Infant Gastrointestinal Tract Research Articles

Validation of a new in vitro dynamic system to simulate infant digestion

Understanding the mechanisms of infant formula disintegration in the infant gastrointestinal tract is a key step for developing new formulas with health benefits for the neonate. For ethical reasons, the access to in vivo data obtained on infants is limited. The use of animal models can be an alternative but these experiments are labour intensive, expensive and results obtained show high inter-individual variability, making their interpretation difficult. The aim of this work was to develop a simple in vitro dynamic gastrointestinal digestion system, for studying infant formula digestion, and to validate it by comparing the kinetics of proteolysis obtained in vitro with in vivo data collected from piglets. Results showed a good correlation between in vitro and in vivo data and confirmed the rapid hydrolysis of caseins in gastric conditions, whereas whey proteins appeared more resistant to digestion.

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.

A pig model of the human gastrointestinal tract

Easy access to next generation sequencing has enabled the rapid analysis of complex microbial populations. To take full advantage of these technologies, animal models enabling the manipulation of human microbiomes and the study of the impact of such perturbations on the host are needed. To this aim we are developing experimentally tractable and clinically relevant pig models of the human adult and infant gastro-intestinal tract. The intestine of germ-free piglets was populated with human adult or infant fecal microbial populations, and the piglets were maintained on solid or milk diet, respectively. Amplicons of 16S rRNA V6 region were deep-sequenced to monitor to what extent the transplanted human microbiomes changed in the pig. Within 24 h of transfer of human fecal microbiome to pigs, bacterial microbiomes rich in Proteobacteria emerged. These populations evolved toward a more diverse composition rich in Bacteroidetes and Firmicutes. In the experiment where infant microbiome was used, the phylogenetic composition of the transplanted bacterial population converged toward that of the human inoculum. A majority of sequences belonged to a relatively small number of operational taxonomic units, whereas at the other end of the abundance spectrum, a large number of rare and transient OTUs were detected. Analysis of fecal and colonic microbiomes originating from the same animal indicate that feces closely replicate the colonic microbiome. We conclude that the pig intestine can be colonized with human fecal microbiomes to generate a realistic model of the human GI tract.

Fortification of Donor Human Milk for Premature Infants

Premature infants at risk for metabolic bone disease may need increased minerals for normal bone mineralization, in which case supplementation of donor human milk (DHM) with calcium (Ca), phosphorus (P), and post‐discharge formula (PDF) is common. The bioavailability of these additives has not been proven. Objectives were to study the effect of Ca, P, and PDF supplementation on the bioaccessibility of calcium and phosphate. A novel in‐vitro model of the premature infant's gastrointestinal tract was developed to simulate effects of digestion, absorption and transport. Calcium and phosphate were measured before and after in‐vitro digestion in DHM with and without added supplements. Bioaccessible calcium was greater in DHM with supplemental calcium and calcium and phosphate together than in DHM alone or with supplemental PDF (p < 0.0001). Bioaccessible phosphate was not significantly different between treatment groups. The addition of minerals rather than PDF provides the most bioaccessible calcium.Funded by San Jose Mothers’ Milk Bank

Postnatally-transmitted HIV-1 Envelope variants have similar neutralization-sensitivity and function to that of nontransmitted breast milk variants

BackgroundBreastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n=13 viruses), five clinically-matched nontransmitting mothers (n=16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses).ResultsThere was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants.ConclusionPostnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies.

Differences in direct pharmacologic effects and antioxidative properties of mature breast milk and infant formulas

ObjectiveEarly-onset and exclusive breast-feeding provides a significant health benefit to infants compared with infant formulas. The aim of this study was to compare mature breast milk with standard infant formulas by examining their effects on non-vascular smooth muscle contraction and their antioxidative properties. MethodsThe pharmacologic effects of breast milk and formulas were examined using a model system of the rat uterine smooth muscle contraction. Electron paramagnetic resonance spin-trapping spectroscopy was used to compare the antioxidative capacities of breast milk (obtained in the ninth week of lactation) with commercial infant formulas against hydroxyl radical production in the Fenton reaction. The activities of superoxide dismutase, glutathione peroxidase, and the sulfhydryl group were determined in the breast milk and infant formulas. ResultsIn contrast to the infant formulas, breast milk exerted a relaxing effect on isolated non-vascular smooth muscle. In general, breast milk showed higher antioxidative activity compared with the infant formulas. In all samples, the generation of hydroxyl radicals led to the formation of carbon-centered and ascorbyl radicals. ConclusionsHuman milk exerts direct pharmacologic relaxation effects and provides better antioxidant protection compared with infant formulas because of the presence of specific enzymatic components, such as human superoxide dismutase. We propose that these effects should be advantageous to an infant's gastrointestinal tract by supporting the normal work of the smooth musculature and maintaining redox homeostasis and may represent one of the mechanisms by which breast-feeding benefits health.

Postnatally-transmitted HIV-1 variants are efficient at dendritic cell trans-infection and sensitive to autologous and heterologous neutralization

Background Postnatal transmission via breastfeeding is a leading cause of infant HIV infection in the developing world. However, only a small minority of breastfed infants born to HIVinfected women become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic differences in the virus Envelope (Env) may play a role in its ability to breech the mucosal barrier in the infant gastrointestinal tract.

Characterization of Bifidobacterium spp. strains for the treatment of enteric disorders in newborns

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastrointestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to nontumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify three Bifidobacterium breve strains and a Bifidobacterium longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. A validation clinical trial involving the selected strains is being planned.

Human Milk Oligosaccharides Promote the Growth of Staphylococci

Human milk oligosaccharides (HMO), which constitute a major component of human milk, promote the growth of particular bacterial species in the infant's gastrointestinal tract. We hypothesized that HMO also interact with the bacterial communities present in human milk. To test this hypothesis, two experiments were conducted. First, milk samples were collected from healthy women (n = 16); culture-independent analysis of the bacterial communities was performed, HMO content was analyzed, and the relation between these factors was investigated. A positive correlation was observed between the relative abundance of Staphylococcus and total HMO content (r = 0.66). In a follow-up study, we conducted a series of in vitro growth curve experiments utilizing Staphylococcus aureus or Staphylococcus epidermidis and HMO isolated from human milk. HMO exhibited stimulatory effects on bacterial growth under various nutritional conditions. Analysis of culture supernatants from these experiments revealed that HMO did not measurably disappear from the culture medium, indicating that the growth-enhancing effects were not a result of bacterial metabolism of the HMO. Instead, stimulation of growth caused greater utilization of amino acids in minimal medium. Collectively, the data provide evidence that HMO may promote the growth of Staphylococcus species in the lactating mammary gland.

A Molecular Basis for Bifidobacterial Enrichment in the Infant Gastrointestinal Tract

Bifidobacteria are commonly used as probiotics in dairy foods. Select bifidobacterial species are also early colonizers of the breast-fed infant colon; however, the mechanism for this enrichment is unclear. We previously showed that Bifidobacterium longum subsp. infantis is a prototypical bifidobacterial species that can readily utilize human milk oligosaccharides as the sole carbon source. MS-based glycoprofiling has revealed that numerous B. infantis strains preferentially consume small mass oligosaccharides, abundant in human milks. Genome sequencing revealed that B. infantis possesses a bias toward genes required to use mammalian-derived carbohydrates. Many of these genomic features encode enzymes that are active on milk oligosaccharides including a novel 40-kb region dedicated to oligosaccharide utilization. Biochemical and molecular characterization of the encoded glycosidases and transport proteins has further resolved the mechanism by which B. infantis selectively imports and catabolizes milk oligosaccharides. Expression studies indicate that many of these key functions are only induced during growth on milk oligosaccharides and not expressed during growth on other prebiotics. Analysis of numerous B. infantis isolates has confirmed that these genomic features are common among the B. infantis subspecies and likely constitute a competitive colonization strategy used by these unique bifidobacteria. By detailed characterization of the molecular mechanisms responsible, these studies provide a conceptual framework for bifidobacterial persistence and host interaction in the infant gastrointestinal tract mediated in part through consumption of human milk oligosaccharides.

Characterization of the Diversity and Temporal Stability of Bacterial Communities in Human Milk

Recent investigations have demonstrated that human milk contains a variety of bacterial genera; however, as of yet very little work has been done to characterize the full diversity of these milk bacterial communities and their relative stability over time. To more thoroughly investigate the human milk microbiome, we utilized microbial identification techniques based on pyrosequencing of the 16S ribosomal RNA gene. Specifically, we characterized the bacterial communities present in milk samples collected from 16 women at three time-points over four weeks. Results indicated that milk bacterial communities were generally complex; several genera represented greater than 5% of the relative community abundance, and the community was often, yet not always, stable over time within an individual. These results support the conclusion that human milk, which is recommended as the optimal nutrition source for almost all healthy infants, contains a collection of bacteria more diverse than previously reported. This finding begs the question as to what role this community plays in colonization of the infant gastrointestinal tract and maintaining mammary health.

Compromised ventilation caused by tracheoesophageal fistula and gastrointestinal endoscope undergoing removal of disk battery on esophagus in pediatric patient -A case report-

Ingestion of disk batteries may have serious complications such as esophageal burn, perforation, and tracheoesophageal fistula, particularly when the battery is caught in the esophagus. Proper placement of the tracheal tube is critical when tracheoesophageal fistula was occurred from esophageal impaction the battery. Endoscopy of upper gastrointestinal tract in infants and children is an important and effective tool for the diagnosis and treatment of foreign body ingestion. But upper gastrointestinal endoscopy in infant and children has very high risk of tracheal compression and airway compromise. We present a case of ventilatory compromise during insertion of the upper gastrointestinal endoscopy in 16-month-old child with tracheoesophageal fistula secondary to disk battery ingestion.

Unique Sugar Metabolic Pathways of Bifidobacteria

Bifidobacteria have many beneficial effects for human health. The gastrointestinal tract, where natural colonization of bifidobacteria occurs, is an environment poor in nutrition and oxygen. Therefore, bifidobacteria have many unique glycosidases, transporters, and metabolic enzymes for sugar fermentation to utilize diverse carbohydrates that are not absorbed by host humans and animals. They have a unique, effective central fermentative pathway called bifid shunt. Recently, a novel metabolic pathway that utilizes both human milk oligosaccharides and host glycoconjugates was found. The galacto-N-biose/lacto-N-biose I metabolic pathway plays a key role in colonization in the infant gastrointestinal tract. These pathways involve many unique enzymes and proteins. This review focuses on their molecular mechanisms, as revealed by biochemical and crystallographic studies.

Robust Vaccine-Elicited Cellular Immune Responses in Breast Milk following Systemic Simian Immunodeficiency Virus DNA Prime and Live Virus Vector Boost Vaccination of Lactating Rhesus Monkeys

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

Inhibition of Human Immunodeficiency Virus Type 1 by Lactic Acid Bacteria from Human Breastmilk

Human breastmilk provides a rich source of commensal lactic acid bacteria (LAB) to the infant during breastfeeding and stimulates abundant growth and colonization of these bacteria at mucosal surfaces in the infant gastrointestinal tract. While conferring critical nutritional and immunologic support to the developing newborn, breastmilk also serves as a vehicle for human immunodeficiency virus type 1 (HIV-1) transmission from mother to child during breastfeeding. Whether breastmilk LAB confer protection against mucosal exposure to HIV-1 in breastfeeding infants is unknown. In the present study, we sought to evaluate LAB isolated from the breastmilk of healthy women for the ability to inhibit HIV-1 infection in vitro. A total of 38 strains of breastmilk bacteria were evaluated in this study. Both heat-killed bacteria and cell-free conditioned supernatants from bacterial cultures were tested for the ability to inhibit infection with HIV-1 using viral isolates with tropism for CCR5 (R5), CXCR4 (X4), or R5/X4 dual-tropism. Significant inhibition of R5-tropic HIV-1 was demonstrated using heat-killed bacteria, most notably among breastmilk strains of Lactobacillus and Pediococcus. Selected strains of breastmilk LAB also demonstrated significant inhibition of HIV-1 infection against virus with tropism for X4 and R5/X4. These results demonstrate for the first time that commensal LAB from human breastmilk inhibit HIV-1 infection in vitro and suggest a possible role for these bacteria in mucosal protection against HIV-1 in the breastfeeding infant.

Probiotics: Are We Ready for Routine Use?

Necrotizing enterocolitis (NEC) is a significant cause of morbidity and mortality among premature infants and affects 7% of very low birth weight infants.1 The cause of NEC is unclear, and its etiology is multifactorial; NEC seems to be the “single final pathway” created by a variety of injuries, including mucosal damage from gastrointestinal ischemia or hypoxia, increased osmolarity from enteral feedings, and the presence of potentially invasive pathogenic bacteria. Few treatments are available that have been proven to either prevent or ameliorate NEC. Feeding fresh human breast milk seems to be one of the more effective approaches to preventing NEC.2,3 The use of enteral antibiotics and other feeding practices may or may not affect NEC.4 Probiotics may be one of the most effective therapies for the prevention of NEC. At birth, an infant's gastrointestinal tract is sterile. Colonization of the gastrointestinal tract starts immediately after birth with the initiation of enteral feedings and is well established within the first few days of life.5 In breastfed infants, Bifidobacterium and lactobacillus predominate, with other enteric organisms being present less frequently.6,–,10 This is not the case for formula-fed infants. In these infants, coliforms, enterococci, and Bacteroides predominately colonize the intestinal tract. Preterm … Address correspondence to Roger F. Soll, MD, Division of Neonatal-Perinatal Medicine, University of Vermont, College of Medicine, Fletcher Allen Health Care, Smith 552A, 111 Colchester Ave, Burlington, VT 05401. E-mail: roger.soll{at}vtmednet.org

Role of gut microbiota in early infant development.

Early colonization of the infant gastrointestinal tract is crucial for the overall health of the infant, and establishment and maintenance of non-pathogenic intestinal microbiota may reduce several neonatal inflammatory conditions. Much effort has therefore been devoted to manipulation of the composition of the microbiota through 1) the role of early infant nutrition, particularly breast milk, and supplementation of infant formula with prebiotics that positively influence the enteric microbiota by selectively promoting growth of beneficial bacteria and 2) oral administration of probiotic bacteria which when administered in adequate amounts confer a health benefit on the host. While the complex microbiota of the adult is difficult to change in the long-term, there is greater impact of the diet on infant microbiota as this is not as stable as in adults. Decreasing excessive use of antibiotics and increasing the use of pre- and probiotics have shown to be beneficial in the prevention of several important infant diseases such as necrotizing enterocolitis and atopic eczema as well as improvement of short and long-term health. This review addresses how the composition of the gut microbiota becomes established in early life, its relevance to infant health, and dietary means by which it can be manipulated.

The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome

Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.

Hyperbilirubinemia in the Late Preterm Infant

Hyperbilirubinemia is the leading cause of rehospitalization in the late preterm infant (Tomashek, Sem in Perinatology. 2006). Immaturity of the infant's gastrointestinal tract and hepatic system, along with immaturity of feeding, leads to an increased risk of hyperbilirubinemia and the need for medical attention. To better understand the risks of hyperbilirubinemia, we will explore the normal bilirubin metabolism and alterations seen in the late preterm. Because the ultimate goal of treatment of hyperbilirubinemia is the prevention of kernicterus, we will also explore the causes of kernicterus and the increased susceptibility in the late preterm.

Alignment-Independent Comparisons of Human Gastrointestinal Tract Microbial Communities in a Multidimensional 16S rRNA Gene Evolutionary Space

We present a novel approach for comparing 16S rRNA gene clone libraries that is independent of both DNA sequence alignment and definition of bacterial phylogroups. These steps are the major bottlenecks in current microbial comparative analyses. We used direct comparisons of taxon density distributions in an absolute evolutionary coordinate space. The coordinate space was generated by using alignment-independent bilinear multivariate modeling. Statistical analyses for clone library comparisons were based on multivariate analysis of variance, partial least-squares regression, and permutations. Clone libraries from both adult and infant gastrointestinal tract microbial communities were used as biological models. We reanalyzed a library consisting of 11,831 clones covering complete colons from three healthy adults in addition to a smaller 390-clone library from infant feces. We show that it is possible to extract detailed information about microbial community structures using our alignment-independent method. Our density distribution analysis is also very efficient with respect to computer operation time, meeting the future requirements of large-scale screenings to understand the diversity and dynamics of microbial communities.