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The intestinal microbiota are important in human growth and development. Microbial composition may yield insights into the temporal development of microbial communities and vulnerabilities to disorders of microbial ecology such as recurrent Clostridium difficile infection. Discoveries of key microbiome features of carbohydrate and amino acid metabolism are lending new insights into possible therapies or preventative strategies for inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). In this review, we summarize the current understanding of the development of the pediatric gastrointestinal microbiome, the influence of the microbiome on the developing brain through the gut-brain axis, and the impact of dysbiosis on disease development. Dysbiosis is explored in the context of pediatric allergy and asthma, recurrent C. difficile infection, IBD, IBS, and metabolic disorders. The central premise is that the human intestinal microbiome plays a vital role in health and disease, beginning in the prenatal period and extending throughout childhood.
The human intestine harbors trillions of microbial cells which form a symbiotic relationship with the host and play a vital role in both health and disease. While the specific microbial composition varies among healthy individuals, the functional repertoire of the microbiome is conserved.
Therefore, the gut microbiota is considered a crucial factor for proper early life development and lifelong health. However, when the balance of the intestinal microbiota becomes disrupted, alterations can lead to immunologic dysregulation and the development of diseases including Clostridium difficile infection,
In this review, we describe the development of the pediatric microbiome, starting in utero and progressing through infancy, childhood, and adolescence. We then discuss the impact of the microbiome on the developing brain and neural function through the gut-brain axis. We conclude with a discussion on the impact of dysbiosis on disease development.
Establishment of Early Life Intestinal Microbiome
Microbial Colonization of the Neonatal Gut
For many years it was believed that the fetus’ in utero environment was sterile, with infant gut colonization beginning at the time of delivery. However, recent work demonstrating the presence of a microbial community in the meconium
Meconium microbiota types dominated by lactic acid or enteric bacteria are differentially associated with maternal eczema and respiratory problems in infants.
it is now clear that microbial colonization of the infant gut may begin prior to birth as additional evidence suggests microbial colonization of the placenta,
collected 320 placental specimens under sterile conditions and found a unique placental microbiome niche, which most closely resembled the human oral microbiome.
Furthermore, a randomized, double blind, placebo-controlled trial demonstrated that maternal probiotic supplementation could affect the expression of Toll-like receptor-related genes in both the placenta and the fetal intestine.
This finding suggested that the fetal intestinal immune gene expression profile could be affected by microbial contact in utero. Similarities between the unique microbiota composition of the placenta and amniotic fluid to that of the infant meconium further suggests a prenatal microbial transfer from mother to fetus.
In addition to potential in utero environmental influences, many factors have been found to contribute to early intestinal colonization, such as gestational age at birth. Studies have shown that the intestinal microbiota of preterm infants differs from that of healthy term infants,
The contribution of mild and moderate preterm birth to infant mortality. Fetal and Infant Health Study Group of the Canadian Perinatal Surveillance System.
These premature neonates are often exposed to prolonged hospitalizations, antibiotics, and formula feeding which may all disrupt the maturation of health-associated microbial communities.
Importantly, alterations in the microbiome of preterm infants have been correlated with increased risk for complications such as necrotizing enterocolitis
Another major influence on the infant gut microbiome is infant diet. Breast-fed infants have microbiota enriched in Lactobacillus, Staphylococcus, and Bifidobacterium, as compared to formula-fed infants with microbiomes dominated by Roseburia, Clostridium, and Anaerostipes.
Formula-fed infants have greater quantities of microbes associated with inflammation, with a more rapid maturation of their microbiome toward that of an adult-type composition.
Human milk oligosaccharides, the 3rd largest component in human milk, are considered prebiotic, with antimicrobial and antiadhesive properties thought to be protective to the infant.
Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligo saccharides of human milk inhibit its binding and infection.
During the first year of an infant's life, the relatively simple neonatal microbiome matures and develops into a more complex microbiome, with a composition more representative of an adult gastrointestinal tract enriched in Bacteroides and Firmicutes.
During the first year of life, the infant's microbiome also gains functionality similar to their mother's gut metagenome, with decreasing interindividual variation over time.
An increased number of bacterial genes relevant for plant polysaccharide metabolism primes the infant microbiome for the adult diet even before the introduction of solid foods.
Once solid foods are introduced, there is a sustained shift in the microbial composition with an increase in Bacteroidetes. Additional modifications include increased short chain fatty acids in the stool, and expression of genes relevant for carbohydrate metabolism, vitamin biosynthesis, and xenobiotic degredation.
During the early infant development period many exposures can influence the progression of the intestinal microbiota. For example, antibiotic treatment during this period of early life development can dramatically alter the intestinal microbiota structure.
Similarly, exposure to less sanitary environments, including contact with household pets and siblings, have significant effects on the developing microbiome.
In fact, the number of older siblings positively correlates with bacterial diversity and richness at 18 months of age, with increasing relative abundances of Firmicutes and Bacteroidetes in infants with more siblings.
Conversely, the microbiome also affects the general health status of the infant or child. A longitudinal comparative study of Malawian twins discordant for kwashiorkor found that the malnourished twin displayed abnormal microbiome signatures compared to the healthy twin.
As proof of concept that the microbiome was a causal factor in the development of kwashiorkor phenotype, frozen fecal communities from the discordant twin pairs were transplanted into gnotobiotic mice. The mice receiving kwashiorkor microbiome exhibited marked weight loss with accompanied perturbations in amino acid, carbohydrate, and intermediary metabolism.
Development of Pediatric and Adolescent Intestinal Microbiota
While some investigators have suggested that the pediatric microbiome reaches a relatively stable, adult-like configuration within the first 3 years of life,
In a study comparing the intestinal microbiota of 1-4 year old children to healthy adults, the adult microbiome had significantly greater diversity (abundance and richness) than young children.
A study comparing the fecal microbiota of adolescents (11-18 years of age) to healthy adults found that the number of detected species was similar between groups, but the relative abundances of genera differentiated adolescents from adults, suggesting that the microbiomes differed, even into adolescence.
compared 7-12-year-old children to adults, and found that similar to adults, the pediatric gut microbiome was largely composed of Bacteroidetes and Firmicutes (Figure 1). However, the relative abundances of these bacteria differed from adults, with relatively lesser abundances of Bacteroidetes and greater abundances of Firmicutes and Actinobacteria.
They also found that while many taxa were shared between pediatric and adult samples, the distribution was significantly different, with children having greater abundances of bacteria belonging to the genera Faecalibacterium, Dialister, Roseburia, Ruminococcus, and Bifidobacterium.
Figure 1Healthy pediatric and adult gastrointestinal tracts differ in relative abundances of gut bacterial taxa. (a) Phylum level relative abundances via 16S rRNA gene sequencing. Genus level relative abundances by (b) 16S sequencing and (c) shotgun metagenomic sequencing. (Adapted from Hollister et al.
also characterized the metagenomic profiles of pediatric and adult microbiomes. Children demonstrated an enrichment of genes which may support ongoing development, including genes involved in vitamin synthesis, de novo folate synthesis, and amino acid metabolism.
Meanwhile, adults were enriched in pathways previously linked to inflammation, including genes involved in oxidative phosphorylation, lipopolysaccharide biosynthesis, flagellar assembly, and steroid hormone biosynthesis.
While the intestinal communities of children shared 35-46% similarity to each other taxonomically, they had substantially greater overlap at the functional level, with >90% similarity in the ortholog groups and pathways.
This difference implies that the functional capacity of microbes present in the pediatric gastrointestinal tract is more highly conserved than microbial composition.
The Gut-Brain Axis
Impact on Brain Development
The human brain undergoes rapid growth during the perinatal period, corresponding to dramatic changes in the maternal microbiota.
Mothers demonstrate an increase in Proteobacteria and Actinobacteria, and a decreased richness as they progress from the first to the third trimester of pregnancy.
While these changes are often correlated with metabolic syndrome in nonpregnant females, in the setting of pregnancy these changes are beneficial in promoting energy storage and allowing for adequate growth of the fetus.
Many studies have demonstrated the importance of the microbiota during brain development, including the microbiome's indirect effect on tryptophan metabolism and serotonin (5-HT) synthesis (Figure 2). 5-HT is known to be crucial to CNS development.
Knock-in mice lacking the tryptophan hydroxylase 2 gene, demonstrated that a lack of brain 5-HT caused improper wiring of the brain that may lead to long-term changes and neurodevelopmental disorders.
When compared to specific pathogen free (SPF) mice, germ-free mice have increased motor activity and decreased anxiety, as well as altered levels of neurotransmitters such as noradrenaline, dopamine, and 5-HT.
Germ-free mice also have a decreased kynurenine: tryptophan ratio compared to conventionally raised mice, which normalizes upon exposure to gut microbiota immediately after weaning.
Furthermore, rats treated with Bifidobacterium infantis showed reduced 5-HIAA concentrations in the frontal cortex, and increased plasma concentrations of tryptophan and kynurenic acid compared to controls.
Figure 2The bidirectional gut-brain axis. The gut-brain axis is a complex interplay between the central nervous system, the neuroendocrine, and neuroimmune systems, the autonomic nervous system, the enteric nervous system, and the microbiota. 5-HT, 5-hydroxytryptamine. DC, dendritic cell. GABA, γ-aminobutyric acid. (Adapted from Collins et al.
Colonic bacteria have an important role of fermenting carbohydrates and proteins to produce metabolites, including short chain fatty acids (SCFA), which are essential for human health.
Therefore, defective microglia were found in mice with altered microbiota, including germ-free mice, mice with temporal eradication of microbiota, and mice with limited microbial complexity.
Germ-free mice display an exaggerated hypothalamic-pituitary-adrenal stress response, which was reversible with exposure to SPF feces during early development.
This study demonstrates the important role that the microbiota play during early postnatal brain development, while brain plasticity may still be preserved.
Bidirectional Gut-Brain Axis and Its Impact on Brain Function
The brain-gut-microbiota axis is a complex interplay between the CNS, the neuroendocrine and neuroimmune systems, the sympathetic and parasympathetic arms of the autonomic nervous system, the enteric nervous system, and the microbiota.
The communication throughout this axis is bidirectional, with brain signals affecting gastrointestinal tract motor, sensory and secretory functions, and simultaneous visceral signaling from the GI tract affecting brain function
Long-term treatment with the probiotic Lactobacillus rhamnosus (JB-1) led to decreased levels of stress-induced corticosteroids, depressive symptoms, and anxiety.
Alterations in GABA production are implicated in depression and anxiety disorders. Importantly these changes were not found in vagotomized mice, implicating the vagus nerve as a direct line of communication between the gut bacteria and the brain.
Another study has implicated the gut microbiome in pain perception. Certain strains of Lactobacillus induce increased expression of µ-opioid and cannabinoid receptors in intestinal epithelial cells, mimicking the analgesic effects of morphine.
Similarly, the brain can affect the composition of the gut microbiota. These effects on the microbiota can be indirect, through changes in motility and secretion, or direct, through signaling molecules released into the gastrointestinal tract via enterochromaffin cells, neurons, and immune cells.
The autonomic nervous system affects motility as well as mucus secretion into the gut lumen, both of which can alter the gastrointestinal environment, thereby changing the bacteria that are present.
For example, exposure to stressful stimuli has been shown to increase permeability of the epithelium, allowing bacterial antigens to cross the epithelium and stimulate an immune response in the mucosa, which in turn alters the microbiome.
This increased permeability is secondary to mast cell degranulation, overproduction of interferon gamma, and decreased expression of mRNA encoding tight junction proteins.
While it is clear that the maintenance of the microbiome is vital for preservation of health, imbalances in the microbiome can shift the microbiome–host relationship from symbiotic to pathogenic. Below, we discuss some examples of communicable and noncommunicable disorders that are associated with a dysbiotic microbiome.
Clostridium difficile Infection
Clostridium difficile infection (CDI) is the leading nosocomial infection in the United States, affecting more than 500,000 people annually.
A complex microbial community in the intestine is vitally important to providing colonization resistance to CDI. Therefore, alterations to the microbiota increase the risk of infection from C. difficile. When comparing patients with CDI to diarrheal and nondiarrheal controls, Schubert et al
determined that Ruminococcaceae, Lachnospiraceae, Bacteroides, and Porphyromonadaceae were absent in patients with CDI, but highly associated with nondiarrheal controls. These compositional changes are even more pronounced in patients with recurrent CDI who have been exposed to multiple courses of antbiotics.
These antibiotic-related changes included decreased taxonomic and functional diversity of the gut microbiome as well as a decreased colonization resistance against invading pathogens.
discovered that exposure to subinhibitory concentrations of certain antibiotics upregulated the expression of genes encoding colonization factors in C. difficile, and increased the adherence of C. difficile to cultured cells.
Proton pump inhibitor (PPI) use also increases the risk of CDI.
Chronic PPI use leads to decreased abundances of Bacteroidetes and increased abundances of Firmicutes at the phylum level, which may predispose patients to CDI.
Therapies aimed at correcting and restoring health-associated complex microbial communities have yielded successful outcomes in treating CDI. Fecal microbiota transplantation (FMT) is around 90% effective at curing recurrent CDI with one or more infusions.
demonstrated that FMT normalizes both bacterial community composition and metabolic capacity. Pre-FMT fecal samples had greater concentrations of primary bile acids and bile salts, while post-FMT samples contained mostly secondary bile acids.
Patients with recurrent CDI yielded disrupted abilities to convert primary bile acids to secondary bile acids. Primary bile acids have been shown to promote germination and growth of C. difficile, while secondary bile acids inhibit this growth.
Therefore, it is possible that the correction of bile acid metabolism, and not just restoration of community structure, is a crucial mechanistic element in the efficacy of FMT against CDI.
However, it is unclear if this dysbiosis plays a role in the etiology of the disease, is a result of the disease, or both. Patients with IBD demonstrated exaggerated immune responses against commensal intestinal microbes, which may be essential for the development of intestinal inflammation.
In support of this theory, studies have shown that treatment with antibiotics can substantially decrease intestinal inflammation and improve IBD symptoms.
demonstrated that the abundance of Fusobacterium species is increased in patients with ulcerative colitis compared with healthy controls. In a separate study, Ohkusa et al
showed that when given as an enema, the isolated human strain of Fusobacterium varium caused UC-like colonic ulcerations in mice, indicating that this bacterium may play a role in the pathogenesis of ulcerative colitis.
Patients with IBD have a variety of changes in the fecal microbiota including decreased abundances of Bacteroides, Bidifdobacterium, Clostridium, and Lactobacillus, as well as increased abundances of Fusobacterium and adherent-invasive Escherichia coli.
Additionally, the functional composition of the gut microbiota is altered in IBD, with one study showing an alteration of 12% of pathways analyzed compared to 2% of genera between IBD and healthy individuals.
Functional alterations in IBD patients include diminished carbohydrate metabolism and decreased production of butyrate and other short chain fatty acids.
Given the importance of the microbiome in IBD, therapies aimed at manipulating the microbiome have gained popularity. While there are some promising studies demonstrating the efficacy of certain antibiotic combinations in treating IBD, more controlled trials are needed.
Similarly, while studies exploring the use of probiotics in IBD have yielded encouraging results, results vary across studies and more randomized controlled trials are needed.
Treatment of relapsing mild-to-moderate ulcerative colitis with the probiotic VSL#3 as adjunctive to a standard pharmaceutical treatment: a double-blind, randomized, placebo-controlled study.
IBS is diagnosed based on the presence of chronic recurrent abdominal pain related to defecation or associated with changes in frequency or form of stool, without accompanying warning signs.
Patient with IBS show reductions in the relative abundance of Bifidobacterium and Lactobacillus, as well as increased abundances in the Firmicutes: Bacteroidetes ratio.
Molecular analysis of faecal and duodenal samples reveals significantly higher prevalence and numbers of Pseudomonas aeruginosa in irritable bowel syndrome.
confirmed differences in microbiome signatures in pediatric IBS compared to healthy controls. Additionally, they were able to classify different subtypes of IBS using a limited set of discriminatory species, with a success rate of 98.5%.
Further support for the role of gut microbiome perturbations in the development of IBS is the persistence of IBS-like symptoms following confirmed bacterial or viral gastroenteritis, a term called postinfectious IBS.
Proposed mechanisms for postinfectious IBS include enteroendocrine cell hyperplasia, elevated T-lymphocytes, and increased gut permeability following infection.
Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome.
To further elucidate the role of the intestinal microbiota in IBS, multiple studies have examined the effects of probiotic supplementation in this disorder. Based on several meta-analyses and systematic reviews, probiotic use for treatment of IBS appears more effective than placebo.
However, many of these studies vary in their specific conclusions, likely due to inadequate sample sizes, weak study design, and the use of various probiotic strains making comparisons difficult.
Another therapeutic avenue for the treatment of IBS has included dietary changes. Fermentable carbohydrates can be difficult to absorb and have been shown to contribute to symptoms in IBS. Consistent with this finding, a low FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) diet has been shown to decrease symptoms in adults with IBS.
Randomised clinical trial: gut microbiome biomarkers are associated with clinical response to a low FODMAP diet in children with the irritable bowel syndrome.
confirmed that this response was also true in pediatric IBS. Additionally, they demonstrated that specific microbial signatures were associated with the efficacy of the FODMAP diet.
Randomised clinical trial: gut microbiome biomarkers are associated with clinical response to a low FODMAP diet in children with the irritable bowel syndrome.
Specifically, FODMAP responders had baseline microbiomes enriched with taxa with greater saccharolytic metabolic capacity and metabolic pathways related to carbohydrate metabolism.
Randomised clinical trial: gut microbiome biomarkers are associated with clinical response to a low FODMAP diet in children with the irritable bowel syndrome.
The development of asthma and allergies has been associated with deviations in the developing microbiota. For example, infants colonized with E. coli were at an increased risk of developing eczema, while infants colonized with C. difficile were at increased risk of all atopic outcomes (eczema, recurrent wheeze, and allergic sensitization).
Antibiotic exposure in the first year of life has also been associated with an increased risk for the development of asthma in children, with this risk increasing in parallel with the number of courses of antibiotics prescribed.
These human findings have been further explored using mouse models of allergy and atopy. Allergic germ-free mice developed more severe disease than conventionally housed controls.
Importantly, this phenotype could be reversed by recolonization of the germ-free mice with conventional microbiota, demonstrating the important and influential role of the microbiota in allergic conditions.
Furthermore, in a mouse model of allergic airway inflammation (asthma), symptoms could be attenuated by exposure to Lactobacillus reuteri, but not Lactobacillus salivarius,
Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the Global Burden of Disease Study 2013.
The patterns of maturation of microbial communities in infancy can affect the relative risk of becoming overweight and obese in later childhood. A recent longitudinal study of more than 900 infants found that mode of delivery and infant gut microbiota (specifically belonging to the Lachnospiraceae family) mediated the association between prepregnancy maternal overweight status and overweight status of children at 1 and 3 years of age.
Another study found that low levels of Bifidobacterium spp. and increased Staphylococcus aureas in infancy were associated with being overweight by age seven.
In this study low dose antibiotic exposure in young mice led to increased adiposity, metabolic hormone levels, and SCFA levels, as well as changes to the hepatic metabolism of lipids and cholesterol.
found that low dose penicillin given at birth can induce sustained effects on body composition and enhance high fat diet-induced obesity in mice. Furthermore, the obese phenotype was transferable to germ-free mice by transfer of low-dose penicillin microbiome,
implicating the microbiome as the driver of this phenotype as opposed to antibiotics.
Autism Spectrum Disorder
While the underlying etiology of autism or autism spectrum disorder is not well understood, the intestinal microbiota is proposed to play a role in the development of autism. Children with autism have dysbiotic fecal microbiota, with greater abundances of Bacteroidetes and lesser abundances of Firmicutes compared to controls.
compared the mucosal microbiome of autistic children with functional abdominal pain to neurotypical children with function abdominal pain, and found distinct microbial signatures in autistic children that correlate with cytokine quantities and tryptophan homeostasis.
Children with regressive (late-onset) autism have increased numbers of fecal clostridial species, as well as the presence of nonspore-forming anaerobes and microaerophilic bacteria, which were absent in control children.
hypothesized that, in some children, antibiotic-induced disruption of the microbiome may facilitate colonization by autism-promoting bacterial species. They tested this hypothesis by treating 10 autistic children with minimally absorbed oral vancomycin, and found that 8 of 10 children had short-term improvement in autistic symptoms.
While the improvements were not long-lasting, this report indicates a potential role for the gut microbiota in the symptomatology of autism spectrum disorder and thus warrants further investigation.
Murine studies have also supported a role of the microbiome in autism. Buffington et al
demonstrated impaired social behavior in the offspring of dams fed a high-fat diet, which were mediated by changes in the offspring's microbiota. While these pups’ microbiota was notable for a significant reduction in L. reuteri, supplementation with this bacterium reversed the observed social deficits.
found that in utero valproic acid exposure resulted in decreased social behavior scores and impacted the gut microbiota of mice, with specific changes in Bacteroidetes and Firmicutes, similar to human autism studies. Together these results establish that in murine models of autism, behavioral alterations have been associated with altered microbial colonization.
Conclusions
In this review, we summarized the current understanding of the development of the pediatric microbiome, the impact of the microbiome on the developing brain and brain function through the gut-brain axis, and the impact of dysbiosis on disease development. The intestinal microbiome is an important factor in human growth and development, and the appropriate balance of microbes throughout life plays a crucial role in the both health and disease. As emerging technology allows us to understand more about the microbiome and its many important functions, we in turn begin to understand the disease processes that the microbes impact. With this deeper knowledge and understanding comes the hope of new therapeutic targets and avenues through which to treat these diseases and promote human health across life stages and ages.
Funding Sources Statement
This work was supported by the National Institutes of Health (U01 CA170930), Texas Medical Center Digestive Disease Center (P30 DK56338), and unrestricted research support from BioGaia AB (Stockholm, Sweden) (J.V.).
References
Human Microbiome Project C
Structure, function and diversity of the healthy human microbiome.
Meconium microbiota types dominated by lactic acid or enteric bacteria are differentially associated with maternal eczema and respiratory problems in infants.
The contribution of mild and moderate preterm birth to infant mortality. Fetal and Infant Health Study Group of the Canadian Perinatal Surveillance System.
Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligo saccharides of human milk inhibit its binding and infection.
Treatment of relapsing mild-to-moderate ulcerative colitis with the probiotic VSL#3 as adjunctive to a standard pharmaceutical treatment: a double-blind, randomized, placebo-controlled study.
Molecular analysis of faecal and duodenal samples reveals significantly higher prevalence and numbers of Pseudomonas aeruginosa in irritable bowel syndrome.
Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome.
Randomised clinical trial: gut microbiome biomarkers are associated with clinical response to a low FODMAP diet in children with the irritable bowel syndrome.
Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the Global Burden of Disease Study 2013.
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