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Advances in Gut Microbiome Research and Relevance to Pediatric Diseases
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Advances in Gut Microbiome Research and Relevance to Pediatric Diseases Lindsey Albenberg, DO, and Judith Kelsen, MD
T
he human microbiota is the collection of microorganisms that reside within and on the surface of humans, and the term “human microbiome” refers to the genes that these microorganisms harbor. Although the human bacterial microbiome is the most commonly described, all 3 kingdoms of life, Archaea, Bacteria, and Eukarya are represented within this dense community. There are distinctive microbial communities among body sites, but by far, the vastest population of microorganisms resides within our gastrointestinal tracts. An estimated 1013 individual bacteria belonging to over 1000 species reside in the mammalian gastrointestinal tract, making it the most densely populated microbial community on Earth.1 Indeed, the collective genome of the human gut microbiome is predicted to be 100-fold greater than that of its human host.2 Although there are over 50 bacterial phyla on Earth, the majority of the bacteria in the human gut belong to 1 of 4 phyla, Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes. As humans, we have evolved to live in a mutualistic relationship with our microbiota. In fact, humans should be viewed as a biologic “supraorganism” where we provide an essential niche for our microbiota and in turn, our microbiota carry out critical physiological functions. For example, within the gut, bacteria participate in fermentation of indigestible carbohydrates to produce short chain fatty acids that are used by the host, biotransformation of conjugated bile acids, synthesis of certain vitamins, degradation of dietary oxalates, hydrolysis of urea by urease activity that participates in host nitrogen balance, and education of the mucosal immune system.3 Indeed, it was not very long ago when the term “gut flora” denoted an obscure and relatively unexplored space. However, advances in genome sequencing technologies and metagenomic analysis methods have led to numerous discoveries regarding community membership and the relationship between the gut microbiota and human health. Subsequently, it has become apparent that aberrations in the composition and functions of the gut microbiota may potentially be fundamental to the development of certain diseases, many of which are very relevant to pediatrics.
CD CF FMT IBD IgE IL iTregs UC
Crohn’s disease Cystic fibrosis Fecal microbiota transplantation Inflammatory bowel diseases Immunoglobulin E Interleukin Induced Tregs Ulcerative colitis
Technological Advances in Gut Microbiome Research Most species of gut-residing organisms are obligate anaerobes, many of which are fastidious and difficult to grow in vitro making traditional culture techniques of limited value in characterizing the composition of the gut microbiota. The development of culture-independent methods, mainly through the use of high-throughput DNA sequencing, has provided a novel means to evaluate the gut microbiota and its relationship to disease. There are 2 primary methods that use deep-sequencing technologies to characterize the microbiome. The first approach, uses small-subunit ribosomal RNA (16S ribosomal RNA) gene sequences (for Archaea and Bacteria), or internal transcribed spacer gene sequences (for Eukaryotes) as stable phylogenetic markers to define the lineages present in a sample.4 Another approach uses shotgun metagenomic sequencing in which the total community DNA is sequenced, thereby allowing for the microbial community structure and genomic representation of the community to be evaluated. The genomic community evaluation provides an understanding of the functions encoded by the genomes of the gut microbiota. 5 Metatranscriptomics and metaproteomics provide a deeper understanding of microbial function through direct evaluation of gene expression.6 These advances in sequencing technologies have allowed investigators to characterize the bacterial composition of the gut throughout different stages of life, a critical step in the study of health and disease.
Development of the Infant Microbiome and the Effect of Early Life Exposures It was once thought that the healthy human fetus develops in an environment that is completely sterile.7 However, there is an evolving body of literature suggesting that even the human placenta harbors a low-abundance commensal (nonpathologic) microbiota with potential biological relevance.8 For example, there may be an association between the composition of the placental microbiota and birth weight in full-term neonates.9 However, this needs to be further explored. Still, it is generally accepted that significant colonization of the infant does
From the Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, PA The authors declare no conflicts of interest. 0022-3476/$ - see front matter. © 2016 Elsevier Inc. All rights reserved. http://dx.doi.org10.1016/j.jpeds.2016.08.044
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THE JOURNAL OF PEDIATRICS • www.jpeds.com not occur until the time of delivery when infants are rapidly exposed to an abundance of organisms.10 There is increasing knowledge regarding the colonization succession of the infant gut and its relevance to health and disease. However, the initial colonization of other body sites remains relatively unexplored. In a study of 10 infants who underwent sampling across multiple body sites within 24 hours of delivery, the microbiota seemed to be homogenously distributed across skin, oral, and gut habitats.10 In contrast, their mothers harbored distinctly different communities at various body sites.10 The initial intestinal colonization pattern depends upon mode of delivery.7 Infants born vaginally are initially colonized by bacterial taxa found in the vagina, such as Lactobacillus and Prevotella, whereas infants who are born by cesarean delivery are initially colonized by bacteria found in the skin microbiota.10 After this primary inoculation, infants are regularly exposed to microbes, and diversity increases rapidly.11 Diversity is a term used to describe the microbial community in terms of richness, or number of microbial species present and also evenness, the proportions in which each species is represented. High diversity has been associated with health and low diversity has been associated with various disease states.12 This highlights the importance of this process of attaining diversity early in life as it likely has important implications for future health. It is also important to recognize that studies of humans around the world have consistently demonstrated that inhabitants of developed countries harbor microbial communities with reduced diversity. This has led to the hypothesis that loss of diversity is secondary to“Westernization”and may be involved in the pathogenesis of various chronic diseases, which have been rapidly increasing in incidence in developing countries.12 In infants, the initial colonization pattern is thought to be chaotic, and a growing body of literature has shown that environmental exposures early in life, including diet, are responsible for these fluctuations. Characterization of the intestinal microbiota in a single infant, over a period of 2.5 years, showed how the bacterial taxa changed with life events, such as illnesses, dietary changes, and antibiotic treatment. Interestingly, the greatest change in the composition of the infant’s intestinal microbiota occurred with the introduction of solid foods. There was also a shift toward a more stable, adult-like microbiota with weaning.11 This particular finding was replicated in a subsequent analysis of fecal samples from 98 fullterm infants where cessation of breastfeeding seemed to drive the transition to a more mature gut microbiome.13 Ultimately, the intestinal microbiota of the young resembles that of the adult by approximately the age of 3 years.14 Within the first year of life, there are significant interindividual differences in the composition of the intestinal microbiota, yet some similarities exist. Similarities among individual infants can be attributed to the major taxonomic groups associated with the infant diet. Multiple studies have established differences in the composition of the intestinal microbiota based on whether infants are breastfed or formula fed.15-17 Indeed, this introduces the concept of a potential association between infant diets, the composition of the intestinal microbiota, and health.
Volume ■■ Delivery mode and dietary factors are clearly not the only determinants of early gut colonization pattern. Genetics18 and other environmental exposures, such as antibiotic usage,19 also play a role. It is clear that appropriate colonization of the infant gut is important for education of the mucosal immune system and optimal gut function.20 Thus, it is logical to think that abnormal microbial exposures leading to dysbiosis, or an abnormal composition of the gut microbiota, play a role in the development of pediatric diseases.
The Relationship between the Gut Microbiome and Pediatric Diseases Pediatric Obesity The prevalence of pediatric obesity has increased in recent decades and represents a serious public health concern. In 20112012, approximately 12.7 million children and adolescents in the US were considered obese.21 Many obese children go on to become obese adults22 who have significant comorbidities such as diabetes, hypertension, atherosclerosis, and certain types of cancer, such as colorectal cancer. There are also, of course, more immediate health consequences for obese children and teens, such as hypertension and dyslipidemia.23 Therefore, it is important to understand underlying biological factors that may be related to the development of obesity, including factors that may be important early in life. For example, rapid infant weight gain in the first year of life has been associated with the development of obesity, and many factors related to weight gain in the first year of life have been identified including maternal obesity, mode of delivery, and early exposure to antibiotics.24 Dietary factors are also thought to be important.25 This raises the question of whether these factors are influencing a single mechanism, namely the development of the infant gut microbiota. The presence of an altered gut microbiota composition in obese individuals compared with lean individuals has been well established in both animal and human studies. This finding was first described by Ley et al26 who reported a decreased abundance of Bacteriodetes and a proportional increased abundance of Firmicutes in the cecal contents of genetically obese mice relative to their lean counterparts. The difference was independent of kinship and sex and importantly, all animals were fed the same diet. A similar shift in the abundance of Bacteriodetes and Firmicutes in the intestinal tract was subsequently demonstrated in obese adults compared with lean, healthy controls.27 Interestingly, these changes seemed modifiable as the relative abundance of Bacteriodetes increased as obese individuals lost weight on a low-calorie diet.26 One important function of the gut microbiota is degradation of otherwise indigestible components of our diet, thus, playing a role in energy balance. As mentioned previously, an example of this is fermentation of indigestible carbohydrates to produce short chain fatty acids. Thus, Turnbaugh et al27 sought to examine whether the microbiota of obese mice were more efficient at extracting energy than the microbiota of lean mice. Indeed, when germ-free mice were inoculated with a microbiota
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harvested from the cecum of obese mice, there was a significantly greater percentage increase in body fat over a 14-day period than when germ-free mice were inoculated with a lean donor microbiota. This difference occurred in the setting of identical calorie consumption among the 2 groups. It is very clear that calorie consumption is critical in the development and treatment of human obesity. However, the development of obesity likely involves increased calorie consumption plus an increased capacity of the microbiota to harvest energy from the diet, and this latter characteristic seems to be transmissible. The knowledge of the potential importance of energy harvest by gut microbes has led to the exploration of the hypothesis that perhaps antibiotics early in life alter the composition of the gut microbiota in a way that alters energy balance and promotes obesity. In fact, multiple epidemiologic studies have established a relationship between antibiotic exposures during infancy with increases in body mass index.24,28,29 For example, as part of the Avon Longitudinal Study of Parents and Children in the United Kingdom,29 exposures to antibiotics during 3 different early-life time windows and the relationship of the exposures to body mass were examined. In this populationbased study of more than 10 000 infants, treatment with antibiotics within the first 6 months of life was associated with increased body mass at 10-38 months of age.29 A mouse model of pediatric antibiotic use (repeated courses of beta-lactams or macrolides at therapeutic dosages) recently suggested a relationship between early life antibiotic exposure, alterations in gut microbiota diversity, and changes in body composition and growth.30 Pulsed antibiotic treatment also led to changes in hepatic gene expression and led to reduced levels of the hormone ghrelin, and these changes persisted even once antibiotics were discontinued.30 It is likely that other environmental factors in infancy contribute to dysbiosis conferring increased risk of obesity, and there is early data from the large, prospective child, parents, and health birth cohort to suggest that early colonization with a particular bacterial taxon, Bacteroides fragilis, may be associated with higher body mass index in children up to 10 years of age.31 With advances in our understanding of the structure and functions of the gut microbiota in infancy and its relationship to obesity, it does not seem implausible that one day the gut microbiota could be manipulated at an early age to prevent obesity. Disorders of the Skin and Atopic Disorders Atopic eczema is one of the most common inflammatory diseases, with increasing prevalence of 2%-3% in the adult population and 10%-30% in infants. It is often associated with other allergic diseases such as asthma, eosinophilic esophagitis, and allergic rhinitis. The presentation can be acute or patients may have recurrent symptoms characterized by erythema, papules, and scaly plaques that can have associated pruritus.32 As with many of the other diseases described in this review, the etiology of atopy is complex, because of an aberrant immune response to environmental factors in the genetically susceptible host. The prominent role of the gut microbiome is supported by the increase in the incidence of atopic disease as-
sociated with residence in or immigration to industrialized nations.33 In addition, the “hygiene hypothesis” suggests that humans living in more industrialized societies are exposed to fewer microbes or less complex microbial communities at an early age. Subsequently, the developing immune system is less able to “tolerate” exposure to the microbial-laden environment and results in an inappropriate immune activation.34 The importance of early life exposures has also been suggested with at least 1 meta-analysis demonstrating an association between delivery by cesarean and a moderate risk increase for allergic rhinitis and asthma.35 Human and animal models have been used to study the role of the gut microbiome in the development of atopy. For example, alterations of the gut microbiome have been demonstrated in children and young adults with eczema and atopy. Patients were shown to have a decreased relative abundance of Bifidobacteria in the feces compared with controls, and this was more pronounced in severe cases.36 Furthermore, an increased relative abundance of Clostridium and decreased relative abundance of Bifidobacteria has been demonstrated in the feces of infants with atopy at 3 weeks of age compared with healthy controls.37 West et al38 similarly found a low relative abundance of Ruminococcaceae at 1 week of age in infants who developed immunoglobulin E (IgE)-associated eczema. Murine models of atopy have demonstrated dysregulation of the IgE-basophil axis secondary to failure of the development of the gut commensal population.39 IgE has been associated with type 1 hypersensitivity anaphylactic reactions and is also responsible for binding mast cells, eosinophils, and basophils characteristic of other atopic conditions. Immune dysregulation can also be see through an exaggerated proinflammatory Th2 response with dominance of cytokine response characterized by interleukin (IL)-4 and IL-13.37 These cytokines are responsible for the keratinocytes in atopy with reduced antimicrobial peptides levels secondary to the Th2 response. Furthermore, T regulatory cells (Tregs) produced in the periphery are known as induced Tregs (iTreg). They are stimulated in the mesenteric lymph nodes, Peyer patches, and colonic lamina propria of mice.40 Interestingly, mice deficient in iTregs spontaneously develop Th2-type response with increase in CD4+T cells, with the production of Il-4, Il-13, and IL-5 cytokines in the mesenteric lymph nodes and both small and large intestine.41 These iTregs deficient animals go on to develop dysbiosis, with a relative increase in the TM7 phylum, and a macrophage and neutrophil infiltration of the airways with allergic phenotype.41 Together, these studies provide evidence for the existence of a gut-allergy axis, and potentially the opportunity to develop strategies aimed at manipulating the gut microbiota in early stages of immune development to prevent the onset of atopy and eczema. One potential preventative strategy could include treatment with probiotic bacterial strains very early on in life, such as in pregnant or breastfeeding mothers or infants. Indeed, results from clinical trials have suggested a role for early life probiotic administration to prevent allergic diseases.42 However, these trials have generally been underpowered and have demonstrated inconsistent results,43 thus, further studies are needed.
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THE JOURNAL OF PEDIATRICS • www.jpeds.com Disorders of the Respiratory Tract The respiratory tract, particularly the lower respiratory tract, is another body site once thought to be sterile in the healthy state. However, with advances in culture-independent techniques, there is evidence to suggest that even the airways of healthy people contain detectable microbes and that the community composition is altered in chronic disease. Still, compared with the gut, relatively little is known regarding the development of the human respiratory microbiome. This may be at least partially related to difficulty obtaining samples from the respiratory tract. Even samples from bronchoscopy, which would seem to be the most precise, are flawed, as the bronchoscope needs to pass through the oropharynx, thus, creating inevitable contamination. However, advanced analytical methods have been proposed to distinguish upper respiratory tract and lower respiratory tract taxa.44 The lung microbiota is now thought to be quite diverse with distinct communities in the upper and lower tracts.45 Overall, our knowledge of the lung microbiota comes from a small number of studies in both children and adults. With technological advances in microbiota analysis, there has been increased interest in the microbial communities in patients with cystic fibrosis (CF). Patients with CF are known to have viscous secretions and chronic airway obstruction, which leads to colonization with pathogenic bacteria. Antimicrobial activities are also compromised.46 Also, traditional culture results and response to antibiotics correlate poorly.46 Dozens of studies have now used advanced methods to attempt to identify microbes, not identified by clinical cultures, which may contribute to progression of lung disease. For example, Coburn et al47 evaluated sputum samples from over 200 patients with CF across various ages and disease stages. There was significant interindividual variability, however, a small, shared core microbiota was suggested. Diversity seemed to decrease with age, and this correlated with more severe lung disease. In addition, with increase in age, there was an increase in dominance of CF-associated pathogens Pseudomonas and Burkholderia. Similar findings were also demonstrated in a study of sputum samples from pediatric patients with CF.48 As with other disorders discussed in this review, early life exposures in pediatric patients with CF may have significant implications. There is evidence to suggest that the nasopharyngeal microbial community is different in young infants with CF compared with healthy controls and that antibiotics early in life alter community composition and perhaps increase the abundance of potentially pathogenic bacteria.49 A limited number of studies have also demonstrated alterations in the composition of the gut microbiota in patients with CF compared with healthy controls.50-52 However, the mechanism(s) and the degree to which abnormal intestinal fluid composition, fat malabsorption, and frequent antibiotic exposures contribute to dysbiosis remain to be elucidated. Importantly, at least 2 studies have demonstrated the presence of a core group of microbes that are shared between oropharyngeal and fecal samples from patients with CF.53,54 Gut colonization of a subset of bacterial genera, including those considered to be potential pathogens, may actually precede their appearance in the
Volume ■■ respiratory tract suggesting a close interrelationship between the microbiota in 2 body sites in patients with CF.53 There may even be an association between gut microbial colonization in early life and respiratory outcomes in these patients.54 This is intriguing as it conceivable that in the future engineering of the gut microbiota in patients with CF may have therapeutic implications. Intestinal Disorders–Inflammatory Bowel Diseases The inflammatory bowel diseases (IBD), comprised of Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases of the gastrointestinal tract. They are due to an aberrant immune response to environmental factors in the genetically susceptible host. The gut microbiota is thought to be a critical environmental factor in the development of IBD. Indeed, there is significant evidence to support the role of gut microbes in the development of IBD. Animal studies of IBD have demonstrated that germ-free animals show little sign of inflammation,55 however, inflammation develops with exposure to microbes.56 Adaptive immune responses to bacterial antigens have been shown to lead to the spontaneous development of colitis through immune activation and/or the loss of immune tolerance in various models.57 From a clinical standpoint, inflammation in CD and UC occur predominately in the terminal ileum (in CD) and colon (both UC and CD) where the greatest concentrations of bacteria are found. Antibiotics can have efficacy in the treatement of IBD.58-60 Furthermore, the fecal flow exacerbates IBD and surgical diversion of the flow amerliorates the disease.61,62 Genetic studies have also provided strong support for the role of the microbiota in the development of IBD.63 Multiple studies have now demonstrated that the composition of the gut microbiota is different in patients with IBD, including decreased diversity in patients with IBD, as well as alterations in the relative abundances of certain bacterial taxa.64 In terms of taxonomic changes, both expansion of potentially pathogenic bacteria and changes in microbial composition (ie, increased or decreased abundance of indicator species) has been described. For example, in CD, the phylum Firmicutes is commonly reduced in proportional abundance,65-70 notably, Faecailbacterium prausnitzii.71-73 Members of the Proteobacteria phylum, such as Enterobacteriaceae,74,75 including Escherichia coli70,76,77 are commonly increased. The E coli isolated in CD is often adherent and invasive phenotype. Myobacterium Avium subpecies Paratuberculosis has also been implicated as a potentially pathogenic organism in the development of IBD. It is the known cause of Johne disease in cattle which, similar to the histologic appearance of human CD, leads to a chronic granulomatous enteritis. There have been multiple studies exploring the role of Myobacterium Avium subpecies Paratuberculosis in CD,78 however, controversy remains whether this organism indeed has a causal role. In contrast to the many studies of the bacterial component of the gut microbiota, there is relatively little known about nonbacterial gut microbial inhabitants such as fungi, archaea, viruses, and bacteriophage. However, with advances in sequencing technology and novel techniques to explore these less commonly investigated
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populations, there is increasing evidence of fungal dysbiosis79,80 in patients with IBD, as well as differences in virus and bacteriophage81-83 populations in both pediatric and adult patients with IBD compared with healthy controls. Presently, the techniques available for studying nonbacterial gut microbial inhabitants are somewhat limited. However, the potential importance of gut fungi, archaea, and viruses in IBD and in child health and disease in general should not be discounted. As these techniques become well established, it is likely that important relationships will be demonstrated between these less wellunderstood populations and the disorders mentioned in this review and others. To date, the majority of the studies examining the gut microbiome in pediatric patients with IBD have included patients with established disease without accounting for clinical variables or the potential effects of medications. However, in 2015, Gevers et al84 published results from a large survey of the stool and biopsy-associated microbiome in newly diagnosed, treatment-naïve children with CD, which advanced our understanding of the microbial dysbiosis in new-onset CD. There were many important findings, including the discovery of a subset of bacterial taxa proportionally increased or decreased in abundance in biopsy samples from patients with CD compared with healthy controls. Interestingly, a few additional less commonly described taxa were suggested as potential biomarkers for disease including increased abundances of Pasteurellaceae (Hemophilus sp), Veillonellaceae, Neisseriaceae, and Fusobacteriaceae, and some were present in higher abundance in patients under the age of 10 years compared with older children. In this study, a small subset of patients were taking an antibiotic at the time of sample collection. Despite the small number of subjects, it seemed that concurrent antibiotics intensified the dysbiosis. This is particularly noteable given the potential association between childhood antibiotic exposure and IBD development.85 Despite significant advances in our knowledge of the dysbiotic gut microbiota that is seen in IBD, several questions remain to be answered. In particular, the question of causation vs correlation needs to be further explored. For example, does the altered composition of the gut microbiota in IBD precede disease development or do factors such as immune dysregulation and/or the harsh inflammatory environment lead to dysbiosis? It is also possible that a combination of these factors are involved, however, mechanistic studies in humans are generally lacking. Twin studies have provided some insight. Noteably, a study of twin pairs discordant for UC revealed a reduction in gut microbiota diversity in the healthy twin similar to the changes seen the affected twin suggesting that alterations in the intestinal microbiota may precede the development of disease.86 Given the potential causal role of the gut microbiota in the pathogenesis of IBD, targeting the microbiota is an attractive therapeutic approach. Unfortunately, evidence for the efficacy of probiotics in the treatment of IBD is currently equivocal.87 There may be some efficacy in UC, particularly in patients who have pouchitis, where probiotics can be an effective strategy to prevent relapse after successful antibiotic treatment.87 A recent
Cochrane review also suggested a potential role for treatment with a particular probiotic, VSL#3, in maintenance of remission of pouchitis and also in prevention of pouchitis following ileal-pouch anal anastomosis for UC.88 Similarly, the evidence for the effectiveness of antibiotics in the treatment of patients with IBD has historically not been robust. However, within the past several years, 2 meta-analyses of randomized controlled trials have documented a small but statistically significant benefit of antibiotics to induce remission in both CD and UC.89,90 In addition, several studies have now shown that antibiotic combination therapy significantly improves rates of remission and also steroid withdrawal in UC.91-93 Fecal microbiota transplantation (FMT) is another microbiota-based therapy that involves collecting stool from a healthy donor, preparing it in one of several ways, and transferring it to a patient with a disease or dysbiotic condition. FMT is known to be a successful therapy for the treatment of C difficile-infection,94 which has led to the hypothesis that perhaps FMT could be successful in other dysbiotic conditions, such as IBD. In pediatric IBD, the use of FMT has shown clinical benefit for a small cohort of 7 out of 9 subjects with CD through nasogastric administration95 but not for subjects with UC.96 As with most pediatric therapies, the long-term consequences of FMT are unknown and should be better understood before implementing in conventional practice. As mechanisms by which the gut microbiota play a role in the pathogenesis of IBD are better elucidated, in turn, this may lead to novel, microbial-based treatment strategies.
Conclusions Thanks to advances in deep sequencing technology and bioinformatics tools allowing for the analysis of large datasets, the importance of the microbiota in maintaining health and its relationship to disease is becoming increasingly recognized. We are also learning more about the importance of the microbiota in the pediatric population in terms of the establishment of a healthy microbiota and how early life exposures may contribute to disease, particularly diseases, such as obesity, atopic disorders, and the inflammatory bowel diseases, which have been rapidly increasing in incidence. Now we are at a crossroads where we need to move from descriptive, crosssectional studies to mechanistic studies, which take into account microbiota structure and function. Indeed, current associations between the microbiota and human disease demonstrate “proof of principle,” but do not prove cause-andeffect. To begin to demonstrate causation, there is a need for additional carefully designed studies in human subjects in whom clinical metadata is extensively collected together with continued investigations in animal models in the era of rapidly advancing broad-based technologies such as DNA sequencing, transcriptomics, proteomics, and metabolomics. It is also critical to design randomized, controlled clinical trials evaluating therapeutic efficacy of microbiota-based interventions, such as prebiotics, probiotics, synbiotics, and antibiotics in
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THE JOURNAL OF PEDIATRICS • www.jpeds.com various disease states while paying special attention to adverse events and effects on community structure in the long term. Future studies may lead to novel therapeutic strategies, as well as help to determine whether the microbiota can be permanently altered in a way that is beneficial to the host recognizing that the window of opportunity may be early in life in our pediatric population. ■
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Submitted for publication Mar 7, 2016; last revision received Jul 5, 2016; accepted Aug 10, 2016 Reprint requests: Lindsey Albenberg, DO, Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital Of Philadelphia, 3401 Civic Center Blvd, Philadelphia, PA 19104. E-mail: [email protected]
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tiple environmental and host genetic factors. Proc Natl Acad Sci USA 2010;107:18933-8. Vangay P, Ward T, Gerber JS, Knights D. Antibiotics, pediatric dysbiosis, and disease. Cell Host Microbe 2015;17:553-64. Houghteling PD, Walker WA. Why is initial bacterial colonization of the intestine important to infants’ and children’s health? J Pediatr Gastroenterol Nutr 2015;60:294-307. Ogden CL, Carroll MD, Kit BK, Flegal KM. Prevalence of childhood and adult obesity in the United States, 2011-2012. JAMA 2014;311:806-14. Guo SS, Roche AF, Chumlea WC, Gardner JD, Siervogel RM. The predictive value of childhood body mass index values for overweight at age 35 y. Am J Clin Nutr 1994;59:810-9. Dietz WH. Health consequences of obesity in youth: childhood predictors of adult disease. Pediatrics 1998;101(3 Pt 2):518-25. Ajslev TA, Andersen CS, Gamborg M, Sorensen TI, Jess T. Childhood overweight after establishment of the gut microbiota: the role of delivery mode, pre-pregnancy weight and early administration of antibiotics. Int J Obes 2011;35:522-9. Arenz S, Ruckerl R, Koletzko B, von Kries R. Breast-feeding and childhood obesity – a systematic review. Int J Obes Relat Metab Disord 2004;28:1247-56. Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. Obesity alters gut microbial ecology. Proc Natl Acad Sci USA 2005;102:11070-5. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006;444:1027-31. Murphy R, Stewart AW, Braithwaite I, Beasley R, Hancox RJ, Mitchell EA, et al. Antibiotic treatment during infancy and increased body mass index in boys: an international cross-sectional study. Int J Obes 2014;38:11159. Trasande L, Blustein J, Liu M, Corwin E, Cox LM, Blaser MJ. Infant antibiotic exposures and early-life body mass. Int J Obes 2013;37:16-23. Nobel YR, Cox LM, Kirigin FF, Bokulich NA, Yamanishi S, Teitler I, et al. Metabolic and metagenomic outcomes from early-life pulsed antibiotic treatment. Nat Commun 2015;6:7486. Scheepers LE, Penders J, Mbakwa CA, Thijs C, Mommers M, Arts IC. The intestinal microbiota composition and weight development in children: the KOALA Birth Cohort Study. Int J Obes 2015;39:16-25. Eyerich K, Eyerich S, Biedermann T. The multi-modal immune pathogenesis of atopic eczema. Trends Immunol 2015;36:788-801. Azad MB, Konya T, Maughan H, Guttman DS, Field CJ, Sears MR, et al. Infant gut microbiota and the hygiene hypothesis of allergic disease: impact of household pets and siblings on microbiota composition and diversity. Allergy Asthma Clin Immunol 2013;9:15. Panzer AR, Lynch SV. Influence and effect of the human microbiome in allergy and asthma. Curr Opin Rheumatol 2015;27:373-80. Bager P, Wohlfahrt J, Westergaard T. Caesarean delivery and risk of atopy and allergic disease: meta-analyses. Clin Exp Allergy 2008;38:634-42. Watanabe S, Narisawa Y, Arase S, Okamatsu H, Ikenaga T, Tajiri Y, et al. Differences in fecal microflora between patients with atopic dermatitis and healthy control subjects. J Allergy Clin Immunol 2003;111:587-91. Penders J, Thijs C, van den Brandt PA, Kummeling I, Snijders B, Stelma F, et al. Gut microbiota composition and development of atopic manifestations in infancy: the KOALA Birth Cohort Study. Gut 2007;56:6617. West CE, Ryden P, Lundin D, Engstrand L, Tulic MK, Prescott SL. Gut microbiome and innate immune response patterns in IgE-associated eczema. Clin Exp Allergy 2015;45:1419-29. Trompette A, Gollwitzer ES, Yadava K, Sichelstiel AK, Sprenger N, NgomBru C, et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat Med 2014;20:159-66. Fujimura KE, Lynch SV. Microbiota in allergy and asthma and the emerging relationship with the gut microbiome. Cell Host Microbe 2015;17:592602. Josefowicz SZ, Niec RE, Kim HY, Treuting P, Chinen T, Zheng Y, et al. Extrathymically generated regulatory T cells control mucosal TH2 inflammation. Nature 2012;482:395-9.
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42. Elazab N, Mendy A, Gasana J, Vieira ER, Quizon A, Forno E. Probiotic administration in early life, atopy, and asthma: a meta-analysis of clinical trials. Pediatrics 2013;132:e666-76. 43. Cuello-Garcia CA, Brozek JL, Fiocchi A, Pawankar R, Yepes-Nunez JJ, Terracciano L, et al. Probiotics for the prevention of allergy: a systematic review and meta-analysis of randomized controlled trials. J Allergy Clin Immunol 2015;136:952-61. 44. Charlson ES, Bittinger K, Chen J, Diamond JM, Li H, Collman RG, et al. Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts. PLoS ONE 2012;7:e42786. 45. Brown PS, Pope CE, Marsh RL, Qin X, McNamara S, Gibson R, et al. Directly sampling the lung of a young child with cystic fibrosis reveals diverse microbiota. Ann Am Thorac Soc 2014;11:1049-55. 46. Chmiel JF, Aksamit TR, Chotirmall SH, Dasenbrook EC, Elborn JS, LiPuma JJ, et al. Antibiotic management of lung infections in cystic fibrosis. I. The microbiome, methicillin-resistant Staphylococcus aureus, gram-negative bacteria, and multiple infections. Ann Am Thorac Soc 2014;11:1120-9. 47. Coburn B, Wang PW, Diaz Caballero J, Clark ST, Brahma V, Donaldson S, et al. Lung microbiota across age and disease stage in cystic fibrosis. Sci Rep 2015;5:10241. 48. Hampton TH, Green DM, Cutting GR, Morrison HG, Sogin ML, Gifford AH, et al. The microbiome in pediatric cystic fibrosis patients: the role of shared environment suggests a window of intervention. Microbiome 2014;2:14. 49. Prevaes SM, de Winter-de Groot KM, Janssens HM, de Steenhuijsen Piters WA, Tramper-Stranders GA, Wyllie AL, et al. Development of the nasopharyngeal microbiota in infants with cystic fibrosis. Am J Respir Crit Care Med 2016;193:504-15. 50. Duytschaever G, Huys G, Bekaert M, Boulanger L, De Boeck K, Vandamme P. Cross-sectional and longitudinal comparisons of the predominant fecal microbiota compositions of a group of pediatric patients with cystic fibrosis and their healthy siblings. Appl Environ Microbiol 2011;77:801524. 51. Scanlan PD, Buckling A, Kong W, Wild Y, Lynch SV, Harrison F. Gut dysbiosis in cystic fibrosis. J Cyst Fibros 2012;11:454-5. 52. Duytschaever G, Huys G, Bekaert M, Boulanger L, De Boeck K, Vandamme P. Dysbiosis of bifidobacteria and Clostridium cluster XIVa in the cystic fibrosis fecal microbiota. J Cyst Fibros 2013;12:206-15. 53. Madan JC, Koestler DC, Stanton BA, Davidson L, Moulton LA, Housman ML, et al. Serial analysis of the gut and respiratory microbiome in cystic fibrosis in infancy: interaction between intestinal and respiratory tracts and impact of nutritional exposures. MBio 2012;3. pii: e00251-12. doi: 10.1128/mBio.00251-12. 54. Hoen AG, Li J, Moulton LA, O’Toole GA, Housman ML, Koestler DC, et al. Associations between gut microbial colonization in early life and respiratory outcomes in cystic fibrosis. J Pediatr 2015;167:138-47, e1-3. 55. Sartor RB. Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat Clin Pract Gastroenterol Hepatol 2006;3:390407. 56. Rath HC, Herfarth HH, Ikeda JS, Grenther WB, Hamm TE Jr, Balish E, et al. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats. J Clin Invest 1996;98:945-53. 57. Sartor RB. Microbial influences in inflammatory bowel diseases. Gastroenterology 2008;134:577-94. 58. Rutgeerts P, Hiele M, Geboes K, Peeters M, Penninckx F, Aerts R, et al. Controlled trial of metronidazole treatment for prevention of Crohn’s recurrence after ileal resection. Gastroenterology 1995;108:1617-21. 59. Rutgeerts P, Van Assche G, Vermeire S, D’Haens G, Baert F, Noman M, et al. Ornidazole for prophylaxis of postoperative Crohn’s disease recurrence: a randomized, double-blind, placebo-controlled trial. Gastroenterology 2005;128:856-61. 60. Sachar DB. Management of acute, severe ulcerative colitis. J Dig Dis 2012;13:65-8. 61. Harper PH, Lee EC, Kettlewell MG, Bennett MK, Jewell DP. Role of the faecal stream in the maintenance of Crohn’s colitis. Gut 1985;26:27984.
62. Rutgeerts P, Goboes K, Peeters M, Hiele M, Penninckx F, Aerts R, et al. Effect of faecal stream diversion on recurrence of Crohn’s disease in the neoterminal ileum. Lancet 1991;338:771-4. 63. Jostins L, Ripke S, Weersma RK, Duerr RH, McGovern DP, Hui KY, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012;491:119-24. 64. Kostic AD, Xavier RJ, Gevers D. The microbiome in inflammatory bowel disease: current status and the future ahead. Gastroenterology 2014;146:1489-99. 65. Vanderploeg R, Panaccione R, Ghosh S, Rioux K. Influences of intestinal bacteria in human inflammatory bowel disease. Infect Dis Clin North Am 2010;24:977-93, ix. 66. Van de Merwe JP, Schroder AM, Wensinck F, Hazenberg MP. The obligate anaerobic faecal flora of patients with Crohn’s disease and their firstdegree relatives. Scand J Gastroenterol 1988;23:1125-31. 67. Walker AW, Sanderson JD, Churcher C, Parkes GC, Hudspith BN, Rayment N, et al. High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and noninflamed regions of the intestine in inflammatory bowel disease. BMC Microbiol 2011;11:7. 68. Manichanh C, Rigottier-Gois L, Bonnaud E, Gloux K, Pelletier E, Frangeul L, et al. Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach. Gut 2006;55:205-11. 69. Gophna U, Sommerfeld K, Gophna S, Doolittle WF, Veldhuyzen van Zanten SJ. Differences between tissue-associated intestinal microfloras of patients with Crohn’s disease and ulcerative colitis. J Clin Microbiol 2006;44:4136-41. 70. Frank DN, St Amand AL, Feldman RA, Boedeker EC, Harpaz N, Pace NR. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc Natl Acad Sci USA 2007;104:13780-5. 71. Martinez-Medina M, Aldeguer X, Gonzalez-Huix F, Acero D, GarciaGil LJ. Abnormal microbiota composition in the ileocolonic mucosa of Crohn’s disease patients as revealed by polymerase chain reactiondenaturing gradient gel electrophoresis. Inflamm Bowel Dis 2006;12:113645. 72. Prescott NJ, Fisher SA, Franke A, Hampe J, Onnie CM, Soars D, et al. A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn’s disease and is independent of CARD15 and IBD5. Gastroenterology 2007;132:1665-71. 73. Swidsinski A, Loening-Baucke V, Vaneechoutte M, Doerffel Y. Active Crohn’s disease and ulcerative colitis can be specifically diagnosed and monitored based on the biostructure of the fecal flora. Inflamm Bowel Dis 2008;14:147-61. 74. Seksik P, Rigottier-Gois L, Gramet G, Sutren M, Pochart P, Marteau P, et al. Alterations of the dominant faecal bacterial groups in patients with Crohn’s disease of the colon. Gut 2003;52:237-42. 75. Baumgart M, Dogan B, Rishniw M, Weitzman G, Bosworth B, Yantiss R, et al. Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum. ISME J 2007;1:403-18. 76. Sartor RB. Therapeutic correction of bacterial dysbiosis discovered by molecular techniques. Proc Natl Acad Sci USA 2008;105:16413-4. 77. Mangin I, Bonnet R, Seksik P, Rigottier-Gois L, Sutren M, Bouhnik Y, et al. Molecular inventory of faecal microflora in patients with Crohn’s disease. FEMS Microbiol Ecol 2004;50:25-36. 78. Feller M, Huwiler K, Stephan R, Altpeter E, Shang A, Furrer H, et al. Mycobacterium avium subspecies paratuberculosis and Crohn’s disease: a systematic review and meta-analysis. Lancet Infect Dis 2007;7:60713. 79. Chehoud C, Albenberg LG, Judge C, Hoffmann C, Grunberg S, Bittinger K, et al. Fungal signature in the gut microbiota of pediatric patients with inflammatory bowel disease. Inflamm Bowel Dis 2015;21: 1948-56. 80. Sokol H, Leducq V, Aschard H, Pham HP, Jegou S, Landman C, et al. Fungal microbiota dysbiosis in IBD. Gut 2016 Feb 3. pii: gutjnl-2015310746. doi:10.1136/gutjnl-2015-310746 [Epub ahead of print].
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THE JOURNAL OF PEDIATRICS • www.jpeds.com 81. Wagner J, Maksimovic J, Farries G, Sim WH, Bishop RF, Cameron DJ, et al. Bacteriophages in gut samples from pediatric Crohn’s disease patients: metagenomic analysis using 454 pyrosequencing. Inflamm Bowel Dis 2013;19:1598-608. 82. Norman JM, Handley SA, Baldridge MT, Droit L, Liu CY, Keller BC, et al. Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell 2015;160:447-60. 83. Wang W, Jovel J, Halloran B, Wine E, Patterson J, Ford G, et al. Metagenomic analysis of microbiome in colon tissue from subjects with inflammatory bowel diseases reveals interplay of viruses and bacteria. Inflamm Bowel Dis 2015;21:1419-27. 84. Gevers D, Kugathasan S, Denson LA, Vazquez-Baeza Y, Van Treuren W, Ren B, et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 2014;15:382-92. 85. Kronman MP, Zaoutis TE, Haynes K, Feng R, Coffin SE. Antibiotic exposure and IBD development among children: a population-based cohort study. Pediatrics 2012;130:e794-803. 86. Lepage P, Hasler R, Spehlmann ME, Rehman A, Zvirbliene A, Begun A, et al. Twin study indicates loss of interaction between microbiota and mucosa of patients with ulcerative colitis. Gastroenterology 2011;141:22736. 87. Sanders ME, Guarner F, Guerrant R, Holt PR, Quigley EM, Sartor RB, et al. An update on the use and investigation of probiotics in health and disease. Gut 2013;62:787-96. 88. Singh S, Stroud AM, Holubar SD, Sandborn WJ, Pardi DS. Treatment and prevention of pouchitis after ileal pouch-anal anastomosis for chronic ulcerative colitis. Cochrane Database Syst Rev 2015;11:CD001176.
Volume ■■ 89. Khan KJ, Ullman TA, Ford AC, Abreu MT, Abadir A, Marshall JK, et al. Antibiotic therapy in inflammatory bowel disease: a systematic review and meta-analysis. Am J Gastroenterol 2011;106:661-73. 90. Wang SL, Wang ZR, Yang CQ. Meta-analysis of broad-spectrum antibiotic therapy in patients with active inflammatory bowel disease. Exp Ther Med 2012;4:1051-6. 91. Terao S, Yamashiro K, Tamura I, Hirano T, Ohkusa T, Kato K. Antibiotic combination therapy for steroid withdrawal in steroid-dependent ulcerative colitis. Digestion 2011;83:198-203. 92. Ohkusa T, Kato K, Terao S, Chiba T, Mabe K, Murakami K, et al. Newly developed antibiotic combination therapy for ulcerative colitis: a double-blind placebo-controlled multicenter trial. Am J Gastroenterol 2010;105:1820-9. 93. Uehara T, Kato K, Ohkusa T, Sugitani M, Ishii Y, Nemoto N, et al. Efficacy of antibiotic combination therapy in patients with active ulcerative colitis, including refractory or steroid-dependent cases. J Gastroenterol Hepatol 2010;25(Suppl 1):S62-6. 94. Gough E, Shaikh H, Manges AR. Systematic review of intestinal microbiota transplantation (fecal bacteriotherapy) for recurrent Clostridium difficile infection. Clin Infect Dis 2011;53:994-1002. 95. Obih C, Wahbeh G, Lee D, Braly K, Giefer M, Shaffer ML, et al. Specific carbohydrate diet for pediatric inflammatory bowel disease in clinical practice within an academic IBD center. Nutrition 2016;32:41825. 96. Suskind DL, Singh N, Nielson H, Wahbeh G. Fecal microbial transplant via nasogastric tube for active pediatric ulcerative colitis. J Pediatr Gastroenterol Nutr 2015;60:27-9.
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Advances in Gut Microbiome Research and Relevance to Pediatric Diseases Lindsey Albenberg, DO, and Judith Kelsen, MD
T
he human microbiota is the collection of microorganisms that reside within and on the surface of humans, and the term “human microbiome” refers to the genes that these microorganisms harbor. Although the human bacterial microbiome is the most commonly described, all 3 kingdoms of life, Archaea, Bacteria, and Eukarya are represented within this dense community. There are distinctive microbial communities among body sites, but by far, the vastest population of microorganisms resides within our gastrointestinal tracts. An estimated 1013 individual bacteria belonging to over 1000 species reside in the mammalian gastrointestinal tract, making it the most densely populated microbial community on Earth.1 Indeed, the collective genome of the human gut microbiome is predicted to be 100-fold greater than that of its human host.2 Although there are over 50 bacterial phyla on Earth, the majority of the bacteria in the human gut belong to 1 of 4 phyla, Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes. As humans, we have evolved to live in a mutualistic relationship with our microbiota. In fact, humans should be viewed as a biologic “supraorganism” where we provide an essential niche for our microbiota and in turn, our microbiota carry out critical physiological functions. For example, within the gut, bacteria participate in fermentation of indigestible carbohydrates to produce short chain fatty acids that are used by the host, biotransformation of conjugated bile acids, synthesis of certain vitamins, degradation of dietary oxalates, hydrolysis of urea by urease activity that participates in host nitrogen balance, and education of the mucosal immune system.3 Indeed, it was not very long ago when the term “gut flora” denoted an obscure and relatively unexplored space. However, advances in genome sequencing technologies and metagenomic analysis methods have led to numerous discoveries regarding community membership and the relationship between the gut microbiota and human health. Subsequently, it has become apparent that aberrations in the composition and functions of the gut microbiota may potentially be fundamental to the development of certain diseases, many of which are very relevant to pediatrics.
CD CF FMT IBD IgE IL iTregs UC
Crohn’s disease Cystic fibrosis Fecal microbiota transplantation Inflammatory bowel diseases Immunoglobulin E Interleukin Induced Tregs Ulcerative colitis
Technological Advances in Gut Microbiome Research Most species of gut-residing organisms are obligate anaerobes, many of which are fastidious and difficult to grow in vitro making traditional culture techniques of limited value in characterizing the composition of the gut microbiota. The development of culture-independent methods, mainly through the use of high-throughput DNA sequencing, has provided a novel means to evaluate the gut microbiota and its relationship to disease. There are 2 primary methods that use deep-sequencing technologies to characterize the microbiome. The first approach, uses small-subunit ribosomal RNA (16S ribosomal RNA) gene sequences (for Archaea and Bacteria), or internal transcribed spacer gene sequences (for Eukaryotes) as stable phylogenetic markers to define the lineages present in a sample.4 Another approach uses shotgun metagenomic sequencing in which the total community DNA is sequenced, thereby allowing for the microbial community structure and genomic representation of the community to be evaluated. The genomic community evaluation provides an understanding of the functions encoded by the genomes of the gut microbiota. 5 Metatranscriptomics and metaproteomics provide a deeper understanding of microbial function through direct evaluation of gene expression.6 These advances in sequencing technologies have allowed investigators to characterize the bacterial composition of the gut throughout different stages of life, a critical step in the study of health and disease.
Development of the Infant Microbiome and the Effect of Early Life Exposures It was once thought that the healthy human fetus develops in an environment that is completely sterile.7 However, there is an evolving body of literature suggesting that even the human placenta harbors a low-abundance commensal (nonpathologic) microbiota with potential biological relevance.8 For example, there may be an association between the composition of the placental microbiota and birth weight in full-term neonates.9 However, this needs to be further explored. Still, it is generally accepted that significant colonization of the infant does
From the Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, PA The authors declare no conflicts of interest. 0022-3476/$ - see front matter. © 2016 Elsevier Inc. All rights reserved. http://dx.doi.org10.1016/j.jpeds.2016.08.044
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THE JOURNAL OF PEDIATRICS • www.jpeds.com not occur until the time of delivery when infants are rapidly exposed to an abundance of organisms.10 There is increasing knowledge regarding the colonization succession of the infant gut and its relevance to health and disease. However, the initial colonization of other body sites remains relatively unexplored. In a study of 10 infants who underwent sampling across multiple body sites within 24 hours of delivery, the microbiota seemed to be homogenously distributed across skin, oral, and gut habitats.10 In contrast, their mothers harbored distinctly different communities at various body sites.10 The initial intestinal colonization pattern depends upon mode of delivery.7 Infants born vaginally are initially colonized by bacterial taxa found in the vagina, such as Lactobacillus and Prevotella, whereas infants who are born by cesarean delivery are initially colonized by bacteria found in the skin microbiota.10 After this primary inoculation, infants are regularly exposed to microbes, and diversity increases rapidly.11 Diversity is a term used to describe the microbial community in terms of richness, or number of microbial species present and also evenness, the proportions in which each species is represented. High diversity has been associated with health and low diversity has been associated with various disease states.12 This highlights the importance of this process of attaining diversity early in life as it likely has important implications for future health. It is also important to recognize that studies of humans around the world have consistently demonstrated that inhabitants of developed countries harbor microbial communities with reduced diversity. This has led to the hypothesis that loss of diversity is secondary to“Westernization”and may be involved in the pathogenesis of various chronic diseases, which have been rapidly increasing in incidence in developing countries.12 In infants, the initial colonization pattern is thought to be chaotic, and a growing body of literature has shown that environmental exposures early in life, including diet, are responsible for these fluctuations. Characterization of the intestinal microbiota in a single infant, over a period of 2.5 years, showed how the bacterial taxa changed with life events, such as illnesses, dietary changes, and antibiotic treatment. Interestingly, the greatest change in the composition of the infant’s intestinal microbiota occurred with the introduction of solid foods. There was also a shift toward a more stable, adult-like microbiota with weaning.11 This particular finding was replicated in a subsequent analysis of fecal samples from 98 fullterm infants where cessation of breastfeeding seemed to drive the transition to a more mature gut microbiome.13 Ultimately, the intestinal microbiota of the young resembles that of the adult by approximately the age of 3 years.14 Within the first year of life, there are significant interindividual differences in the composition of the intestinal microbiota, yet some similarities exist. Similarities among individual infants can be attributed to the major taxonomic groups associated with the infant diet. Multiple studies have established differences in the composition of the intestinal microbiota based on whether infants are breastfed or formula fed.15-17 Indeed, this introduces the concept of a potential association between infant diets, the composition of the intestinal microbiota, and health.
Volume ■■ Delivery mode and dietary factors are clearly not the only determinants of early gut colonization pattern. Genetics18 and other environmental exposures, such as antibiotic usage,19 also play a role. It is clear that appropriate colonization of the infant gut is important for education of the mucosal immune system and optimal gut function.20 Thus, it is logical to think that abnormal microbial exposures leading to dysbiosis, or an abnormal composition of the gut microbiota, play a role in the development of pediatric diseases.
The Relationship between the Gut Microbiome and Pediatric Diseases Pediatric Obesity The prevalence of pediatric obesity has increased in recent decades and represents a serious public health concern. In 20112012, approximately 12.7 million children and adolescents in the US were considered obese.21 Many obese children go on to become obese adults22 who have significant comorbidities such as diabetes, hypertension, atherosclerosis, and certain types of cancer, such as colorectal cancer. There are also, of course, more immediate health consequences for obese children and teens, such as hypertension and dyslipidemia.23 Therefore, it is important to understand underlying biological factors that may be related to the development of obesity, including factors that may be important early in life. For example, rapid infant weight gain in the first year of life has been associated with the development of obesity, and many factors related to weight gain in the first year of life have been identified including maternal obesity, mode of delivery, and early exposure to antibiotics.24 Dietary factors are also thought to be important.25 This raises the question of whether these factors are influencing a single mechanism, namely the development of the infant gut microbiota. The presence of an altered gut microbiota composition in obese individuals compared with lean individuals has been well established in both animal and human studies. This finding was first described by Ley et al26 who reported a decreased abundance of Bacteriodetes and a proportional increased abundance of Firmicutes in the cecal contents of genetically obese mice relative to their lean counterparts. The difference was independent of kinship and sex and importantly, all animals were fed the same diet. A similar shift in the abundance of Bacteriodetes and Firmicutes in the intestinal tract was subsequently demonstrated in obese adults compared with lean, healthy controls.27 Interestingly, these changes seemed modifiable as the relative abundance of Bacteriodetes increased as obese individuals lost weight on a low-calorie diet.26 One important function of the gut microbiota is degradation of otherwise indigestible components of our diet, thus, playing a role in energy balance. As mentioned previously, an example of this is fermentation of indigestible carbohydrates to produce short chain fatty acids. Thus, Turnbaugh et al27 sought to examine whether the microbiota of obese mice were more efficient at extracting energy than the microbiota of lean mice. Indeed, when germ-free mice were inoculated with a microbiota
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harvested from the cecum of obese mice, there was a significantly greater percentage increase in body fat over a 14-day period than when germ-free mice were inoculated with a lean donor microbiota. This difference occurred in the setting of identical calorie consumption among the 2 groups. It is very clear that calorie consumption is critical in the development and treatment of human obesity. However, the development of obesity likely involves increased calorie consumption plus an increased capacity of the microbiota to harvest energy from the diet, and this latter characteristic seems to be transmissible. The knowledge of the potential importance of energy harvest by gut microbes has led to the exploration of the hypothesis that perhaps antibiotics early in life alter the composition of the gut microbiota in a way that alters energy balance and promotes obesity. In fact, multiple epidemiologic studies have established a relationship between antibiotic exposures during infancy with increases in body mass index.24,28,29 For example, as part of the Avon Longitudinal Study of Parents and Children in the United Kingdom,29 exposures to antibiotics during 3 different early-life time windows and the relationship of the exposures to body mass were examined. In this populationbased study of more than 10 000 infants, treatment with antibiotics within the first 6 months of life was associated with increased body mass at 10-38 months of age.29 A mouse model of pediatric antibiotic use (repeated courses of beta-lactams or macrolides at therapeutic dosages) recently suggested a relationship between early life antibiotic exposure, alterations in gut microbiota diversity, and changes in body composition and growth.30 Pulsed antibiotic treatment also led to changes in hepatic gene expression and led to reduced levels of the hormone ghrelin, and these changes persisted even once antibiotics were discontinued.30 It is likely that other environmental factors in infancy contribute to dysbiosis conferring increased risk of obesity, and there is early data from the large, prospective child, parents, and health birth cohort to suggest that early colonization with a particular bacterial taxon, Bacteroides fragilis, may be associated with higher body mass index in children up to 10 years of age.31 With advances in our understanding of the structure and functions of the gut microbiota in infancy and its relationship to obesity, it does not seem implausible that one day the gut microbiota could be manipulated at an early age to prevent obesity. Disorders of the Skin and Atopic Disorders Atopic eczema is one of the most common inflammatory diseases, with increasing prevalence of 2%-3% in the adult population and 10%-30% in infants. It is often associated with other allergic diseases such as asthma, eosinophilic esophagitis, and allergic rhinitis. The presentation can be acute or patients may have recurrent symptoms characterized by erythema, papules, and scaly plaques that can have associated pruritus.32 As with many of the other diseases described in this review, the etiology of atopy is complex, because of an aberrant immune response to environmental factors in the genetically susceptible host. The prominent role of the gut microbiome is supported by the increase in the incidence of atopic disease as-
sociated with residence in or immigration to industrialized nations.33 In addition, the “hygiene hypothesis” suggests that humans living in more industrialized societies are exposed to fewer microbes or less complex microbial communities at an early age. Subsequently, the developing immune system is less able to “tolerate” exposure to the microbial-laden environment and results in an inappropriate immune activation.34 The importance of early life exposures has also been suggested with at least 1 meta-analysis demonstrating an association between delivery by cesarean and a moderate risk increase for allergic rhinitis and asthma.35 Human and animal models have been used to study the role of the gut microbiome in the development of atopy. For example, alterations of the gut microbiome have been demonstrated in children and young adults with eczema and atopy. Patients were shown to have a decreased relative abundance of Bifidobacteria in the feces compared with controls, and this was more pronounced in severe cases.36 Furthermore, an increased relative abundance of Clostridium and decreased relative abundance of Bifidobacteria has been demonstrated in the feces of infants with atopy at 3 weeks of age compared with healthy controls.37 West et al38 similarly found a low relative abundance of Ruminococcaceae at 1 week of age in infants who developed immunoglobulin E (IgE)-associated eczema. Murine models of atopy have demonstrated dysregulation of the IgE-basophil axis secondary to failure of the development of the gut commensal population.39 IgE has been associated with type 1 hypersensitivity anaphylactic reactions and is also responsible for binding mast cells, eosinophils, and basophils characteristic of other atopic conditions. Immune dysregulation can also be see through an exaggerated proinflammatory Th2 response with dominance of cytokine response characterized by interleukin (IL)-4 and IL-13.37 These cytokines are responsible for the keratinocytes in atopy with reduced antimicrobial peptides levels secondary to the Th2 response. Furthermore, T regulatory cells (Tregs) produced in the periphery are known as induced Tregs (iTreg). They are stimulated in the mesenteric lymph nodes, Peyer patches, and colonic lamina propria of mice.40 Interestingly, mice deficient in iTregs spontaneously develop Th2-type response with increase in CD4+T cells, with the production of Il-4, Il-13, and IL-5 cytokines in the mesenteric lymph nodes and both small and large intestine.41 These iTregs deficient animals go on to develop dysbiosis, with a relative increase in the TM7 phylum, and a macrophage and neutrophil infiltration of the airways with allergic phenotype.41 Together, these studies provide evidence for the existence of a gut-allergy axis, and potentially the opportunity to develop strategies aimed at manipulating the gut microbiota in early stages of immune development to prevent the onset of atopy and eczema. One potential preventative strategy could include treatment with probiotic bacterial strains very early on in life, such as in pregnant or breastfeeding mothers or infants. Indeed, results from clinical trials have suggested a role for early life probiotic administration to prevent allergic diseases.42 However, these trials have generally been underpowered and have demonstrated inconsistent results,43 thus, further studies are needed.
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THE JOURNAL OF PEDIATRICS • www.jpeds.com Disorders of the Respiratory Tract The respiratory tract, particularly the lower respiratory tract, is another body site once thought to be sterile in the healthy state. However, with advances in culture-independent techniques, there is evidence to suggest that even the airways of healthy people contain detectable microbes and that the community composition is altered in chronic disease. Still, compared with the gut, relatively little is known regarding the development of the human respiratory microbiome. This may be at least partially related to difficulty obtaining samples from the respiratory tract. Even samples from bronchoscopy, which would seem to be the most precise, are flawed, as the bronchoscope needs to pass through the oropharynx, thus, creating inevitable contamination. However, advanced analytical methods have been proposed to distinguish upper respiratory tract and lower respiratory tract taxa.44 The lung microbiota is now thought to be quite diverse with distinct communities in the upper and lower tracts.45 Overall, our knowledge of the lung microbiota comes from a small number of studies in both children and adults. With technological advances in microbiota analysis, there has been increased interest in the microbial communities in patients with cystic fibrosis (CF). Patients with CF are known to have viscous secretions and chronic airway obstruction, which leads to colonization with pathogenic bacteria. Antimicrobial activities are also compromised.46 Also, traditional culture results and response to antibiotics correlate poorly.46 Dozens of studies have now used advanced methods to attempt to identify microbes, not identified by clinical cultures, which may contribute to progression of lung disease. For example, Coburn et al47 evaluated sputum samples from over 200 patients with CF across various ages and disease stages. There was significant interindividual variability, however, a small, shared core microbiota was suggested. Diversity seemed to decrease with age, and this correlated with more severe lung disease. In addition, with increase in age, there was an increase in dominance of CF-associated pathogens Pseudomonas and Burkholderia. Similar findings were also demonstrated in a study of sputum samples from pediatric patients with CF.48 As with other disorders discussed in this review, early life exposures in pediatric patients with CF may have significant implications. There is evidence to suggest that the nasopharyngeal microbial community is different in young infants with CF compared with healthy controls and that antibiotics early in life alter community composition and perhaps increase the abundance of potentially pathogenic bacteria.49 A limited number of studies have also demonstrated alterations in the composition of the gut microbiota in patients with CF compared with healthy controls.50-52 However, the mechanism(s) and the degree to which abnormal intestinal fluid composition, fat malabsorption, and frequent antibiotic exposures contribute to dysbiosis remain to be elucidated. Importantly, at least 2 studies have demonstrated the presence of a core group of microbes that are shared between oropharyngeal and fecal samples from patients with CF.53,54 Gut colonization of a subset of bacterial genera, including those considered to be potential pathogens, may actually precede their appearance in the
Volume ■■ respiratory tract suggesting a close interrelationship between the microbiota in 2 body sites in patients with CF.53 There may even be an association between gut microbial colonization in early life and respiratory outcomes in these patients.54 This is intriguing as it conceivable that in the future engineering of the gut microbiota in patients with CF may have therapeutic implications. Intestinal Disorders–Inflammatory Bowel Diseases The inflammatory bowel diseases (IBD), comprised of Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases of the gastrointestinal tract. They are due to an aberrant immune response to environmental factors in the genetically susceptible host. The gut microbiota is thought to be a critical environmental factor in the development of IBD. Indeed, there is significant evidence to support the role of gut microbes in the development of IBD. Animal studies of IBD have demonstrated that germ-free animals show little sign of inflammation,55 however, inflammation develops with exposure to microbes.56 Adaptive immune responses to bacterial antigens have been shown to lead to the spontaneous development of colitis through immune activation and/or the loss of immune tolerance in various models.57 From a clinical standpoint, inflammation in CD and UC occur predominately in the terminal ileum (in CD) and colon (both UC and CD) where the greatest concentrations of bacteria are found. Antibiotics can have efficacy in the treatement of IBD.58-60 Furthermore, the fecal flow exacerbates IBD and surgical diversion of the flow amerliorates the disease.61,62 Genetic studies have also provided strong support for the role of the microbiota in the development of IBD.63 Multiple studies have now demonstrated that the composition of the gut microbiota is different in patients with IBD, including decreased diversity in patients with IBD, as well as alterations in the relative abundances of certain bacterial taxa.64 In terms of taxonomic changes, both expansion of potentially pathogenic bacteria and changes in microbial composition (ie, increased or decreased abundance of indicator species) has been described. For example, in CD, the phylum Firmicutes is commonly reduced in proportional abundance,65-70 notably, Faecailbacterium prausnitzii.71-73 Members of the Proteobacteria phylum, such as Enterobacteriaceae,74,75 including Escherichia coli70,76,77 are commonly increased. The E coli isolated in CD is often adherent and invasive phenotype. Myobacterium Avium subpecies Paratuberculosis has also been implicated as a potentially pathogenic organism in the development of IBD. It is the known cause of Johne disease in cattle which, similar to the histologic appearance of human CD, leads to a chronic granulomatous enteritis. There have been multiple studies exploring the role of Myobacterium Avium subpecies Paratuberculosis in CD,78 however, controversy remains whether this organism indeed has a causal role. In contrast to the many studies of the bacterial component of the gut microbiota, there is relatively little known about nonbacterial gut microbial inhabitants such as fungi, archaea, viruses, and bacteriophage. However, with advances in sequencing technology and novel techniques to explore these less commonly investigated
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populations, there is increasing evidence of fungal dysbiosis79,80 in patients with IBD, as well as differences in virus and bacteriophage81-83 populations in both pediatric and adult patients with IBD compared with healthy controls. Presently, the techniques available for studying nonbacterial gut microbial inhabitants are somewhat limited. However, the potential importance of gut fungi, archaea, and viruses in IBD and in child health and disease in general should not be discounted. As these techniques become well established, it is likely that important relationships will be demonstrated between these less wellunderstood populations and the disorders mentioned in this review and others. To date, the majority of the studies examining the gut microbiome in pediatric patients with IBD have included patients with established disease without accounting for clinical variables or the potential effects of medications. However, in 2015, Gevers et al84 published results from a large survey of the stool and biopsy-associated microbiome in newly diagnosed, treatment-naïve children with CD, which advanced our understanding of the microbial dysbiosis in new-onset CD. There were many important findings, including the discovery of a subset of bacterial taxa proportionally increased or decreased in abundance in biopsy samples from patients with CD compared with healthy controls. Interestingly, a few additional less commonly described taxa were suggested as potential biomarkers for disease including increased abundances of Pasteurellaceae (Hemophilus sp), Veillonellaceae, Neisseriaceae, and Fusobacteriaceae, and some were present in higher abundance in patients under the age of 10 years compared with older children. In this study, a small subset of patients were taking an antibiotic at the time of sample collection. Despite the small number of subjects, it seemed that concurrent antibiotics intensified the dysbiosis. This is particularly noteable given the potential association between childhood antibiotic exposure and IBD development.85 Despite significant advances in our knowledge of the dysbiotic gut microbiota that is seen in IBD, several questions remain to be answered. In particular, the question of causation vs correlation needs to be further explored. For example, does the altered composition of the gut microbiota in IBD precede disease development or do factors such as immune dysregulation and/or the harsh inflammatory environment lead to dysbiosis? It is also possible that a combination of these factors are involved, however, mechanistic studies in humans are generally lacking. Twin studies have provided some insight. Noteably, a study of twin pairs discordant for UC revealed a reduction in gut microbiota diversity in the healthy twin similar to the changes seen the affected twin suggesting that alterations in the intestinal microbiota may precede the development of disease.86 Given the potential causal role of the gut microbiota in the pathogenesis of IBD, targeting the microbiota is an attractive therapeutic approach. Unfortunately, evidence for the efficacy of probiotics in the treatment of IBD is currently equivocal.87 There may be some efficacy in UC, particularly in patients who have pouchitis, where probiotics can be an effective strategy to prevent relapse after successful antibiotic treatment.87 A recent
Cochrane review also suggested a potential role for treatment with a particular probiotic, VSL#3, in maintenance of remission of pouchitis and also in prevention of pouchitis following ileal-pouch anal anastomosis for UC.88 Similarly, the evidence for the effectiveness of antibiotics in the treatment of patients with IBD has historically not been robust. However, within the past several years, 2 meta-analyses of randomized controlled trials have documented a small but statistically significant benefit of antibiotics to induce remission in both CD and UC.89,90 In addition, several studies have now shown that antibiotic combination therapy significantly improves rates of remission and also steroid withdrawal in UC.91-93 Fecal microbiota transplantation (FMT) is another microbiota-based therapy that involves collecting stool from a healthy donor, preparing it in one of several ways, and transferring it to a patient with a disease or dysbiotic condition. FMT is known to be a successful therapy for the treatment of C difficile-infection,94 which has led to the hypothesis that perhaps FMT could be successful in other dysbiotic conditions, such as IBD. In pediatric IBD, the use of FMT has shown clinical benefit for a small cohort of 7 out of 9 subjects with CD through nasogastric administration95 but not for subjects with UC.96 As with most pediatric therapies, the long-term consequences of FMT are unknown and should be better understood before implementing in conventional practice. As mechanisms by which the gut microbiota play a role in the pathogenesis of IBD are better elucidated, in turn, this may lead to novel, microbial-based treatment strategies.
Conclusions Thanks to advances in deep sequencing technology and bioinformatics tools allowing for the analysis of large datasets, the importance of the microbiota in maintaining health and its relationship to disease is becoming increasingly recognized. We are also learning more about the importance of the microbiota in the pediatric population in terms of the establishment of a healthy microbiota and how early life exposures may contribute to disease, particularly diseases, such as obesity, atopic disorders, and the inflammatory bowel diseases, which have been rapidly increasing in incidence. Now we are at a crossroads where we need to move from descriptive, crosssectional studies to mechanistic studies, which take into account microbiota structure and function. Indeed, current associations between the microbiota and human disease demonstrate “proof of principle,” but do not prove cause-andeffect. To begin to demonstrate causation, there is a need for additional carefully designed studies in human subjects in whom clinical metadata is extensively collected together with continued investigations in animal models in the era of rapidly advancing broad-based technologies such as DNA sequencing, transcriptomics, proteomics, and metabolomics. It is also critical to design randomized, controlled clinical trials evaluating therapeutic efficacy of microbiota-based interventions, such as prebiotics, probiotics, synbiotics, and antibiotics in
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THE JOURNAL OF PEDIATRICS • www.jpeds.com various disease states while paying special attention to adverse events and effects on community structure in the long term. Future studies may lead to novel therapeutic strategies, as well as help to determine whether the microbiota can be permanently altered in a way that is beneficial to the host recognizing that the window of opportunity may be early in life in our pediatric population. ■
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Submitted for publication Mar 7, 2016; last revision received Jul 5, 2016; accepted Aug 10, 2016 Reprint requests: Lindsey Albenberg, DO, Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital Of Philadelphia, 3401 Civic Center Blvd, Philadelphia, PA 19104. E-mail: [email protected]
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THE JOURNAL OF PEDIATRICS • www.jpeds.com 81. Wagner J, Maksimovic J, Farries G, Sim WH, Bishop RF, Cameron DJ, et al. Bacteriophages in gut samples from pediatric Crohn’s disease patients: metagenomic analysis using 454 pyrosequencing. Inflamm Bowel Dis 2013;19:1598-608. 82. Norman JM, Handley SA, Baldridge MT, Droit L, Liu CY, Keller BC, et al. Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell 2015;160:447-60. 83. Wang W, Jovel J, Halloran B, Wine E, Patterson J, Ford G, et al. Metagenomic analysis of microbiome in colon tissue from subjects with inflammatory bowel diseases reveals interplay of viruses and bacteria. Inflamm Bowel Dis 2015;21:1419-27. 84. Gevers D, Kugathasan S, Denson LA, Vazquez-Baeza Y, Van Treuren W, Ren B, et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 2014;15:382-92. 85. Kronman MP, Zaoutis TE, Haynes K, Feng R, Coffin SE. Antibiotic exposure and IBD development among children: a population-based cohort study. Pediatrics 2012;130:e794-803. 86. Lepage P, Hasler R, Spehlmann ME, Rehman A, Zvirbliene A, Begun A, et al. Twin study indicates loss of interaction between microbiota and mucosa of patients with ulcerative colitis. Gastroenterology 2011;141:22736. 87. Sanders ME, Guarner F, Guerrant R, Holt PR, Quigley EM, Sartor RB, et al. An update on the use and investigation of probiotics in health and disease. Gut 2013;62:787-96. 88. Singh S, Stroud AM, Holubar SD, Sandborn WJ, Pardi DS. Treatment and prevention of pouchitis after ileal pouch-anal anastomosis for chronic ulcerative colitis. Cochrane Database Syst Rev 2015;11:CD001176.
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