Precision modulation of dysbiotic adult microbiomes with a human-milk-derived synbiotic reshapes gut microbial composition and metabolites.

Manipulation of the gut microbiome using live biotherapeutic products shows promise for clinical applications but remains challenging to achieve. Here, we induced dysbiosis in 56 healthy volunteers using antibiotics to test a synbiotic comprising the infant gut microbe, Bifidobacterium longum subspecies infantis (B. infantis), and human milk oligosaccharides (HMOs). B. infantis engrafted in 76% of subjects in an HMO-dependent manner, reaching a relative abundance of up to 81%. Changes in microbiome composition and gut metabolites reflect altered recovery of engrafted subjects compared with controls. Engraftment associates with increases in lactate-consuming Veillonella, faster acetate recovery, and changes in indolelactate and p-cresol sulfate, metabolites that impact host inflammatory status. Furthermore, Veillonella co-cultured in vitro and in vivo with B. infantis and HMO converts lactate produced by B. infantis to propionate, an important mediator of host physiology. These results suggest that the synbiotic reproducibly and predictably modulates recovery of a dysbiotic microbiome.

Human milk oligosaccharides modulate the intestinal microbiome of healthy adults.

Human milk contains over 200 distinct oligosaccharides, which are critical to shaping the developing neonatal gut microbiome. To investigate whether a complex mixture of human milk oligosaccharides (HMOs) would similarly modulate the adult gut microbiome, HMO-Concentrate derived from pooled donor breast milk was administered orally to 32 healthy adults for 7 days followed by 21 days of monitoring. Fecal samples were collected for 16S rRNA gene sequencing, shotgun metagenomics, and metabolomics analyses. HMO-Concentrate induced dose-dependent Bifidobacterium expansion, reduced microbial diversity, and altered microbial gene content. Following HMO cessation, a microbial succession occurred with diverse taxonomic changes-including Bacteroides expansion-that persisted through day 28. This was associated with altered microbial gene content, shifts in serum metabolite levels, and increased circulating TGFß and IL-10. Incubation of cultured adult microbiota with HMO-Concentrate induced dose-dependent compositional shifts that were not recapitulated by individual HMOs or defined mixtures of the 10 most abundant HMOs in HMO-Concentrate at their measured concentrations. These findings support that pooled donor HMOs can exert direct effects on adult gut microbiota and that complex mixtures including low abundance HMOs present in donor milk may be required for maximum effect.Registration: ClinicalTrials.gov NCT05516225.

Dosing a synbiotic of human milk oligosaccharides and B. infantis leads to reversible engraftment in healthy adult microbiomes without antibiotics.

Predictable and sustainable engraftment of live biotherapeutic products into the human gut microbiome is being explored as a promising way to modulate the human gut microbiome. We utilize a synbiotic approach pairing the infant gut microbe Bifidobacterium longum subspecies infantis (B. infantis) and human milk oligosaccharides (HMO). B. infantis, which is typically absent in adults, engrafts into healthy adult microbiomes in an HMO-dependent manner at a relative abundance of up to 25% of the bacterial population without antibiotic pretreatment or adverse effects. Corresponding changes in metabolites are detected. Germ-free mice transplanted with dysbiotic human microbiomes also successfully engraft with B. infantis in an HMO-dependent manner, and the synbiotic augments butyrate levels both in this in vivo model and in in vitro cocultures of the synbiotic with specific Firmicutes species. Finally, the synbiotic inhibits the growth of enteropathogens in vitro. Our findings point to a potential safe mechanism for ameliorating dysbioses characteristic of numerous human diseases.

Beyond Necrotizing Enterocolitis: Other Clinical Advantages of an Exclusive Human Milk Diet.

OBJECTIVE: Articles previously published by Sullivan et al. and Cristofalo et al. were reanalyzed using the proportion of cow milk-based nutrition received to determine whether that affected clinical outcomes during hospitalization for infants birth weight 500-1250 g. Abrams et al. showed in the same cohort incidences of necrotizing enterocolitis (NEC), NEC requiring surgery and sepsis increased proportionally to the amount of dietary cow milk. METHODS: The data from the two studies conducted under essentially the same protocol were combined yielding a cohort of 260 infants receiving a diet ranging from 0% to 100% cow milk. Data analysis utilized negative binomial regression which mitigates differences between subjects in terms of their time on study by incorporating that number into the statistical model. The percent of cow milk-based nutrition was the only predictor investigated. RESULTS: For all outcomes the larger the amount of cow's milk in the diet the greater the number of days of that intervention required. A trend toward statistical significance was seen for ventilator days; however, only parenteral nutrition (PN) days and days to full feeds achieved statistical significance. CONCLUSIONS: Incorporation of any cow milk-based nutrition into the diet of extremely premature infants correlates with more days on PN and a longer time to achieve full feeds. There was a nonstatistically significant trend toward increased ventilator days. These represent additional clinical consequences of the use of any cow milk-based protein in feeding EP infants.

Premature Infants 750-1,250 g Birth Weight Supplemented with a Novel Human Milk-Derived Cream Are Discharged Sooner.

OBJECTIVE: Infants may benefit from early nutritional intervention to decrease hospital stay. To evaluate the effects of adding a human milk (HM)-derived cream (Cream) product to a standard feeding regimen in preterm infants. MATERIALS AND METHODS: In a prospective multicenter randomized study, infants with birth weights 750-1,250 g were assigned to a Control or Cream group. The Control group received a standard feeding regimen consisting of mother's own milk or donor HM with donor HM-derived fortifier. The Cream group received the standard feeding regimen along with an additional HM-derived cream supplement when the HM they received was <20 kcal/oz. Primary outcomes of this secondary analysis included comorbidities, length of stay (LOS), and postmenstrual age (PMA) at discharge. RESULTS: We enrolled 75 infants (Control n = 37, Cream n = 38) with gestational age 27.7 ± 1.8 weeks and birth weight 973 ± 145 g (mean ± SD). After adjusting for gestational age, birth weight, and presence of bronchopulmonary dysplasia (BPD), the Cream group had a decreased PMA at discharge (39.9 ± 4.8 versus 38.2 ± 2.7 weeks, p = 0.03) and LOS (86 ± 39 versus 74 ± 22 days, p = 0.05). For 21 infants with BPD, these values trended toward significance for PMA at discharge (44.2 ± 6.1 versus 41.3 ± 2.7 weeks, p = 0.08) and LOS (121 ± 49 versus 104 ± 23 days, p = 0.08). CONCLUSIONS: Very preterm infants who received an HM-derived cream supplement were discharged earlier. Infants with BPD may have benefited the most.

Randomized trial of human milk cream as a supplement to standard fortification of an exclusive human milk-based diet in infants 750-1250 g birth weight.

OBJECTIVE: To evaluate whether premature infants who received an exclusive human milk (HM)-based diet and a HM-derived cream supplement (cream) would have weight gain (g/kg/d) at least as good as infants receiving a standard feeding regimen (control). STUDY DESIGN: In a prospective noninferiority, randomized, unmasked study, infants with a birth weight 750-1250 g were randomly assigned to the control or cream group. The control group received mother's own milk or donor HM with donor HM-derived fortifier. The cream group received a HM-derived cream supplement if the energy density of the HM tested <20 kcal/oz using a near infrared HM analyzer. Infants were continued on the protocol until 36 weeks postmenstrual age. Primary outcomes included growth velocities and amount of donor HM-derived fortifier used. The hypothesis of noninferiority was established if the lower bound of the one-sided 95% CI for the difference in weight velocities exceeded -3 g/kg/day. RESULTS: There were no differences between groups in baseline demographics for the 78 infants studied except racial distribution (P = .02). The cream group (n = 39) had superior weight (14.0 ± 2.5 vs 12.4 ± 3.0 g/kg/d, P = .03) and length (1.03 ± 0.33 vs 0.83 ± 0.41 cm/wk, P = .02) velocity compared with the control group (n = 39). There were no significant differences in amount of fortifier used between study groups. The 1-sided 95% lower bound of the CI for the difference in mean velocity (cream-control) was 0.38 g/kg/d. CONCLUSIONS: Premature infants who received HM-derived cream to fortified HM had improved weight and length velocity compared with the control group. HM-derived cream should be considered an adjunctive supplement to an exclusive HM-based diet to improve growth rates in premature infants.

Greater mortality and morbidity in extremely preterm infants fed a diet containing cow milk protein products.

BACKGROUND: Provision of human milk has important implications for the health and outcomes of extremely preterm (EP) infants. This study evaluated the effects of an exclusive human milk diet on the health of EP infants during their stay in the neonatal intensive care unit. SUBJECTS AND METHODS: EP infants <1,250 g birth weight received a diet consisting of either human milk fortified with a human milk protein-based fortifier (HM) (n=167) or a diet containing variable amounts of milk containing cow milk-based protein (CM) (n=93). Principal outcomes were mortality, necrotizing enterocolitis (NEC), growth, and duration of parenteral nutrition (PN). RESULTS: Mortality (2% versus 8%, p=0.004) and NEC (5% versus 17%, p=0.002) differed significantly between the HM and CM groups, respectively. For every 10% increase in the volume of milk containing CM, the risk of sepsis increased by 17.9% (p<0.001). Growth rates were similar between groups. The duration of PN was 8 days less in the subgroup of infants receiving a diet containing <10% CM versus ≥10% CM (p<0.02). CONCLUSIONS: An exclusive human milk diet, devoid of CM-containing products, was associated with lower mortality and morbidity in EP infants without compromising growth and should be considered as an approach to nutritional care of these infants.

Long term healthcare costs of infants who survived neonatal necrotizing enterocolitis: a retrospective longitudinal study among infants enrolled in Texas Medicaid.

BACKGROUND: Infants who survive advanced necrotizing enterocolitis (NEC) at the time of birth are at increased risk of having poor long term physiological and neurodevelopmental growth. The economic implications of the long term morbidity in these children have not been studied to date. This paper compares the long term healthcare costs beyond the initial hospitalization period incurred by medical and surgical NEC survivors with that of matched controls without a diagnosis of NEC during birth hospitalization. METHODS: The longitudinal healthcare utilization claim files of infants born between January 2002 and December 2003 and enrolled in the Texas Medicaid fee-for-service program were used for this research. Propensity scoring was used to match infants diagnosed with NEC during birth hospitalization to infants without a diagnosis of NEC on the basis of gender, race, prematurity, extremely low birth weight status and presence of any major birth defects. The Medicaid paid all-inclusive healthcare costs for the period from 6 months to 3 years of age among children in the medical NEC, surgical NEC and matched control groups were evaluated descriptively, and in a generalized linear regression framework in order to model the impact of NEC over time and by birth weight. RESULTS: Two hundred fifty NEC survivors (73 with surgical NEC) and 2,909 matched controls were available for follow-up. Medical NEC infants incurred significantly higher healthcare costs than matched controls between 6-12 months of age (mean incremental cost = US$ 5,112 per infant). No significant difference in healthcare costs between medical NEC infants and matched controls was seen after 12 months. Surgical NEC survivors incurred healthcare costs that were consistently higher than that of matched controls through 36 months of age. The mean incremental healthcare costs of surgical NEC infants compared to matched controls between 6-12, 12-24 and 24-36 months of age were US$ 18,274, 14,067 (p < 0.01) and 8,501 (p = 0.06) per infant per six month period, respectively. These incremental costs were found to vary between sub-groups of infants born with birth weight < 1,000g versus ≥ 1,000g (p < 0.05). CONCLUSIONS: The all-inclusive healthcare costs of surgical NEC survivors continued to be substantially higher than that of matched controls through the early childhood development period. These results can have important treatment and policy implications. Further research in this topic is needed.

Randomized trial of exclusive human milk versus preterm formula diets in extremely premature infants.

OBJECTIVE: To compare the duration of parenteral nutrition, growth, and morbidity in extremely premature infants fed exclusive diets of either bovine milk-based preterm formula (BOV) or donor human milk and human milk-based human milk fortifier (HUM), in a randomized trial of formula vs human milk. STUDY DESIGN: Multicenter randomized controlled trial. The authors studied extremely preterm infants whose mothers did not provide their milk. Infants were fed either BOV or an exclusive human milk diet of pasteurized donor human milk and HUM. The major outcome was duration of parenteral nutrition. Secondary outcomes were growth, respiratory support, and necrotizing enterocolitis (NEC). RESULTS: Birth weight (983 vs 996 g) and gestational age (27.5 vs 27.7 wk), in BOV and HUM, respectively, were similar. There was a significant difference in median parenteral nutrition days: 36 vs 27, in BOV vs HUM, respectively (P = .04). The incidence of NEC in BOV was 21% (5 cases) vs 3% in HUM (1 case), P = .08; surgical NEC was significantly higher in BOV (4 cases) than HUM (0 cases), P = .04. CONCLUSIONS: In extremely preterm infants given exclusive diets of preterm formula vs human milk, there was a significantly greater duration of parenteral nutrition and higher rate of surgical NEC in infants receiving preterm formula. This trial supports the use of an exclusive human milk diet to nourish extremely preterm infants in the neonatal intensive care unit.

An exclusive human milk-based diet in extremely premature infants reduces the probability of remaining on total parenteral nutrition: a reanalysis of the data.

BACKGROUND: We have previously shown that an exclusively human milk-based diet is beneficial for extremely premature infants who are at risk for necrotizing enterocolitis (NEC). However, no significant difference in the other primary study endpoint, the length of time on total parenteral nutrition (TPN), was found. The current analysis re-evaluates these data from a different statistical perspective considering the probability or likelihood of needing TPN on any given day rather than the number of days on TPN. This study consisted of 207 premature infants randomized into three groups: one group receiving a control diet of human milk, formula and bovine-based fortifier ("control diet"), and the other two groups receiving only human milk and human milk-based fortifier starting at different times in the enteral feeding process (at feeding volumes of 40 or 100 mL/kg/day; "HM40" and "HM100", respectively). The counting process Cox proportional hazards survival model was used to determine the likelihood of needing TPN in each group. RESULTS: The two groups on the completely human-based diet had an 11-14 % reduction in the likelihood of needing nutrition via TPN when compared to infants on the control diet (p = 0.0001 and p = 0.001, respectively for the HM40 and HM100 groups, respectively). This was even more pronounced if the initial period of TPN was excluded (p < 0.0001 for both the HM40 and HM100 groups). CONCLUSIONS: A completely human milk-based diet significantly reduces the likelihood of TPN use for extremely premature infants when compared to a diet including cow-based products. This likelihood may be reduced even further when the human milk fortifier is initiated earlier in the feeding process. TRIAL REGISTRATION: This study was registered at http://www.clinicaltrials.gov reg. # NCT00506584.

An exclusively human milk-based diet is associated with a lower rate of necrotizing enterocolitis than a diet of human milk and bovine milk-based products.

OBJECTIVE: To evaluate the health benefits of an exclusively human milk-based diet compared with a diet of both human milk and bovine milk-based products in extremely premature infants. STUDY DESIGN: Infants fed their own mothers' milk were randomized to 1 of 3 study groups. Groups HM100 and HM40 received pasteurized donor human milk-based human milk fortifier when the enteral intake was 100 and 40 mL/kg/d, respectively, and both groups received pasteurized donor human milk if no mother's milk was available. Group BOV received bovine milk-based human milk fortifier when the enteral intake was 100 mL/kg/d and preterm formula if no mother's milk was available. Outcomes included duration of parenteral nutrition, morbidity, and growth. RESULTS: The 3 groups (total n = 207 infants) had similar baseline demographic variables, duration of parenteral nutrition, rates of late-onset sepsis, and growth. The groups receiving an exclusively human milk diet had significantly lower rates of necrotizing enterocolitis (NEC; P = .02) and NEC requiring surgical intervention (P = .007). CONCLUSIONS: For extremely premature infants, an exclusively human milk-based diet is associated with significantly lower rates of NEC and surgical NEC when compared with a mother's milk-based diet that also includes bovine milk-based products.

Macronutrient analysis of a nationwide sample of donor breast milk.

Banked donor milk may be a reasonable substitute for mother's milk for human infants. No data on the macronutrient composition of banked donor milk have been reported. This study determined the composition of donated milk from a large number of banked donor milk samples and compared it to the reported values for macronutrients in mature breast milk. During a 9-month sampling period (May 2006 through February 2007) from a nationwide milk bank network, 415 sequential samples from 273 unique donors were analyzed for fat, protein, and lactose content, as well as energy density. Descriptive statistics were computed, including mean, standard deviation, coefficient of variation, median, and range. Percentiles were determined from the empirical distribution of the data. A ninety-five percent confidence interval was computed using standard, large sample (Gaussian) methods. Banked donor milk mean values (in weight/volume) were found to be 1.16%+/-0.25% for protein, 3.22%+/-1.00% for fat, 7.80%+/-0.88% for lactose, and mean total energy was 65+/-11 kcal/dL. Banked donor milk macronutrient content was found to differ from the values reported in the literature for mature human milk. Unformulated banked donor milk alone, similar to mother's milk alone, does not have sufficient macronutrient content or energy density to sustain a very-low-birth-weight preterm infant. Fortification could make up for these shortcomings, perhaps making formulated banked donor milk a better choice for preterm infants than bovine-based formulas when mother's milk is unavailable.

Effects of a human milk-derived human milk fortifier on the antibacterial actions of human milk.

OBJECTIVES: To compare the effects of a human breastmilk-derived fortifier on the antibacterial activity of milk obtained from lactating mothers delivering prematurely with the effects of a powdered fortifier on the same milk. STUDY DESIGN: Human milk samples were obtained after the first week of postnatal life from 10 lactating mothers, who had delivered prematurely. A bovine milk-based powdered fortifier and a human breastmilk-based frozen fortifier were evaluated. All mothers were healthy and they were not on any medications, although they were taking prenatal vitamins during lactation. The effects of each fortifier on the antimicrobial activity of milk toward Enterobacter sakazaki (ES), Escherichia coli, Clostridium difficile (CD), and Shigella soneii (SS) were evaluated by both the filter paper method and the growth inhibition method. RESULTS: Human milk inhibited the growth of all of the test organisms. This antibacterial activity was almost totally inhibited by the addition of the bovine protein-based human milk fortifier, while it remained unaffected by the addition of the human breastmilk-based fortifier. CONCLUSIONS: Breastmilk from women who have delivered preterm has antibacterial activity that can be affected by the addition of bovine-based fortifier, but not by the addition of a human breastmilk-based fortifier.

Antimicrobial and antiviral effect of high-temperature short-time (HTST) pasteurization applied to human milk.

In the United States, concerns over the transmission of infectious diseases have led to donor human milk generally being subjected to pasteurization prior to distribution and use. The standard method used by North American milk banks is Holder pasteurization (63 degrees C for 30 minutes). The authors undertook an experiment to validate the effects of a high-temperature short-time (HTST) pasteurization process (72 degrees C for 16 seconds) on the bioburden of human milk. It was concluded that HTST is effective in the elimination of bacteria as well as of certain important pathogenic viruses.

Effect of environmental conditions on unpasteurized donor human milk.

As a result of concerns over the transmission of infectious diseases by donor milk, as well as the possible loss of nutritional value of donor milk through exposure to a variety of environmental conditions, the practice in the United States has been to discard unpasteurized donor milk that has thawed or sat for several hours at room temperature or in the refrigerator rather than (re)freezing it. We undertook an experiment to measure the effects of ambient temperature conditions and refreezing on the bioburden and nutritional content of human milk. We conclude that unpasteurized human milk is robust and can be used after storage under certain conditions.