Synthetic human milk oligosaccharides (HMOs) are used to supplement infant formula despite limited understanding of their impact on the post-weaned developing gut microbiota. Here, we assess the influence of 0.5 g/L 2-fucosyllactose (2’FL) and 4.0 g/L pooled HMOs (pHMOs) on the composition and activity of cultured fecal-derived microbial communities from seven healthy young children. Exposure to pHMOs induced significant shifts in both the microbial community composition and metabolic output, including an increased abundance of several genera, notably Bacteroides, and the production of health-associated metabolites. In contrast, 2’FL alone did not lead to substantial changes in the communities. A total of 330 bacterial isolates, spanning 157 species, were cultured from these communities and individually evaluated for their responses to HMOs. Over 100 non-Bifidobacterium species showed enhanced growth upon pHMOs treatment and a high degree of intraspecies variation in HMO metabolism was observed. Our study provides valuable insight into the health-enhancing properties of HMOs while highlighting the need for future research into the efficacy of incorporating individual structures into infant formula, particularly when aiming to modulate the gut microbiota.

BACKGROUND:Infant cognitive development is influenced by maternal factors that range from obesity to early feeding and breast milk composition. Animal studies suggest a role for human milk oligosaccharide (HMO), 2'-fucosyllactose (2'FL), on learning and memory, yet no human studies have examined its impact on infant cognitive development relative to other HMOs and maternal factors.OBJECTIVE:To determine the impact of 2'FL from breast milk feeding on infant cognitive development at 24 months of age relative to maternal obesity and breast milk feeding frequency.METHODS AND MATERIALS:Hispanic mother-infant pairs (N = 50) were recruited across the spectrum of pre-pregnancy BMI. Breast milk was collected at 1 and 6 months, and feedings/day were reported. Nineteen HMOs were analyzed using high-performance liquid chromatography, with initial interest in 2'FL. Infant cognitive development score was assessed with the Bayley-III Scale at 24 months. Linear regressions were used for prediction, and bootstrapping to determine mediation by 2'FL.RESULTS:Maternal pre-pregnancy BMI was not related to feedings/day or HMOs, but predicted poorer infant cognitive development (β = -0.31, P = 0.03). Feedings/day (β = 0.34) and 2'FL (β = 0.59) at 1 month predicted better infant cognitive development (both P≤ 0.01). The association of feedings/day with infant cognitive development was no longer significant after further adjustment for 2'FL (estimated mediation effect = 0.13, P = 0.04). There were no associations of feedings/day and 2'FL at 6 months with infant cognitive development.CONCLUSIONS:Our findings suggest that maternal factors influence infant cognitive development through multiple means. Though maternal obesity may be a separate negative influence, greater frequency of breast milk feeding at 1 month contributed to infant cognitive development through greater exposure to 2'FL relative to other HMOs. The influence of 2'FL was not significant at 6 months, indicating that early exposure to 2'FL may be a critical temporal window for positively influencing infant cognitive development.

Background: Breastfeeding modulates infant growth and protects against the development of obesity. However, whether or not maternal variation in human milk components, such as human milk oligosaccharides (HMOs), is associated with programming of child growth remains unknown. Objective: Our objective was to determine the association between maternal HMO composition and child growth during the first 5 y of life. In addition, the association between maternal prepregnancy BMI and HMO composition was assessed. Methods: Human milk samples from 802 mothers were obtained from a prospective population-based birth cohort study, Steps to healthy development of Children (STEPS), conducted in Turku, Finland. HMO composition in these milk samples was analyzed by HPLC. Child growth data from 3 mo to 5 y were collected from municipal well-baby clinics and linked to maternal HMO composition data to test for associations. Results: Maternal HMO composition 3 mo after delivery was associated with height and weight during the first 5 y of life in children of secretor mothers. Specifically, HMO diversity and the concentration of lacto-N-neo-tetraose (LNnT) were inversely associated and that of 2'-fucosyllactose (2'FL) was directly associated with child height and weight z scores in a model adjusted for maternal prepregnancy BMI, mode of delivery, birthweight z score, sex, and time. Maternal prepregnancy BMI was associated with HMO composition. Conclusions: The association between maternal HMO composition and childhood growth may imply a causal relation, which warrants additional testing in preclinical and clinical studies, especially since 2'FL and LNnT are among the HMOs now being added to infant formula. Furthermore, altered HMO composition may mediate the impact of maternal prepregnancy BMI on childhood obesity, which warrants further investigation to establish the cause-and-effect relation.

OBJECTIVES Establishment and development of the infant gastrointestinal microbiome (GIM) varies cross-culturally and is thought to be influenced by factors such as gestational age, birth mode, diet, and antibiotic exposure. However, there is little data as to how the composition of infants' households may play a role, particularly from a cross-cultural perspective. Here, we examined relationships between infant fecal microbiome (IFM) diversity/composition and infants' household size, number of siblings, and number of other household members. MATERIALS AND METHODS We analyzed 377 fecal samples from healthy, breastfeeding infants across 11 sites in eight different countries (Ethiopia, The Gambia, Ghana, Kenya, Peru, Spain, Sweden, and the United States). Fecal microbial community structure was determined by amplifying, sequencing, and classifying (to the genus level) the V1-V3 region of the bacterial 16S rRNA gene. Surveys administered to infants' mothers identified household members and composition. RESULTS Our results indicated that household composition (represented by the number of cohabitating siblings and other household members) did not have a measurable impact on the bacterial diversity, evenness, or richness of the IFM. However, we observed that variation in household composition categories did correspond to differential relative abundances of specific taxa, namely: Lactobacillus, Clostridium, Enterobacter, and Klebsiella. DISCUSSION This study, to our knowledge, is the largest cross-cultural study to date examining the association between household composition and the IFM. Our results indicate that the social environment of infants (represented here by the proxy of household composition) may influence the bacterial composition of the infant GIM, although the mechanism is unknown. A higher number and diversity of cohabitants and potential caregivers may facilitate social transmission of beneficial bacteria to the infant gastrointestinal tract, by way of shared environment or through direct physical and social contact between the maternal-infant dyad and other household members. These findings contribute to the discussion concerning ways by which infants are influenced by their social environments and add further dimensionality to the ongoing exploration of social transmission of gut microbiota and the "old friends" hypothesis.

BackgroundSince malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites.MethodsPlasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium.ResultsHere the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher K-M values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to alsobe active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity.ConclusionThe characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites' redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.

Human milk oligosaccharides (HMOs) are complex carbohydrates that are highly abundant in and, in their complexity, unique to human milk. Accumulating evidence indicates that exposure to HMOs in the postnatal period affects immediate as well as long-term infant health and development. However, studies reported in the 1970s showed that HMOs already appear in maternal urine and blood during pregnancy and as early as the first trimester. In this pilot study we aimed to determine whether or not HMOs also appear in amniotic fluid. We enrolled women during pregnancy and collected their urine and amniotic fluid at birth as well as their milk 4 days postpartum. We analyzed the samples by high-performance liquid chromatography (HPLC) and mass spectrometry and identified several HMOs including 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, and 6'-sialyllactose to be present in different relative abundancies in all three tissues. This is the first report that HMOs appear in amniotic fluid and that the fetus is already exposed to HMOs in utero, warranting future research to investigate the immediate and long-term implications on fetal and infant health and development.

Preterm infants who receive human milk instead of formula are 6- to 10-times less likely to develop necrotizing enterocolitis (NEC), one of the most common and devastating intestinal disorders that affects 5-10% of all very-low-birth-weight infants. Combined data from in vitro tissue culture models, in vivo preclinical studies in animal models, as well human mother-infant cohort studies support the hypothesis that human milk oligosaccharides (HMOs), complex sugars that are highly abundant in human milk but not in infant formula, contribute to the beneficial effects of human milk feeding in reducing NEC. The almost 20-year long journey of testing this hypothesis took an interesting turn during HMO in vivo efficacy testing and structure elucidation, suggesting that the original hypothesis may indeed be correct and specific HMO reduce NEC risk, however, the underlying mechanisms are likely different than originally postulated.

Human milk oligosaccharides (HMOs) evade digestion and remain largely intact while transiting the infant gastrointestinal tract. Thus, HMOs are available to interact with endogenous microbiota that are capable of colonizing the nursing neonate following birth. This chapter reviews the scientific evidence for HMOs enriching specific microbial populations such as bifidobacteria that are proficient at metabolizing milk oligosaccharides. In addition, HMOs simultaneously restrict potential pathogens by providing decoy lectin-binding sites that mimic similar structures decorating host epithelia. In aggregate, HMOs guide the composition of the infant microbiome by both positively and negatively modulating microbial populations. This suggests an adaptation innovated in humans and perhaps other mammals to promote microbial community function to benefit of infant health.

Elucidation of the biological roles and more detailed structure–function relationships of human milk oligosaccharides (HMOs) requires access to homogeneous, defined HMO samples. Various approaches, including chemical synthesis, enzymatic/chemoenzymatic synthesis, and fermentation processes, have been developed to support this goal. Examples of scalable industrial processes demonstrating each approach are presented. These strategies, alone and in combination, may be deployed to address the problem of routine access to HMOs for biological and nutritional research and applications.

Objective Necrotising enterocolitis (NEC) is one of the most common and often fatal intestinal disorders in preterm infants. Markers to identify at-risk infants as well as therapies to prevent and treat NEC are limited and urgently needed. NEC incidence is significantly lower in breast-fed compared with formula-fed infants. Infant formula lacks human milk oligosaccharides (HMO), such as disialyllacto-N-tetraose (DSLNT), which prevents NEC in neonatal rats. However, it is unknown if DSLNT also protects human preterm infants. Design We conducted a multicentre clinical cohort study and recruited 200 mothers and their very low birthweight infants that were predominantly human milk-fed. We analysed HMO composition in breast milk fed to infants over the first 28 days post partum, matched each NEC case with five controls and used logistic regression and generalised estimating equation to test the hypothesis that infants who develop NEC receive milk with less DSLNT than infants who do not develop NEC. Results Eight infants in the cohort developed NEC (Bell stage 2 or 3). DSLNT concentrations were significantly lower in almost all milk samples in NEC cases compared with controls, and its abundance could identify NEC cases prior to onset. Aggregate assessment of DSLNT over multiple days enhanced the separation of NEC cases and control subjects. Conclusions DSLNT content in breast milk is a potential non-invasive marker to identify infants at risk of developing NEC, and screen high-risk donor milk. In addition, DSLNT could serve as a natural template to develop novel therapeutics against this devastating disorder.

Necrotising enterocolitis (NEC) is one of the most frequent and fatal intestinal disorders in preterm infants and has very limited treatment options. Breast-fed infants are at a 6-10-fold lower NEC risk than formula-fed infants, and we have previously shown that human milk oligosaccharides (HMO) improved survival and reduced pathology in a rat NEC model. The HMO disialyllacto-N-tetraose (DSLNT) was most effective, and sialylation was shown to be essential for its protective effect. Galacto-oligosaccharides (GOS), currently added to some infant formula, but not containing sialic acid, had no effect. In addition to DSLNT, our previous work also showed that the neutral HMO fraction, which contains high concentrations of 2'-fucosyllactose (2'FL), slightly improved pathology scores. Here, we assessed the in vivo efficacy of 2'FL, as well as of GOS that we enzymatically sialylated (Sia-GOS). Neonatal rats were randomised into the following study groups - dam-fed (DF), formula-fed (FF), FF containing pooled HMO (10 mg/ml), GOS (8 mg/ml), Sia-GOS (500 µm) or 2'FL (2 mg/ml) - and subjected to the established NEC protocol. The DF and HMO groups had the lowest pathology scores with mean values of 0·67 (sd 0·34) and 0·90 (sd 0·47), respectively. The FF group had significantly elevated pathology scores of 2·02 (sd 0·63). Although the addition of GOS to the formula had no protective effect and generated scores of 2·00 (sd 0·63), the addition of Sia-GOS or 2'FL significantly lowered pathology scores to 1·32 (sd 0·56) (P<0·0034) and 1·43 (sd 0·51) (P<0·0040), respectively. The results warrant further studies to investigate the underlying mechanisms and to assess safety and efficacy in human neonates.

Human milk is the optimal source of nutrition for the nursing infant. Classically, the nutrients (water, protein, lipid, carbohydrate, vitamins, and minerals) were studied as the critical components of milk serving the growth needs of the infant for optimum growth. However, human milk contains factors other than the classically defined nutrients for which researchers are investigating potential roles in infant and maternal health, development, and well-being. The symposium addressed some of the exciting factors being studied, including microbes and maternal cells found within milk. Drs. Michelle McGuire and Juan M. Rodríguez addressed the presence of a bacterial community in human milk produced by healthy and mastitic mothers, potential sources of those bacteria, and the impact of milk-derived bacteria on the nursing infant. Drs. Donna Geddes, Peter Hartmann, and Foteini Hassiotou discussed the potential importance of maternal cells. For years, immune cells were known to be present in human milk, but recent evidence suggests that their impact is as much on the infant as on the health of the lactating mammary gland. Finally, the existence of highly plastic stem cells in human milk opens doors for previously unforeseen developmental “training” of the nursing infant.