The intestinal microbiota consists of approximately 1013 to 1014 microorganisms, a global microbial community dominated by bacteria, mainly strict anaerobes, but also including viruses, protozoa, archaea, and fungi (1). Although several studies have investigated the bacterial communities in infants, our understanding of the microbiota in children of different ages remains limited because only 20% of the bacterial species have been successfully cultured (2). The limitation of these quantitative and qualitative descriptions reflects the methodological difficulty encountered when trying to delineate and enumerate the constituents of a normal microbiota. Traditional culture-based analyses are adequate only for the minority of the gut microbes that are amenable to cultivation. As new culture-independent techniques, such as genome sequencing and metagenomic techniques, become available and less expensive, the likelihood of a more comprehensive analysis will increase (3). Although gut microbiota comprises a wide variety of bacterial species and strains whose composition and density vary along the gastrointestinal (GI) tract, it is largely defined by 2 bacterial phylotypes, Bacterioidetes and Firmicutes, with Proteonacteria, Acinobacteria, Fusobacteria, and Verrucomicrobia phyla present in relatively low numbers. The size of 1 microbial metagenome, also called microbiome, is 150 times larger than the human genome and encodes 100 times more genes than our own genome. This extensive gene catalogue may enable us to study potential associations between microbial genes and human phenotypes and even environmental factors throughout our lifetime. Indeed, the microbiota acts as a personalized organ that can be modified by diet, lifestyle, prebiotics, probiotics, and antibiotics.
COLONIZATION OF THE GI TRACT
The GI tract of a normal fetus has traditionally been thought of as sterile; however, studies suggest that the fetal intestine may be exposed to microbes via swallowing of colonized amniotic fluid. Nevertheless, colonization of the infant gut commences at birth, when delivery exposes the infant to a complex microflora with aerobic or facultative bacteria, such as enterobacteria, enterococci, and staphylococci, colonizing the GI tract immediately (4). During growth, the intestinal milieu changes, creating the conditions for the proliferation of anaerobic bacteria. The numbers and diversity of strict anaerobes such as Bifidobacterium, Clostridium, and Bacteriodes increase later as a result of diet and environment and after weaning. At age 1 year, a complex adult-like microbiota is evident. A complex microbiota dominated by obligate anaerobes provides a strong barrier against the establishment and proliferation of new bacterial strains, a phenomenon termed “colonization resistance.”
An infant's gestational age at birth (preterm or full-term infants) and mode of delivery (vaginal birth or caesarean section [CS]) seem to have significant effects on the intestinal microbiota. Preterm infants admitted to the neonatal intensive care unit, where they are exposed to increased use of antibiotics, parenteral nutrition, and the like, show delayed colonization and a limited number of bacterial species, often selected based upon their resistance to broad-spectrum antibiotics (5). When infants are born by CS, they are deprived of contact with their mother's vaginal and intestinal microbiota and have lower numbers of Bifidobacteria, Bacteroides, and Escherichia coli compared with vaginally delivered infants (6). Other bacteria expand in CS-delivered infants as a result of reduced competition. Infants living under poor sanitary and crowded conditions in developing countries are colonised earlier than infants in affluent Western societies by E coli and other Enterobacteriaceae, enterococci, and lactobacilli (7). They also have a larger number of species and a more rapid turnover of bacterial strains. It is possible that the establishment of a complex microbiota proceeds faster in developing counties than in developed countries. Because of the differences in composition between human milk and standard infant formula, there is evidence in the literature that GI flora composition differs substantially in breast- and formula-fed infants (8).
Studies using quantitative real-time polymerase chain reaction assays on infants’ fecal samples show that Bifidobacteria are the most prevalent bacteria found in the flora of both feeding groups, but their amount is significantly higher in breast-fed than in formula-fed infants (9). Instead, the amount of E coli and bacteroides is significantly higher in formula-fed than in breast-fed infants (10). The gut microbiota is a major stimulus for the immune system, and a late acquisition of gut bacteria or a reduced complexity of the microbiota may delay immune maturation.
MICROBIOTA AND HEALTH BENEFIT
Despite a significant interpersonal variation, there seems to be a balance among bacterial species in a microbial stable core that confers health benefits. An alteration in beneficial bacteria by infection, disease, diet, and antibiotics can negatively influence the well-being of the individual. Altered microbiota can revert to the stable diversity established in infancy once the initial modifying factor has subsided. Within the GI tract, different microbiota has a mutually beneficial relationship with their host. The microbiota plays a critical role in supporting normal digestion; for example, human milk oligosaccharides (11) and host metabolism promote bacterial fermentation of unused energy substrates of dietary fibre to short-chain fatty acids. Short-chain fatty acids play an important role in appetite regulation through increasing anorexigenic enteroendocrine hormones such as peptide YY and glucagon-like peptide-1 and through modulation of energy storage, extracting calories from otherwise unavailable oligosaccharides (12). Conventionally reared mice have 40% higher body fat content than germfree mice, despite lower food consumption (13). It is also now known that the microbiota is essential for normal peristalsis. Normal intestinal motility requires coordination between the extrinsic neurons, enteric motor neurons, interstitial cells of Cajal and smooth muscle cells. The enteric nervous system (ENS) is a complex integrative division of the autonomic nervous system that is capable of controlling GI function. The abnormal motility of germ-free mice probably reflects a combination of the lack of a mature enteroendocrine system, changes in neurotransmission, and immaturity of the mucosal immune system (14). The prevention of colonization by pathogens is achieved in large part through the resident microbiota by competing for nutrients and receptors, producing antimicrobial compounds, and triggering the expression of multiple antimicrobial factors in Paneth cells (15).
Based upon different animal studies, it is clear that the microbiota is involved in the maintenance of barrier function, inducing increased epithelial cell proliferation and enhancing intestinal epithelial integrity through translocation of the tight junction protein zonula occludens 1 and upregulation of genes involved in desmosome maintenance (16). The gut microbiota contains a host of inflammatory components, and its presence evokes low-grade inflammation, influencing the entire organism. Germ-free studies revealed that the microbiota is essential for the development of the gut-associated lymphoid tissue and plays an important role in shaping the immunological response through the stimulation of the synthesis and secretion of immunoglobulin A and through the production of a balanced T-helper cell response (optimal equilibrium between TH1 and TH2-like immunity) (17). Germ-free animals have few immune cells in the intestinal lamina propria and few intraepithelial lymphocytes. Their Peyer's patches and mesenteric lymph nodes are small and lack germinal centers. Reduced microbial stimulation during infancy results in slower postnatal maturation of the immune system, with difficulty in the capacity to differentiate potentially dangerous from harmless antigens (18). Pathogen or commensal recognition by T-cell receptors results in a cascade of different events and different outcomes in triggering immune responses and intestinal neural pathways (19). The health benefits of human microbiota are as follows:
* Supports normal digestion
* Modulates normal intestinal peristalsis
* Prevents colonisation by pathogens
* Maintains barrier function
* Evokes low grade of inflammation
* Shapes the immunologic response
The gut–brain interaction is a complex bidirectional communication system that exists between the central nervous system (CNS) and the GI tract (20). This interaction has been reflected in the form of a revised nomenclature to the more inclusive term “brain–gut–microbiota axis” and there is now a sustained research effort to establish how communication along this axis contributes to both normal and pathological conditions.
The gut–brain axis integrates cognitive and emotional centres in the CNS with the neuroendocrine and neuroimmune systems, the sympathetic and parasympathetic arms of the autonomic nervous system, the hypothalamic–pituitary–adrenal axis, the ENS, also called the “little brain,” and the intestinal microbiota. Through this bidirectional complex network, the CNS and the gut are intimately connected. Signals from the brain influence the motor, sensory, and secretory functions of the GI tract by releasing neuropeptides and hormones, and conversely visceral messages from the GI tract can influence brain function, mood, and behaviour (21,22). There is evidence of a beneficial effect of using probiotics and prebiotics in hepatic encephalopathy (23), and there are numerous animal studies that indicate a CNS change in responses to alteration in the composition of gut microbes. One approach that is being used to study the role of microbiota on the host's health is the use of germ-free animals. Germ-free mice, which are animals devoid of any bacterial contamination, offer the possibility of studying the impact of the complete absence of microbiota on GI functions and gut–brain axis–related functions. The cross-talk among the gut microbiota, the immune system, and the gut–brain axis also seems to play an important role in the modulation of the stress response. Microbiota communicates with gut–brain axis through different mechanisms and multiple routes, as follows:
* Direct interaction with mucosal cell (endocrine message) through the release of bacterial substances, fermentation products such as short-chain fatty acids, and indirectly stimulating the production of intestinal neuroendocrine factors
* Via immune cells (immune message) through recognition of pathogen-associated molecular patterns by Toll-like receptors, which modulate the expression of factors, such as cytokines and chemokines, that recruit and change the phenotype and function of immune and inflammatory cells. Mast cells are important effectors of the gut–brain axis, which translate the stress signals into the release of a wide range of neurotransmitters and proinflammatory cytokines. Neurons, astrocytes, and microglial cells express membrane surface receptors that are specific to the molecular products of immune cells, which underlie brain cellular responses to immunological signals.
* Via contact with neural endings (neuronal message) through increasing expression of GABA receptors, by inducing the expression of opioid and cannabinoid receptors in intestinal epithelial cells; via elevation in the plasma of tryptophan, a precursor to serotonin, which is a key neurotransmitter within the gut–brain axis; among other pathways.
Obviously, multiple mechanisms are possible and further studies will clarify both neural and humoral routes through which the intestinal communal microflora may influence ENS and CNS signalling (Fig. 1). Taken together, it is clear that microbiota can modulate various aspects of the gut–brain axis; however, these effects are bacterial strain dependent and care must be taken in extrapolating data obtained from one organism to another.
A disturbance in the primary colonization or in the balance of normal intestinal microflora (or the host response to this) has been shown to play a critical role in the pathogenesis of a wide variety of intestinal and extraintestinal disorders. Bacterial colonization of the intestine plays a major role in the postnatal development and maturation of the immune nervous and endocrine systems. These processes are key factors underpinning CNS signalling and suggest a role for microbiota in the modulation of mood and behaviour (24). Microbiota plays an important role in the modulation of the hypothalamic–pituitary–adrenal axis, activated in response to a variety of physical and psychological stressors (25). One of the important coordinators of the endocrine, behavioural, and immune responses to stress is corticothropin-releasing factor (CRF). CRF has a potent effect on gut via modulation of inflammation, increase of gut permeability, contribution to visceral hypersensitivity, and modulation of the gut motility (26). Stressors in GF mice induce an exaggerated release of CRF with an abnormal activation of the hypothalamic–pituitary–adrenal axis involved in stress response. The pituitary gland responds to CRF by releasing adrenocorticotropic hormone to stimulate the adrenal glands’ secretion of cortisol. This abnormal stress response in GF mice is partially reversed by bacterial recolonisation (27). Other authors report in GF mice a reduction in anxiety behaviour and an upregulation in the expression of brain-derived neurotrophic factor, a protein involved in multiple aspects of cognitive and emotional behaviours through the modulation of new neuron and synapse growth and differentiation. A strategy using antibiotic-induced dysbiosis of the microbiota resulted in mice displaying less anxiety-like behaviour and altered protein levels of brain-derived neurotrophic factor. The discontinuation of the antibiotic cocktail restored the normal behavioural profile of the animals (28). Similar perturbation of the microbiota by administration of pathogen bacteria has been shown to increase anxiety-like behaviour and produce stress-induced memory dysfunction, reverted by daily administration of a probiotic cocktail.
The human brain achieves its nearly complete neuronal capacity by birth, but brain development does not cease at birth. Rather, during infancy the brain establishes the myriad synaptic connections that provide the essential substrate for functional brain networks that underlie perception, cognition, and action. A recent study revealed that the bacterial content of the gut can modulate brain developmental pathways (29). This regulation has explicit time constraints, with a critical developmental window in the early postnatal period during which gut microbiota may modulate synaptogenesis through changes in the expression of genes whose products influence neurotransmitter modulation in the nervous system. The microbial colonization process modulates signalling mechanisms that affect neuronal circuits involved in motor and sensitive control and can influence the neural network responsible for controlling stress responsiveness.
Although the microbiota exerts a broad influence on brain functions, the converse is also true. The brain can alter the microbiota through modulation of intestinal secretion, permeability, and motility, removing excessive bacteria from the lumen and preventing bacterial overgrowth (30). Signalling molecules released into the gut lumen from cells in the lamina propria that are under the control of the CNS can result in changes in GI motility and secretion as well as intestinal permeability, thus altering the GI environment in which the bacteria reside (29).
There is evidence that exposure to stress may be responsible for the dysregulation of the gut–brain axis, thus leading to the different diseases of the gut. Changes in bidirectional interplay between the microbiota and the brain have been implicated in the pathophysiology of functional GI disorders, such as infant colic or irritable bowel syndrome (31) and in the pathogenesis of other GI diseases, such as inflammatory bowel disease, food antigen-related adverse responses, peptic ulcer, and gastroesophageal reflux disease (32).
ROLE OF PROBIOTICS IN THE GUT–BRAIN AXIS
An aberrant gut microbial composition, such as an inadequate lactobacilli level and an increased concentration of coliforms in the first months of life, may play an important role in the pathogenesis of GI stress-related disorders. Indeed, intestinal colonization by lactobacilli may be a prerequisite for normal gut–brain axis function. Growing evidence supports that the gut–brain axis is responsive in humans to a variety of nutritional interventions, including the administration of probiotics. Gut microbiota may modulate the sensory response to pain and some probiotics may inhibit hypersensitivity and perhaps intestinal permeability (33). The postulated mechanisms of action of probiotics on stressed GI mucosa include improvement of the barrier function of the epithelium, suppression of the growth and binding of pathogenic bacteria, positive effect on visceral hypersensitivity, and immunomodulatory effect. Although the exact mechanisms are not well understood, evidence is mounting to support the role of probiotic intervention in reducing anxiety and stress response, with some probiotics having the potential to lower inflammatory cytokines and decrease oxidative stress and others altering the expression of the GABA receptor in the CNS (34). Interestingly, vagotomy prevented this effect of probiotics, suggesting that parasympathetic innervation plays an important role in the transmission of information from gut to brain and also in homeostasis associated with the immune system.
It is now known that specific microbes alter pain pathways in the intestine, inducing the expression of opioid and cannabinoid receptors in intestinal epithelial cells and mediating analgesic functions in the gut, similar to the effects of morphine (35). A role for the gut microbiota also has been proposed in decreasing visceral hypersensitivity through the modulation of dorsal root ganglion single-unit activity to colorectal distension (36). The benefit of supplementation with L reuteri has been reported in infants and experimental data suggest that the effect of probiotics may be related to the influence on immune response, gut motility, and pain transmission and perception (37). Despite the high number of studies using probiotics, several questions remain unanswered, such as the optimal dose, the role of combination therapy, strain-specific activity, stability within the GI tract, possible development of antibiotic resistance, and the duration of therapy.
The brain and the intestine constantly communicate with each other and the gut microbiota seems to play a critical role in this interaction. There is a time window when the gut microbiota may affect the structure and the function of the brain. Administration of probiotics may lead to behavioural changes in humans, but this conclusion warrants further investigation.
1. Turnbaugh PJ, Ley RE, Hamady M, et al. The human microbiome project. Nature 2007; 449:804–810.
2. Eckburg PB, Bik EM, Bernstein CN, et al. Diversity of the human intestinal microbial flora. Science 2005; 308:1635–1638.
3. Qin J, Li R, Raes J, et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 2010; 464:59–65.
4. Palmer C, Bik EM, DiGiulio DB, et al. Development of the human infant intestinal microbiota. PLoS Biol 2007; 5:e177.
5. Neu J. Gastrointestinal development and meeting the nutritional needs of premature infants. Am J Clin Nutr 2007; 85:629S–634S.
6. Dominguez-Bello MG, Costello EK, Contreras M, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A 2010; 107:11971–11975.
7. Adlerberth I, Wold AE. Establishment of the gut microbiota in Western infants. Acta Paediatr 2009; 98:229–238.
8. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, et al. Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 2000; 30:61–67.
9. Martin R, Langa S, Reviriego C, et al. Human milk is a source of lactic acid bacteria for the infant gut. J Pediatr 2003; 143:754–758.
10. Penders J, Thijs C, Vink C, et al. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics 2006; 118:511–521.
11. Marcobal A, Barboza M, Sonnenburg ED, et al. Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways. Cell Host Microbe 2011; 10:507–514.
12. Bird AR, Conlon MA, Christophersen CT, et al. Resistant starch, large bowel fermentation and a broader perspective of prebiotics and probiotics. Benef Microbes 2010; 1:423–431.
13. Backhed F, Manchester JK, Semenkovich CF, et al. Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proc Natl Acad Sci U S A 2007; 104:979–984.
14. Indrio F, Riezzo G, Raimondi F, et al. The effects of probiotics on feeding tolerance, bowel habits, and gastrointestinal motility in preterm newborns. J Pediatr 2008; 152:801–806.
15. James SM, Tagg JR. A search within the genera Streptococcus, Enterococcus and Lactobacillus for organisms inhibitory to mutants streptococci. Microb Ecol Health 1988; 1:153–162.
16. Sharma R, Young C, Neu J. Molecular modulation of intestinal epithelial barrier: contribution of microbiota. J Biomed Biotechnol 2010; 2010:305879.
17. Hrncir T, Stepankova R, Kozakova H, et al. Gut microbiota and lipopolysaccharide content of the diet influence development of regulatory T cells: studies in germ-free mice. BMC Immunol 2008; 9:65.
18. Jadcherla SR, Peng J, Chan CY, et al. Significance of gastroesophageal refluxate in relation to physical, chemical, and spatiotemporal characteristics in symptomatic intensive care unit neonates. Pediatr Res 2011; 70:192–198.
19. Abreu MT. Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition shapes intestinal function. Nat Rev Immunol 2010; 10:131–144.
20. Rhee SH, Pothoulakis C, Mayer EA. Principles and clinical implications of the brain-gut-enteric microbiota axis. Nat Rev Gastroenterol Hepatol 2009; 6:306–314.
21. Collins SM, Bercik P. The relationship between intestinal microbiota and the central nervous system in normal gastrointestinal function and disease. Gastroenterology 2009; 136:2003–2014.
22. Romijn JA, Corssmit EP, Havekes LM, et al. Gut-brain axis. Curr Opin Clin Nutr Metab Care 2008; 11:518–521.
23. Shukla S, Shukla A, Mehboob S, et al. Meta-analysis: the effects of gut flora modulation using prebiotics, probiotics and synbiotics on minimal hepatic encephalopathy. Aliment Pharmacol Ther 2011; 33:662–671.
24. McLean PG, Bergonzelli GE, Collins SM, et al. Targeting the microbiota-gut-brain axis to modulate behavior: which bacterial strain will translate best to humans? Proc Natl Acad Sci U S A 2012; 109:E174.
25. Konturek PC, Brzozowski T, Konturek SJ. Stress and the gut: pathophysiology, clinical consequences, diagnostic approach and treatment options. J Physiol Pharmacol 2011; 62:591–599.
26. Larauche M. Novel insights in the role of peripheral corticotropin-releasing factor and mast cells in stress-induced visceral hypersensitivity. Neurogastroenterol Motil 2012; 24:201–205.
27. Bale TL, Lee KF, Vale WW. The role of corticotropin-releasing factor receptors in stress and anxiety. Integr Comp Biol 2002; 42:552–555.
28. Bercik P, Denou E, Collins J, et al. The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice. Gastroenterology 2011; 141:599–609.
29. Diamond B, Huerta PT, Tracey K, et al. It takes guts to grow a brain: increasing evidence of the important role of the intestinal microflora in neuro- and immune-modulatory functions during development and adulthood. Bioessays 2011; 33:588–591.
30. Diaz HR, Wang S, Anuar F, et al. Normal gut microbiota modulates brain development and behavior. Proc Natl Acad Sci U S A 2011; 108:3047–3052.
31. Lin HC. Small intestinal bacterial overgrowth: a framework for understanding irritable bowel syndrome. JAMA 2004; 292:852–858.
32. Cobrin GM, Abreu MT. Defects in mucosal immunity leading to Crohn's disease. Immunol Rev 2005; 206:277–295.
33. Ahrne S, Hagslatt ML. Effect of lactobacilli on paracellular permeability in the gut. Nutrients 2011; 3:104–117.
34. Bravo JA, Forsythe P, Chew MV, et al. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci U S A 2011; 108:16050–16055.
35. Rousseaux C, Thuru X, Gelot A, et al. Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors. Nat Med 2007; 13:35–37.
36. Verdu EF, Bercik P, Verma-Gandhu M, et al. Specific probiotic therapy attenuates antibiotic induced visceral hypersensitivity in mice. Gut 2006; 55:182–190.
37. Ma X, Mao YK, Wang B, et al. Lactobacillus reuteri ingestion prevents hyperexcitability of colonic DRG neurons induced by noxious stimuli. Am J Physiol Gastrointest Liver Physiol 2009; 296:G868–G875.
© 2013 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,