NNIW88 - Intestinal Microbiome: Functional Aspects in Health and Disease

The complex and diverse intestinal bacterial microflora is now recognized as important in promoting human health. An altered gut microflora, referred to as dysbiosis, is increasingly recognized as having an etiologic role in a variety of conditions, including functional gastrointestinal disorders: colic in infants and irritable bowel syndrome in older children and adults. Probiotics are defined as live microorganisms that, when ingested in sufficient amounts, restore microbial homeostasis and have a benefit on health. Randomized controlled trials indicate that certain probiotics are effective in a variety of intestinal conditions, including functional gastrointestinal disorders such as colic and irritable bowel syndrome.

Human milk oligosaccharides are key components of human milk and appear in various compositions and concentrations in all human milks. In regulatory sense human milk oligosaccharides are classified as novel foods or novel food ingredients requiring safety assessment. In addition, if any health messages are intended to be used also health claim regulations apply. This chapter reviews the regulatory settings and studies human milk oligosaccharides are required to fulfill to be able to enter markets in European Union or United States or elsewhere. Examples include Lacto-N-neotetraose and 2-fucosyllactose with safety assessment in European Union and United States.

The composition of an infant’s gut microbiome can impact their immediate and long-term health. Bifdobacteria play a major role in structuring the gut microbiome of breastfed infants due to their ability to consume oligosaccharides found in human milk. However, recent studies have revealed that bifidobacteria are often absent in the gut microbiome of breastfed infants in some locations.

This lack of colonization may be due either to differences in the environmental conditions in the gastrointestinal tract of uncolonized infants which prohibit the growth of bifidobacteria or a dearth of sources from which infants may acquire these specialized bacterial species. Potential mechanisms by which these broad factors may lead to lower colonization of infants by bifidobacteria are discussed herein.

Environmental conditions which may select against bifidobacteria include low rates/duration of breastfeeding, milk glycan composition, and antimicrobial use. Routes of colonization by bifidobacteria which may be disrupted include maternal transfer via vaginal birth, fecal-oral routes, or via breast milk itself. A careful contemplation of the conditions experienced by bifidobacteria over human evolutionary history may lead to further hypotheses as to the causative factors of the differential colonization by this foundation genus in some contemporary locations.

It is a great success that biotechnological means are available today to produce amounts of single human milk oligosaccharides (HMOs) in a purity which allows performing metabolic and functional studies even in humans. As recent data indicate that there is a link between the Lewis blood group and the secretor status of an individual and certain inflammatory diseases, this review will also focus on the metabolic fate of secretor- and Lewis blood group-specific components. We conclude that there is no simple urinary or fecal excretion pattern of HMOs, although the pattern in urine often reflects the mother’s secretor/nonsecretor status.

However, there are deviations for single HMOs which deserve special attention. In feces, the variation in excretion is much higher than in urine, which may be caused by variations in the infant’s intestinal microbiota. A gradual decrease in HMO excretion with time as proposed earlier does not take place as even after 7 months of exclusive breastfeeding often intact HMOs can be detected in feces and urine. In addition, we found that whenever oligosaccharides were detected in feces, LNT, the major core structure of HMOs, was present. Hence, our data do not support speculations that LNT is a preferable source for the microbiota.

Milk proteins are a complex and diverse source of biological activities. Beyond their function, intact milk proteins also act as carriers of encrypted functional sequences that, when released as peptides, exert biological functions, including antimicrobial and immunomodulatory activity, which could contribute to the infant’s competitive success. Research has now revealed that the release of these functional peptides begins within the mammary gland itself.

A complex array of proteases produced in mother’s milk has been shown to be active in the milk, releasing these peptides. Moreover, our recent research demonstrates that these milk proteases continue to digest milk proteins within the infant’s stomach, possibly even to a larger extent than the infant’s own proteases.

As the neonate has relatively low digestive capacity, the activity of milk proteases in the infant may provide important assistance to digesting milk proteins. The coordinated release of these encrypted sequences is accomplished by selective proteolytic action provided by an array of native milk proteases and infant-produced enzymes. The task for scientists is now to discover the selective advantages of this protein-protease-based peptide release system.

Gut microbiota plays an important role in host health maintenance and disease pathogenesis. The development of a stable and diverse gut microbiota is essential to various host physiologic functions such as immunoregulation, pathogen prevention, energy harvest, and metabolism. At the same time, a dysbiotic gut microbiota associated with disease is altered in structure and function, and often characterized by a decrease in species richness and proliferation of pathogenic bacterial taxa.

As a shared substrate between the host and the gut microbiota, diet significantly impacts the health and disease states of the host both directly and through gut microbial metabolite production. This is demonstrated in the examples of short-chain fatty acid and trimethylamine production via bacterial metabolism of dietary complex carbohydrates and choline, respectively. In disorders related to mucosal immune dysregulation such as inflammatory bowel disease, the dysbiotic gut microbiota and diet contribute to its pathogenesis.

Reversal of dysbiosis through fecal microbiota transplantation and dietary interventions may thus represent important strategies to modify the gut microbiota and its metabolite production for health maintenance as well as disease prevention and management.

Obesity is globally the most prevalent nutritional disorder. Multifaceted therapeutic approaches are called for to halt the cascade from neonatal adiposity/high birth weight to childhood excessive weight gain/adult obesity with comorbidities. Recent experimental and clinical data provide one new target for interventions aiming to close this vicious circle: the microbiota.

An aberrant gut microbiota, dysbiosis, induces immune and metabolic disturbances both locally and, consequent upon impaired gut barrier function, also systemic low-grade inflammation, which is causally linked to insulin resistance. The gut microecology could thus fill the gap between energy intake and expenditure by processing nutrients and regulating their access to and storage in the body, producing chemicals of hormonal nature and controlling the secretion of proinflammatory mediators locally and systemically.

Conversely, being highly sensitive to environmental impacts, particularly to early feeding, the compositional development of the gut microbiota may prove the target of choice in efforts to reduce the risk of obesity. It has been demonstrated that a lower number of bifidobacteria precedes the development of obesity, and a dearth of butyrate-producing bacteria and an overall richness of bacteria increase the risk of metabolic disease; moreover, recognition that practices known to disrupt the early gut microbiota, e.g., cesarean section delivery and antibiotic exposure, contribute to obesity, encourages to pursue this line of research.

Necrotizing enterocolitis (NEC) is an acquired gastrointestinal inflammatory condition with significant mortality and morbidity in preterm very low birth weight infants. The interplay between toll-like receptors, bacterial endotoxins, developmentally regulated excessive proinflammatory responses of the immature innate immune system, hypoxia, ischemia, reperfusion, free radicals, and the presence of substrates and bacterial endotoxins is thought to play an important role in the pathogenesis of NEC. The association (cause?) of various microbes (bacteria, viruses, and fungi) with NEC has intrigued researchers for many years.

Availability of newer molecular methods (e.g., 16S ribosomal RNA gene-specific primers/pyrosequencing of fecal DNA) is expected to improve our understanding of the role of gut microbiota in the pathogenesis of NEC. Recent studies employing such methods to assess fecal microbiota are reviewed. Current evidence suggests that dysbiosis of the gut microbiota precedes the development of NEC in preterm infants.

Further research is required to understand the significance of changes in the gut microbiome over the early postnatal period including the relative abundance of Gammaproteobacteria and the paucity of strict anaerobic bacteria that precedes NEC in preterm infants. Assessing the reproducibility of previous findings in large prospective studies with standardized methodology (e.g. sample processing, PCR primer, and DNA extraction) is important.

Antibiotics are often prescribed inappropriately to infants and young children, with potentially adverse effects on the developing gut microbiota and related metabolic processes. We review evidence from 17 epidemiologic studies suggesting that antibiotic exposure during critical periods of early development may influence weight gain and the development of obesity.

Complementary research in both humans and rodents indicates that gut microbiota play a key role in this process, although further research is needed to confirm and characterize the causal mechanisms involved. Obesity is a complex and multifactorial condition; thus, a multipronged prevention strategy will be required to curb the current obesity epidemic. Evidence to date suggests this strategy should include the judicious use of antibiotics, especially in early life when the developing gut microbiota is particularly susceptible to perturbations with long-lasting implications for metabolic programming and obesity risk.

From epidemiological studies and studies done evaluating microbiomes in infants, there is a strong signal that the infants born by elective cesarean section (C-section) develop microbiota that differs from those babies born by vaginal delivery.

Epidemiological studies show increased odds ratios for the development of immunological disorders such as type 1 diabetes, celiac disease, asthma, allergic diseases as well as metabolic diseases such as obesity in babies born by C-section. These are interesting associations, and if supported by additional studies that rigorously control for confounding factors, they will have major public health implications.

Such studies represent major challenges because the confounding factors are numerous. The fact that provision of vaginal bacteria to C-sectiondelivered babies using a mouth swab that may actually transmit these bacteria to the infant is of interest and supports the concept that this can be done to alter the infant microbiota. However, significant caution needs to be taken, and alternative approaches that are safe as well as effective need to be considered; follow-up studies showing efficacy as well as safety need to be evaluated in the long term.

In the last 5 years, interest in the interactions among the gut microbiome, brain, and behavior has exploded. Preclinical evidence supports a role of the gut microbiome in behavioral responses associated with pain, emotion, social interactions, and food intake. Limited, but growing, clinical evidence comes primarily from associations of gut microbial composition and function to behavioral and clinical features and brain structure and function. Converging evidence suggests that the brain and the gut microbiota are in bidirectional communication.

Observed dysbiotic states in depression, chronic stress, and autism may reflect altered brain signaling to the gut, while altered gut microbial signaling to the brain may play a role in reinforcing brain alterations. On the other hand, primary dysbiotic states due to Western diets may signal to the brain, altering ingestive behavior. While studies performed in patients with depression and rodent models generated by fecal microbial transfer from such patients suggest causation, evidence for an influence of acute gut microbial alterations on human behavioral and clinical parameters is lacking. Only recently has an open-label microbial transfer therapy in children with autism tentatively validated the gut microbiota as a therapeutic target. The translational potential of preclinical findings remains unclear without further clinical investigation.

Epigenetics can be defined as stable, potentially heritable changes in cellular phenotype caused by mechanisms other than alterations in the underlying DNA sequence. DNA methylation is amongst the most intensely studied epigenetic mechanisms and has been shown to play a major role in regulating fundamental aspects of cell biology including cellular differentiation, organ development, and cell type-specific gene expression. Importantly, it is becoming increasingly clear that epigenetic mechanisms operate at the interface between the genetic code and our environment and are able to mediate environmental changes into stable phenotypic alterations.

Given existing evidence supporting the important effects of environmental factors (e.g., diet, nutrition, and infections) on human health, epigenetic mechanisms provide a plausible mechanistic framework for the development of many multifactorial diseases including inflammatory bowel disease (IBD). Impaired function of the intestinal epithelium has been implicated in IBD pathogenesis, yet underlying mechanisms remain ill defined. The work of our group focuses on investigating the role of DNA methylation in regulating cellular function of the human intestinal epithelium during gastrointestinal health and IBD. In addition to performing an analysis of primary human intestinal epithelium, we utilize human intestinal organoid culture systems allowing us to perform functional analysis in a patient-derived ex vivo model.

We now know that the fetus does not reside in a sterile intrauterine environment but is exposed to commensal bacteria from the maternal gut which cross the placenta and infiltrate the amniotic fluid. This exposure to colonizing bacteria continues at birth and during the first year of life, and it has a profound influence on lifelong health. Why is this important?

Cross talk with colonizing bacteria in the developing neonatal intestine helps in the initial adaptation of the infant to extrauterine life, particularly in acquiring immune homeostasis, and provides protection against disease expression (e.g., allergy, autoimmune disease, and obesity) later in life. Colonizing intestinal bacteria are critical to the development of host defense during the neonatal period.

Disrupted colonization (dysbiosis) due to cesarean section delivery, perinatal antibiotics, or premature delivery may adversely affect the development of host defense mechanisms in the gut and predispose to inflammation leading to increased susceptibility to disease later in life. Clinical evidence suggests that babies born by cesarean section have higher incidence rates of allergy, type 1 diabetes, and obesity.

Infants given repeated antibiotic regimens are more likely to have asthma as adolescents. This observation helps to explain the disease paradigm shift in children from developed countries

Early-life interaction with indigenous intestinal microbes is a prerequisite for healthy immune and metabolic maturation. Human infants acquire their gut microbiota predominantly from the mother. A considerable inoculum of microbes is received by the neonate during vaginal delivery.

Recent observations suggest that human gut colonization may be initiated prenatally by microbes in amniotic fluid, but the significance of this phenomenon remains unknown. After birth, neonatal gut colonization is guided by human milk factors, which selectively promote the growth of specific microbes, as well as by live microbes present in human milk. Aberrant gut colonization in early life has been associated with an increased risk of noncommunicable diseases in later life.

Epidemiological and experimental studies suggest a causal relationship between early-life gut microbiota perturbations and disease risk. Perinatal antibiotic exposure, cesarean section delivery, postnatal antibiotic administration, and formula feeding, which may disrupt intestinal microecology, have been associated with disease development in later life. The modulation of gut microbiota in the perinatal period by pre- and probiotics, for example, may offer a means to reduce the risk of chronic diseases.

In recent years, microbiome research has revealed multiple essential roles of the microorganisms residing within the human body in host metabolism, immunity, and overall health. Numerous physiological and pathological states, including obesity and the metabolic syndrome, have been correlated with microbial changes, termed dysbiosis. Our microbiomes change in response to our environment, diet, weight, hormones, and other factors. It is, therefore, not surprising that there are also significant changes in the microbiome during pregnancy when dramatic weight gain and metabolic and immunological changes occur.

In this review, we summarize the known changes in microbial composition throughout pregnancy at a variety of body sites, including the gut, vagina, oral cavity, and placenta, and we describe several studies that have linked pregnancy complications with microbial changes. Unlike the case of certain disease states, such as obesity, where dysbiosis is considered to have negative effects, we believe that the microbial alterations observed during pregnancy are vital for a healthy pregnancy.

While more research in this field is required to reveal specific mechanisms and pathways regulating these alterations, the microbial changes during pregnancy are likely coordinated with the immune, endocrine, and metabolic states.