined to the soil ecosystem [74] and animal guts [75]; we initiated its application to the human intestinal environment [76]. Although still in its infancy, this approach provides major promises for improved understanding of microbe-food, microbe-host and microbe-microbe interactions. The microbiomic exploration of the human intestinal microbiota is currently ongoing thanks to several programs such as the European Commission-funded program MetaHIT (http://locus.jouy.inra.fr/metahit/), and the French Agency for Research-funded program GMGE Micro-Obes (http:// www.inra.fr/micro_obes), as well as the NIH Roadmap programs (http://nihroadmap.nih.gov/hmp/) and many others worldwide. At the international level these programs are structured within the International Human Microbiome Consortium (IHMC) co-chaired by the NIH and the European Commission. These programs will deliver a huge mass of information that will in turn enable the identification of conserved and variable genomic and functional traits of the ecosystem, the description of those specific to the gut environment and bearing the best diagnostic and/or prognostic potential, reconstruction of the metabolic food-chain of the microbial community, and the initial description of ecotypes and modelling of their relationships in a systems’ecology endeavour.

Conclusion The application of molecular ecology tools to the intestinal microbiota has resulted in very significant improvements in our understanding of this ecosystem in terms of composition and dynamics of species diversity. The single gene approach based on ribosomal RNA as a universal phylogenetic marker had nevertheless overlooked the functions microorganisms do exert in their environment. It has become possible to sequence combined genomes of complex microbial communities, giving access to their potential activities. The next steps towards environmental transcriptomics, expressed proteins, activities and metabolites are being taken. The global functional exploration of the human intestinal microbiota is hence underway, with perspectives as large and fascinating as these of the human genome itself.

Conflicts of interests None

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