Publications

BACKGROUND: Preterm infants routinely receive enteral iron supplementation to support growth, replace phlebotomy losses, and prevent iron deficiency. However, concerns regarding potential harms, including those on the gut microbiome, have contributed to recommendations for lower dosing.

OBJECTIVES: This study aimed to compare the effects of 2 enteral iron doses on gut health in very-low-birth-weight preterm infants. We hypothesized that higher iron dose would increase abundances of pathogenic bacteria, intestinal inflammation, and barrier dysfunction.

METHODS: This randomized, double-blind clinical trial assigned preterm infants born <1500 g to receive either the recommended dose, 2 mg/kg/d, or a higher dose of 6 mg/kg/d of total enteral iron. The primary outcome was the fecal microbiome after 2 wk on iron, assessed by metagenomic sequencing. Secondary outcomes included biomarkers of intestinal inflammation and barrier function (fecal calprotectin, urinary claudin-3, and urinary intestinal fatty acid-binding protein). Iron status, adverse events, and auditory brainstem response latencies at 36 wk postmenstrual age were also evaluated.

RESULTS: Among 151 randomly assigned infants who received study iron (77 low dose; 74 high dose), bacterial diversity, individual taxa, virulence potential, bacterial overgrowth, and iron-related functional genes were not significantly different between the treatment groups. In the subgroup analysis of singletons, treatment groups demonstrated significant differences in temporal shifts in overall bacterial community structure. Infants receiving 2 mg/kg/d had higher posttreatment urinary claudin-3 concentrations, indicating possible differences in intestinal permeability, and a higher prevalence of iron deficiency than those receiving 6 mg/kg/d. Other biomarkers, clinical outcomes, adverse events, and auditory latencies did not differ between groups.

CONCLUSIONS: Enteral iron supplementation at 6 mg/kg/d was associated with improved iron status and lower intestinal barrier dysfunction, without evidence of harms on gut microbiome compared with the recommended 2 mg/kg/d dose. These findings do not support concerns regarding gut microbiome disruption as a justification for lower iron dosing in preterm infants. This trial was registered at clinicaltrials.gov as NCT04497012.

Group B Streptococcus (Streptococcus agalactiae; GBS) is a leading cause of neonatal sepsis worldwide. As a pathobiont of the intestinal tract, it is capable of translocating across barriers leading to invasive disease. Neonatal susceptibility to invasive disease stems from immature intestinal barriers. GBS intestinal colonization induces major transcriptomic changes in the intestinal epithelium related to barrier function. Butyrate, a microbial metabolite produced by fermentation of dietary fiber, bolsters intestinal barrier function against enteric pathogens, and these effects can be transferred in utero via the placenta to the developing fetus. Our aim was to determine if butyrate mitigates GBS disruption of intestinal barriers. We used human intestinal epithelial cell (IEC) lines to evaluate the impact of butyrate on GBS-induced cell death and GBS adhesion and invasion. IECs and human fetal tissue-derived enteroids were used to evaluate monolayer permeability. We evaluated the impact of maternal butyrate treatment (mButyrate) using our established mouse model of neonatal GBS intestinal colonization and late-onset sepsis. We found that butyrate reduces GBS-induced cell death, GBS invasion, monolayer permeability, and translocation in vitro. In mice, mButyrate decreases GBS intestinal burden in offspring. Our results demonstrate the importance of bacterial metabolites, such as butyrate, in their potential to bolster epithelial barrier function and mitigate neonatal sepsis risk.IMPORTANCEGroup B Streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. It is a commensal of the intestines that can translocate across barriers leading to sepsis in vulnerable newborns. With the rise in antibiotic-resistant strains and no licensed vaccine, there is an urgent need for preventative strategies. Butyrate, a short-chain fatty acid metabolized in the gut, enhances barrier function against pathogens. Importantly, butyrate is transferred in utero, conferring these benefits to infants. Here, we demonstrate that butyrate reduces GBS colonization and epithelial invasion. These effects were not microbiome-driven, suggesting butyrate directly impacts epithelial barrier function. Our results highlight the potential impact of maternal dietary metabolites, like butyrate, as a strategy to mitigate neonatal sepsis risk.

Studies have demonstrated the importance of the gut microbiota during pregnancy, and there is emerging literature on the postpartum maternal gut microbiota. The primary objective of this paper was to synthesize the literature on the postpartum gut microbiome composition and diversity measured in stool samples from healthy mothers of predominantly term infants. The secondary objectives were (1) to identify biological and environmental factors that influence postpartum maternal gut microbiota and (2) to assess health conditions and clinical intermediate measures associated with postpartum gut microbiota changes in all mothers. Electronic searches were conducted November 9, 2020 and updated July 25, 2021 without publication time limits on PubMed, Embase, CINHAL, Scopus, Cochrane Library, BioArchives, and OpenGrey.eu. Primary research on maternal gut microbiota in the postpartum (up to one year after childbirth) were eligible. Postpartum gut microbiota comparisons to pregnancy or non-pregnancy gut microbiota were of interest, therefore, studies examining these in addition to the postpartum were included. Studies were excluded if they were only conducted in animals, infants, pregnancy, or microbiome of other body locations (e.g., vaginal). Data extraction of microbial composition and diversity were completed and synthesized narratively. Studies were assessed for risk of bias. A total of 2512 articles were screened after deduplication and 27 were included in this review. Of the 27 included studies, 22 addressed the primary objective. Firmicutes was the predominant phylum in the early (<6 weeks) and late postpartum (6 weeks to 1 year). In early postpartum, Bacteroides was the predominant genus. Findings from longitudinal assessments of alpha and beta diversity from the early to the late postpartum varied. Nineteen of the 27 studies assessed biological and environmental factors influencing the postpartum gut microbial profile changes. Timing of delivery, probiotic supplementation, triclosan exposure, and certain diets influenced the postpartum gut microbiota. Regarding health conditions and intermediate clinical measures assessed in 8 studies; inflammatory bowel disease, postpartum depression, early-onset preeclampsia, gestational diabetes, excessive gestational weight gain, and anthropometric measures such as body mass index and waist-to-hip ratio were related to gut microbiota changes. There is limited data on the maternal postpartum gut microbiota and how it influences maternal health. We need to understand the postpartum maternal gut microbiome, establish how it differs from non-pregnancy and pregnancy states, and determine biological and environmental influencers. Future research of the gut microbiome's significance for the birthing parent in the postpartum could lead to a new understanding of how to improve maternal short and long-term health.

Background: Human milk diet, preferably mother's own milk (MOM) over donor milk (DM), is recommended for preterm infants. Expression of MOM in proximity to preterm infants, especially during or immediately after skin-to-skin contact (SSC), is associated with greater milk production. However, the correlation between SSC and MOM production during hospital admission in preterm infants has not yet been studied. Our study investigated the relationship between SSC and MOM production and consumption in preterm infants during the first postnatal month of life. Materials and Methods: This was a prospective cohort study. Mothers and their preterm infants born at <35 weeks by gestational age (GA) and eligible for SSC within the first 5 postnatal days were eligible for the study. Mothers were given a binder to document pumped breast milk volumes and SSC sessions. Pumped breast milk volumes, enteral feeding type and volume, and SSC duration and frequency were collected daily over the first 28 days of life, along with demographic, perinatal, and feeding data from electronic medical records (EMR). Results: Mean birth GA and weight were 30 ± 3 weeks and 1,443 ± 576 g, respectively. SSC duration was inversely correlated with GA and weight. The SSC duration was positively correlated with ingested MOM volume after correcting for birth GA. The SSC duration was predictive of increased volumes of pumped MOM. Conclusion: Our findings suggest that SSC duration is associated with improved MOM production and consumption. SSC can be a useful tool to increase MOM exposure and improve long-term health outcomes in preterm infants.

BACKGROUND: Preterm (PT) infants harbor a different gut microbiome than full-term infants. Multiple factors affect gut microbial colonization of PT infants, including low gestational age, high rates of Cesarean section, exposure to antibiotics, and diet. Human milk, whether it's mother's own milk (MOM) or donor human milk, is the preferred feeding mode for PT infants but needs to be fortified to achieve adequate nutrient content. Infant formulas are introduced at later stages if human milk is insufficient or unavailable. How these dietary exposures affect the gut microbiome of PT infants is poorly understood.

OBJECTIVES: The goal of this study was to evaluate the metagenomic potential of the fecal microbiome of PT infants consuming MOM with bovine milk-based fortifier compared with PT formula alone.

METHODS: Forty-two stool samples, from 27 infants consuming MOM or formula (21 samples in each group) were included. Twelve infants had repeated sampling (2-3 samples). Shotgun genomic DNA sequencing was performed and analyzed using MetaPhlAn and HUMAnN2. Multivariate regression analysis, adjusting by the repeated sampling, was used to identify the features that differed between PT infants consuming MOM compared with formula.

RESULTS: The primary function of the fecal microbiome of PT infants was characterized by a high abundance of biosynthesis pathways. A set of core features was identified; these belonged to pathways for amino acid metabolism and vitamin K-2 biosynthesis. Five pathways significantly differed between the MOM and formula group. Pathways for fatty acid and carbohydrate degradation were significantly higher in the MOM group. Taxonomically, members of the phylum Actinobacteria and the genus Bifidobacterium were higher in PT infants exposed to MOM.

CONCLUSIONS: This study provides insight into the influence of feeding MOM compared with infant formula on the structure and function of the fecal microbiome of PT infants.

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

BACKGROUND: Preterm infants are at high risk for growth failure and childhood weight problems due to the disruption of normal intrauterine growth and nutrition. Early nutritional support and microbiome acquisition can play an important role in childhood growth.

OBJECTIVE: Our study examined potential postnatal indicators, including gut bacterial compositions, macronutrients, and catch-up growth, of growth pattern from infancy into early childhood.

METHODS: This is a retrospective study of preterm infants born < 35 weeks who were followed up in the university complex care clinic from 2012-2018. Weight and length z-scores at birth, 1, 2, 4, 6, 12 and 15 months, and body mass index (BMI) and length z-scores from 2 to 5 years of age were collected. Catch-up growths were calculated by changes in z-scores and divided into early (birth-4 months) and late (4-18 months). Postnatal nutritional data and fecal samples were collected. Fecal microbiome data obtained from 16S RNA V4 sequencing was analyzed against clinical and growth data using a regression model.

RESULTS: 160 infants included in the final analysis had birth weight and gestational age of 1,149 ± 496 grams and 28 ± 3 weeks. Early weight gain positively correlated with length z-scores but not with BMI at 2 years of age. BMI at 2 years of age strongly correlated with BMI at 3, 4, and 5 years of age. Postnatal abundance of Gammaproteobacteria was negatively associated with early growth while Bacteroides and Lactobacillus were positively associated with childhood BMI.

CONCLUSION: Our findings suggest that optimal postnatal nutrition promoted early catch-up growth in weight as well as improved linear growth without influence on childhood BMI. Postnatal gut microbial colonization, which is a modifiable factor, was associated with childhood growth in preterm infants.

OBJECTIVE: Feeding intolerance (FI) is a common presentation of necrotizing enterocolitis (NEC) and sepsis. NEC and sepsis are associated with hematological changes, but these changes alone are not reliable biomarkers for early diagnosis. This study examined whether the combination of hematological indices and FI can be used as an early diagnostic tool for NEC or sepsis.

STUDY DESIGN: This retrospective cohort study included infants born at <1,500 g or <30 weeks who had symptoms of FI. The exclusion criteria were congenital or chromosomal disorders, thrombocytopenia or platelet transfusion before the onset of FI, and history of bowel resection. We compared the hematological indices from infants with pathologic FI (due to NEC or sepsis) to infants with benign FI.

RESULTS: During the study period, 211 infants developed FI; 185 met the inclusion criteria. Infants with pathologic FI (n = 90, 37 cases with NEC and 53 with sepsis) had lower birth gestational age and weight compared with 95 infants with benign FI (n = 95). Pathologic FI was associated with lower platelet count (median 152 × 103/μL vs. 285 × 103/μL, p < 0.001) and higher immature-to-total neutrophil (I/T) ratio (median 0.23 vs. 0.04, p < 0.001) at the onset of FI. Pathologic FI was also associated with a decrease in baseline platelets compared with an increase in benign FI. For diagnosis of pathologic FI, a decrease ≥10% in platelets from baseline had a sensitivity and specificity of 0.64 and 0.73, respectively, I/T ratio ≥0.1 had a sensitivity and specificity of 0.71 and 0.78, respectively, and the combination of both parameters had a sensitivity and specificity of 0.50 and 0.97, respectively.

CONCLUSION: FI caused by NEC or sepsis was associated with a decrease in platelets from baseline, and a lower platelet level and higher I/T ratio at the onset of FI. These findings can help clinicians in the management of preterm infants with FI.

KEY POINTS: · FI is a common presentation of NEC and sepsis in preterm infants.. · FI due to NEC or sepsis is associated with changes in platelets and I/T ratio.. · These changes could be useful as early markers for diagnosis..

OBJECTIVES: The aim of the study was to compare the intestinal microbiome in very low birth weight (VLBW) infants who received different enteral iron supplementation (EIS) doses.

STUDY DESIGN: Longitudinal stool collection in 80 VLBW infants were conducted up to 2 months postnatally in a prospective study. The 16S rRNA regions V4 was used to calculate microbiome compositions and the Piphillin software was used for bacterial functional prediction. Linear mixed effect models and Wilcoxon rank-sum tests were performed to examine the relationships between initial EIS dosage and stool microbiome and bacterial functional potential.

RESULTS: There were 105 samples collected before and 237 collected after EIS started from infants with birth gestational age and weight of 28.1 ± 2.4 weeks and 1103 ± 210 g, respectively. The average postnatal age at start of EIS was 17.9 ± 6.9 days and the average initial EIS dose was 4.8 ± 1.1 mg · kg-1 · day-1. Infants who were started on ≥6 mg · kg-1 · day-1 had higher abundances of Proteus and Bifidobacterium and a lower alpha diversity than those started on lower doses (P < 0.05). Infants given higher EIS doses had higher bacterial predicted functional potentials for ferroptosis and epithelial invasion after 2 weeks post EIS.

CONCLUSIONS: Higher EIS dosage is linked to higher abundances of Proteus and Bifidobacterium, and a less diverse microbiome and higher predicted potential of bacterial epithelial invasion. These observational findings should be further studied in a randomized study to elucidate the optimal dosage of EIS in VLBW infants.