The developing intestinal microbiome and its relationship to health and disease in the neonate

Abstract

The intestinal microbiota normally exists in a commensal and/or symbiotic relationship with the host. In the past few years, emerging technologies derived largely from the Human Genome Project have been applied to evaluating the intestinal microbiota and new discoveries using these techniques have prompted new initiatives such as the Human Microbiome Roadmap designed to evaluate the role of the intestinal microbiome in health and disease. In this review, we wish to focus on some new developments in this area and discuss some of the effects of medical manipulations such as antibiotics, probiotics, prebiotics and C-section versus vaginal delivery on the intestinal microbiota.

Introduction

The intestinal microbiota normally exists in a commensal and/or symbiotic relationship with the host.1 It is a complex ecosystem shaped by millennia of evolution, with host–bacterial associations that promote a delicate equilibrium with the capability to modulate immune responses and promote health.2, 3 In the past few years, emerging technologies derived largely from the Human Genome Project have been applied to evaluating the intestinal microbiota and new discoveries using these techniques have prompted new initiatives such as the Human Microbiome Roadmap designed to evaluate the role of the intestinal microbiome in health and disease.4, 5

Our current state of knowledge suggests that the neonatal intestinal microbiota is highly variable in its composition and depends on factors such as gestational age, mode of delivery, type of feeding and environmental exposure, as well as on other medical interventions.6, 7, 8 During early development, the microbiota undergoes changes and has major roles in nutrition and in the developing immune system. The nutritional role includes synthesis of vitamin K, short chain fatty acids and biotin. Their role in the developing human immune system is also well known.9, 10, 11, 12

Previous studies have demonstrated that germ-free animals do not develop normal lymph node architecture, with extensive defects in the development of gut-associated lymphoid tissue, arrested capillary network development in the gut and reduced antibody production.11, 12, 13, 14 It is becoming increasingly evident that the microbes of the intestine may be involved in the so-called ‘hygiene hypothesis’ wherein a lack of exposure to a variety of microorganisms, especially during early life, may result in the development of diseases such as type 1 diabetes, asthma, allergies, celiac disease, inflammatory bowel disease and obesity.15, 16, 17, 18 In the case of allergies, a lack of stimulation of Th1-cell responses predisposes to the development of Th2 cell responses and to Th17 cells that induce production of IL-17.19 In this review, we wish to focus on some new developments in this area and discuss some of the effects of medical manipulations such as antibiotics, probiotics, prebiotics and C-section versus vaginal delivery on the intestinal microbiota.

Functions of the intestinal microbiota

In the neonate and especially in the preterm baby the intestinal microbiota has important roles in the metabolism, nutrition, immunological functions and defense against pathogens. Alterations in the microbiota lead to dysbiosis and diseases in the neonate, as well as late in childhood (Table 1). Intestinal bacteria have a key role in promoting the early development of the gut's mucosal immune system, both in terms of its physical components and function, and continue to have a role later in life.20 Bacteria stimulate the gut-associated lymphoid tissue to produce antibodies to pathogens.11, 12, 14, 21 The immune system recognizes and fights harmful bacteria, but does not react against helpful species (as well as the host's own organs)—a tolerance developed in infancy, and sometimes termed the ‘old friends’ hypothesis.22 This hypothesis appears to be a synthesis of the hygiene hypothesis that proposes that these microorganisms that have evolved with humans have an essential role in the establishment of the immune system wherein the microorganisms and the host have evolved a co-dependence.

Table 1 Intestinal microbiota in the neonate

Basic laboratory-based research supplements the epidemiological studies. Recent findings have shown that gut bacteria have a role in the expression of Toll-like receptors (TLRs) in the various intestinal cell types, including the epithelium.23 TLRs are one of the two classes of pattern recognition receptors that provide the intestine the ability to discriminate between pathogenic and commensal bacteria. These pattern recognition receptors identify the pathogens that have crossed the mucosal barriers and trigger a set of responses that take action against the pathogen, which involve three main immunosensory cells—surface enterocytes, M cells and dendritic cells.24 The other classes of pattern recognition receptors are known as the nucleotide-binding oligomerization domain/caspase recruitment domain isoforms, which are cytoplasmic proteins that recognize endogenous or microbial molecules or stress responses and form oligomers that activate inflammatory caspases. Initiating this pathway results in the cleavage and activation of important inflammatory cytokines and/or activation of the nuclear factor-κB signaling pathway with sub-sequent production of inflammatory molecules.24, 25

In the neonate it is important to develop a better understanding of the effects of the intestinal microbiota on secretory IgA, TLRs and other PRRs, as their abundance and activation will greatly determine the development of bacterial invasion and activation of the inflammatory cascade that leads to diseases such as necrotizing enterocolitis (NEC) or systemic inflammation associated with multiple organ dysfunction. The intestinal flora has developed a pattern of recognition or selective ignorance wherein the ‘old friends’ bacteria do not normally cause the activation of this immunological response. Rather, they will elicit tolerance from the host to the bacteria themselves but the host will also exhibit enhanced tolerance to self and thus decrease the likelihood for autoimmunity.22

Bacteria can also influence the phenomenon known as oral tolerance, in which the immune system is less sensitive to an antigen (including those produced by gut bacteria) once it has been ingested. This tolerance, mediated in part by the gastrointestinal immune system and in part by the liver, can reduce an overreactive immune response such as those found in allergies and autoimmune disease.26

Intestinal microecology of the fetus and newborn

During birth and rapidly thereafter, bacteria from the mother and from the surrounding environment colonize the infant's gut. During vaginal delivery, the contact with the mother's vaginal and intestinal flora is an important source for the start of the infant's colonization with a predominance of Lactobacillus, Prevotella and other Bifidobacterium.8, 27 During cesarean delivery, direct contact of the mouth of the newborn with vaginal and intestinal microbiota is absent, and non-maternally derived environmental bacteria have an important role in the infants’ intestinal colonization, which has a less diverse flora and a bacterial community similar to those found on the skin surface dominated by Staphylococcus and with a delayed intestinal colonization by Lactobacillus, Bifidobacterium and Bacteroides.8, 27 Some authors have suggested that the composition of the very first human microbiota could have lasting effects on the intestine.8 Table 2 summarizes the factors that modulate the intestinal microbiota in the neonate. For example, Gronlund et al.28 showed that the primary gut flora in infants born by cesarean delivery may be disturbed for up to 6 months after birth. Another study using culture-based techniques showed that the mode of delivery was associated with differences in intestinal microbes 7 years after delivery.29

Table 2 Factors that modulate the intestinal microbiota in the neonate

Of major importance is the fact that there is accumulating evidence that intestinal microbiota has an important role in the postnatal development of the immune system.30 Thus, if the intestinal flora develops differently depending on the mode of delivery, the postnatal development of the immune system might also be different. Available epidemiological data show that atopic diseases, asthma, type 1 diabetes and food allergies appear more often in infants after cesarean delivery than after vaginal delivery.31, 32, 33, 34 However, most mothers undergoing C-section deliveries also are treated with antibiotics. Studies in adults using non-culture-based analysis of the intestinal microbiota show that antibiotics may perturb the gastrointestinal tract for years.35, 36 Whether these have a role in perturbing the newborn intestinal microecology in C-section versus vaginal delivery remains unknown but could be a confounding factor when one evaluates the epidemiology studies showing higher allergy, asthma, type 1 diabetes and celiac disease in C-section versus vaginally delivered infants. The composition of enteric microbiota in the early days of life thus seems to be a very important factor for achieving and maintaining good health in the years to come. Thus, it is fundamental to identify more thoroughly the intestinal ecosystem during the early developmental stages.

Technological considerations

Most of the current understanding of intestinal colonization in neonates is derived from studies using culture-based techniques. One of the first comprehensive non-culture-based studies of intestinal microbes in 14 healthy term infants, using a ribosomal DNA microarray-based approach, showed that the composition and temporal patterns of the microbial communities varied widely from baby to baby, suggesting a broader definition of ‘healthy colonization’ than previously recognized.37 By 1 year of age, the profile of microbial communities begins to converge toward a profile characteristic of the adult gastrointestinal tract.37 In addition, the composition and temporal patterns of the development of intestinal microbes in a pair of fraternal twins were very similar, suggesting that genetic factors also function to shape intestinal microbial development.37 Another fascinating aspect of this study was the lack of Bifidobacteria detected using these techniques. These are considered to be a major microbe in the use of probiotics and prebiotics, and their absence in this study raises many questions on the importance of Bifidobacteria in normal intestinal development, or a deficiency in the capability of these techniques to detect these microbes.

Although a commonly held belief is that the intestinal tract of the fetus is sterile, recent studies using a combination of culture and non-culture-based techniques suggest that many preterm infants are exposed to microbes found in the amniotic fluid, even without a history of rupture of membranes or culture-positive chorioamnionitis.38 This has led to speculation that the microbial ecology of the swallowed amniotic fluid may have a role not only in the fetal intestinal physiology and inflammation but also perhaps in premature labor.39 Studies of microbial ecology in premature infants using non-culture-based techniques remain few, but this is an area of active investigation. One study used high-throughput 16S-based techniques to analyze intestinal microbial ecology in premature neonates in 23 neonates born at 23–32 weeks’ gestational age.40 Surprisingly, microbial DNA was detected in meconium, suggesting an intrauterine origin. Differences in diversity were detected in infants whose mothers intended to breast feed (but had not yet fed their babies), babies born to mothers with chorioamnionitis and in babies born at <30 weeks’ gestation. A 16S ribosomal RNA sequence analysis detected Citrobacter-like sequences only in cases with NEC (three of four) and an increased frequency of Enterococcus-like sequences in cases and Klebsiella in control subjects.40

In another study, designed to determine differences in microbial patterns that may be critical to the development of NEC, microbial analysis from fecal samples from NEC patients distinctly clustered separately from controls.41 Patients with NEC had less diversity, an increased abundance of Gammaproteobacteria and a decrease in other bacterial species, and had received a higher mean number of antibiotics in previous days. These results, as well as others,42 suggested that NEC is associated with a severe lack of microbiota diversity that may accentuate the impact of single-dominant microorganisms favored by empiric and widespread use of antibiotics.

The use of these non-culture-based technologies will very likely lead to important discoveries in diseases such as NEC and autoimmune diseases and may even lead to clues regarding the etiology of premature labor.

Other manipulations of the gastrointestinal microbial ecology: antibiotics, probiotics and prebiotics

Antibiotics

In the United States, a large number of mothers giving birth prematurely are treated with antibiotics. In addition, most (nearly 90%) very low birth weight infants are treated with a course of broad-spectrum antibiotics. Two studies so far have suggested an increased incidence of NEC related to this practice. Some studies in adults have shown the detrimental effects that a full course of antibiotics has in the intestinal microbiota and microbiome.41, 42 Another study showed a decrease in the diversity of the intestinal microflora and a predominance of ‘less desirable’ bacteria with highly resistant clones as a result of antibiotic exposure that did not recover after 2 years post exposure.35 Further, the abundance of specific resistance genes to antibiotics was greater in those patients treated with antibiotics.35, 36

Probiotics

World Health Organization defines a probiotic as ‘a live microorganism which when administered in adequate amounts confers a health benefit on the host.’ Since Metchnikov suggested the use of live microorganisms to modify gut flora and promote health, it has been recognized that microbial components of certain foods may indeed promote health. Accordingly, over the past two decades there has been an increased interest in the potential benefits accruing from the use of certain types of probiotics. Prospective randomized trials have evaluated the effects of different probiotics on the prevention of NEC.43, 44, 45 Recently, a multicenter trial of probiotics suggested a beneficial preventive effect against NEC, yet with no decrease in NEC-related mortality. However, there was a trend for a higher incidence of sepsis in infants receiving probiotics,44 especially in those with a birthweight <750 g, thus warranting caution, despite recent recommendations for the routine use of probiotics based on a meta-analysis of the current data.45 Furthermore, probiotic products in the United States have not been subjected to rigorous manufacturing quality control or Food and Drug Administration approval. Different probiotics have different mechanisms of action and all of them have not yet been clearly elucidated. Previous studies have shown that some probiotics are more beneficial than others.46, 47 For example, not all probiotics have the same effect when treating acute infectious diarrhea, inflammatory bowel disease or antibiotic-associated diarrhea.48 Probiotics that have shown some beneficial effects are Lactobacillus sp and Bifidobacterium sp, both commonly found in higher proportion in the intestinal microbiota of breast-fed infants compared with formula-fed infants. Probiotics in part are thought to exert their effect by modulating the intestinal microflora, reducing intestinal permeability and decreasing proinflammatory cytokines and increasing anti-inflammatory cytokines. Despite encouraging data of the beneficial effects of some probiotics, more studies are needed to determine which probiotic is appropriate for a given disease or neonatal population. We also need to attain a better understanding of their mechanism of action and also determine the appropriate dose and safety (both short and long term) in the neonate.

Prebiotics

Prebiotics are non-digestible food ingredients such as oligosaccharides that facilitate changes in the composition and or activity in the intestinal microflora with a beneficial effect for the host. Some prebiotic agents include the oligosaccharides inulin, galactose oligosaccharide, fructose oligosaccharide and lactulose. Oligosaccharides are commonly found in human milk.49, 50 Supplementation with prebiotics has been proposed as a strategy to enhance the growth of potentially beneficial intestinal microbes. Although these compounds appear to alter the consistency and frequency of stools, their efficacy in prevention of NEC or other diseases in the newborn is unclear. Human milk oligosaccharides are being proposed as alternatives to the plant-based and synthetic prebiotic agents.51 The theoretical benefit of such preparations has been reviewed;50 however, at this time, not enough information is available to support a benefit in the prevention of NEC or promotion of the overall health in preterm infants.

Microbial components that modulate inflammation

Studies on epithelial cells and in an infant-formula-fed rodent model suggest that dead microbes may be as effective as live microbes in modulating excessive inflammatory stimuli.52, 53, 54 It is also possible that specific microbial components affecting TLR signaling could also be effective.55 Some reports using NEC rodent models suggest that the expression of TLR-4 may be critical in the pathogenesis of NEC.56, 57 Whether low-grade stimulation of these receptors or signaling pathways may induce a tolerance or have a protective effect requires further elucidation. The role and therapeutic potential of pharmaceutical or dietary interventions that may alter accessibility of colonizing bacteria to receptors require additional investigation.

Considerations for the future

Traditionally, the study of intestinal microbiota in the neonate has been conducted using cultured-based techniques. Currently, several fascinating molecular techniques such as denaturing gradient gel electrophoresis, terminal-restriction fragment-length polymorphism and automated ribosomal intergenic spacer analysis are being used for the analysis of intestinal microbial diversity. Real-time quantitative PCR can be used for more specific microbial DNA quantification.58, 59, 60, 61, 62, 63 A more comprehensive analysis is carried out using the 454 pyrosequencing 16S ribosomal RNA method, which is currently one of the best techniques for identification of microbes that cannot be analyzed or isolated by traditional culture-based methods. This technology is available and is currently being used to study and better understand the composition, function and effects of medical interventions in the intestinal microbiota. Analysis of entire microbial population sequences also allows for evaluation of the functional characteristics of the microbial population via functional metagenomics.64, 65, 66, 67

It is important to understand that, although manipulation of the intestinal microbial ecology represents a promising therapeutic intervention for various diseases, it may also present the potential to do harm. Despite the progress made in the care of the newborn, and especially in the care of sick preterm babies, more studies are needed to understand the effects of therapeutic interventions in the intestinal microbiota. Precautions to minimize negative alterations in the intestinal microbiota with current medical practices (for example, antibiotics) and the use of bioactive agents such as probiotics and prebiotics are warranted in the neonate.

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Torrazza, R., Neu, J. The developing intestinal microbiome and its relationship to health and disease in the neonate. J Perinatol 31, S29–S34 (2011). https://doi.org/10.1038/jp.2010.172

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Keywords

  • intestinal microbiota
  • microbiome
  • probiotics
  • prebiotics
  • necrotizing enterocolitis
  • preterm

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