Platelets and Immature Neutrophils in Preterm Infants with Feeding Intolerance.

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..

Enteric dysbiosis and fecal calprotectin expression in premature infants.

BACKGROUND: Premature infants often develop enteric dysbiosis with a preponderance of Gammaproteobacteria, which has been related to adverse clinical outcomes. We investigated the relationship between increasing fecal Gammaproteobacteria and mucosal inflammation, measured by fecal calprotectin (FC). METHODS: Stool samples were collected from very-low-birth weight (VLBW) infants at ≤2, 3, and 4 weeks' postnatal age. Fecal microbiome was surveyed using polymerase chain reaction amplification of the V4 region of 16S ribosomal RNA, and FC was measured by enzyme immunoassay. RESULTS: We enrolled 45 VLBW infants (gestation 27.9 ± 2.2 weeks, birth weight 1126 ± 208 g) and obtained stool samples at 9.9 ± 3, 20.7 ± 4.1, and 29.4 ± 4.9 days. FC was positively correlated with the genus Klebsiella (r = 0.207, p = 0.034) and its dominant amplicon sequence variant (r = 0.290, p = 0.003), but not with the relative abundance of total Gammaproteobacteria. Klebsiella colonized the gut in two distinct patterns: some infants started with low Klebsiella abundance and gained these bacteria over time, whereas others began with very high Klebsiella abundance. CONCLUSION: In premature infants, FC correlated with relative abundance of a specific pathobiont, Klebsiella, and not with that of the class Gammaproteobacteria. These findings indicate a need to define dysbiosis at genera or higher levels of resolution.

Skin to Skin Contact Correlated with Improved Production and Consumption of Mother's Own Milk.

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.

Maternal gut microbiota in the postpartum Period: A Systematic review.

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.

Metagenomic profile of the fecal microbiome of preterm infants consuming mother's own milk with bovine milk-based fortifier or infant formula: a cross-sectional study.

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.

Postnatal growth and gut microbiota development influenced early childhood growth 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.

A Pilot Study To Establish an In Vitro Model To Study Premature Intestinal Epithelium and Gut Microbiota Interactions.

Intestinal microbiota has emerged as an important player in the health and disease of preterm infants. The interactions between intestinal flora and epithelium can lead to local injury and systemic diseases. A suitable in vitro cell model is needed to enhance our understanding of these interactions. In this study, we exposed fetal epithelial cell cultures (FHs-74 int cells, human, ATCC CCL 241) to sterile fecal filtrates derived from stool collected from preterm infants at <2 and at 3 to 4 weeks of age. We measured the cytokine levels from the culture media after 4, 24, and 48 h of exposure to the fecal filtrates. We analyzed the 16S rRNA V4 gene data of the fecal samples and transcriptome sequencing (RNA-seq) data from the fetal epithelial cells after 48 h of exposure to the same fecal filtrates. The results showed correlations between inflammatory responses (both cytokine levels and gene expression) and the Proteobacteria-to-Firmicutes ratio and between fecal bacterial genera and epithelial apoptosis-related genes. Our in vitro cell model can be further developed and applied to study how the epithelium responds to different microbial flora from preterm infants. Combining immature epithelial cells and preterm infant stool samples into one model allows us to investigate disease processes in preterm infants in a way that had not been previously reported. IMPORTANCE The gut bacterial flora influences the development of the immune system and long-term health outcomes in preterm infants. Studies of the mechanistic interactions between the gut bacteria and mucosal barrier are limited to clinical observations, animal models, and in vitro cell culture models for this vulnerable population. Most in vitro cell culture models of microbe-host interactions use single organisms or adult origin cell lines. Our study is innovative and significant in that we expose immature epithelial cells derived from fetal tissues to fecal filtrates from eight stool samples from four preterm infants to study the role of intestinal epithelial cells. In addition, we analyzed epithelial gene expression to examine multiple cellular processes simultaneously. This model can be developed into patient-derived two- or three-dimensional cell cultures exposed to their own fecal material to allow better prediction of patient physiological responses to support the growing field of precision medicine.

A systematic review of the factors influencing microbial colonization of the preterm infant gut.

Prematurity coupled with the necessary clinical management of preterm (PT) infants introduces multiple factors that can interfere with microbial colonization. This study aimed to review the perinatal, physiological, pharmacological, dietary, and environmental factors associated with gut microbiota of PT infants. A total of 587 articles were retrieved from a search of multiple databases. Sixty studies were included in the review after removing duplicates and articles that did not meet the inclusion criteria. Review of this literature revealed that evidence converged on the effect of postnatal age, mode of delivery, use of antibiotics, and consumption of human milk in the composition of gut microbiota of PT infants. Less evidence was found for associations with race, sex, use of different fortifiers, macronutrients, and other medications. Future studies with rich metadata are needed to further explore the impact of the PT exposome on the development of the microbiota in this high-risk population.

Human Milk-Based or Bovine Milk-Based Fortifiers Differentially Impact the Development of the Gut Microbiota of Preterm Infants.

Background: Preterm infants are exposed to different dietary inputs during their hospitalization in the neonatal intensive care unit (NICU). These include human milk (HM), with a human milk-based (HMF) or a bovine milk-based (BMF) fortifier, or formula. Milk consumption and the type of fortification will cause changes in the gut microbiota structure of preterm infants. This study aimed to characterize the gut microbiota of PT infant according to the type of feeding and the type of HM fortification and its possible association with infant's growth. Methods: Ninety-seven infants born ≤33 wks of gestation or <1,500 g were followed during the hospitalization period in the NICU after birth until discharge. Clinical and dietary information was collected, including mode of delivery, pregnancy complications, mechanical ventilation, use of antibiotics, weight, and type and amount of milk consumed. To characterize the gut microbiota composition, weekly stool samples were collected from study participants. The V3-V4 region of the 16S rRNA bacterial gene was Sequenced using Illumina MiSeq technology. Results: After birth, black maternal race, corrected gestational age (GA) and exposure to pregnancy complications, had a significant effect on gut microbial diversity and the abundance of Enterococcus, Veillonella, Bifidobacterium, Enterobacter, and Bacteroides. Over the course of hospitalization, corrected GA and exposure to chorioamnionitis remained to have an effect on gut microbial composition. Two different enterotypes were found in the gut microbiota of preterm infants. One enriched in Escherichia-Shigella, and another enriched in uncharacterized Enterobacteriaceae, Klebsiella and Clostridium sensu stricto 1. Overall, HM and fortification with HMF were the most common feeding strategies. When consuming BMF, PT infants had higher growth rates than those consuming HMF. Milk and type of fortification were significantly associated with the abundance of Clostridium sensu stricto 1, Bifidobacterium and Lactobacillus. Conclusions: This observational study shows the significant association between milk consumption and the exposure to HMF or BMF fortification in the fecal microbiota composition of preterm infants. Additionally, these results show the effect of other perinatal factors in the establishment and development of PT infant's gut microbiota.

The development of intestinal dysbiosis in anemic preterm infants.

OBJECTIVE: Anemia and Proteobacteria-dominant intestinal dysbiosis in very low birth weight (VLBW) infants have been linked to necrotizing enterocolitis, a severe gut inflammatory disease. We hypothesize that anemia of prematurity is related to the development of intestinal dysbiosis. STUDY DESIGN: Three hundred and forty-two weekly stool samples collected prospectively from 80 VLBW infants were analyzed for bacterial microbiomes (with 16S rRNA). Linear mixed-effects model was used to determine the relationships between the onsets of anemia and intestinal dysbiosis. RESULTS: Hematocrit was associated with intestinal microbiomes, with lower Hct occurring with increased Proteobacteria and decreased Firmicutes. Infants with a hematocrit <30% had intestinal microbiomes that diverged toward Proteobacteria dominance and low diversity after the first postnatal month. The microbiome changes were also related to the severity of anemia. CONCLUSIONS: This finding supports a potential microbiological explanation for anemia as a risk factor for intestinal dysbiosis in preterm infants.

Contributors to Dysbiosis in Very-Low-Birth-Weight Infants.

The objective of this commentary was to analyze the causes and outcomes of gut microbiome dysbiosis in preterm infants who are born at very low birth weight (VLBW). The intrauterine development of VLBW infants is interrupted abruptly with preterm birth and followed by extrauterine, health-threatening conditions and sequelae. These infants develop intestinal microbial dysbiosis characterized by low diversity, an overall reduction in beneficial and/or commensal bacteria, and enrichment of opportunistic pathogens of the Gammaproteobacteria class. The origin of VLBW infant dysbiosis is not well understood and is likely the result of a combination of immaturity and medical care. We propose that these factors interact to produce inflammation in the gut, which further perpetuates dysbiosis. Understanding the sources of dysbiosis could result in interventions to reduce gut inflammation, decrease enteric pathology, and improve health outcomes for these vulnerable infants.

Longitudinal Microbiome Composition and Stability Correlate with Increased Weight and Length of Very-Low-Birth-Weight Infants.

The microbiomes of 83 preterm very-low-birth-weight (VLBW) infants and clinical covariates were analyzed weekly over the course of their initial neonatal intensive care unit (NICU) stay, with infant growth as the primary clinical outcome. Birth weight significantly correlated with increased rate of weight gain in the first 6 weeks of life, while no significant relationship was observed between rate of weight gain and feeding type. Microbial diversity increased with age and was significantly correlated with weight gain and percentage of the mother's own milk. As expected, infants who received antibiotics during their NICU stay had significantly lower alpha diversity than those who did not. Of those in the cohort, 25 were followed into childhood. Alpha diversity significantly increased between NICU discharge and age 2 years and between age 2 years and age 4 years, but the microbial alpha diversity of 4-year-old children was not significantly different from that of mothers. Infants who showed improved length over the course of their NICU stay had significantly more volatile microbial beta diversity results than and a significantly decreased microbial maturity index compared with infants who did not; interestingly, all infants who showed improved length during the NICU stay were delivered by Caesarean section. Microbial beta diversity results were significantly different between the time of the NICU stay and all other time points (for children who were 2 or 4 years old and mothers when their children were 2 or 4 years old). IMPORTANCE Preterm infants are at greater risk of microbial insult than full-term infants, including reduced exposure to maternal vaginal and enteric microbes, higher rates of formula feeding, invasive procedures, and administration of antibiotics and medications that alter gastrointestinal pH. This investigation of the VLBW infant microbiome over the course of the neonatal intensive care unit (NICU) stay, and at ages 2 and 4 years, showed that the only clinical variables associated with significant differences in taxon abundance were weight gain during NICU stay (Klebsiella and Staphylococcus) and antibiotic administration (Streptococcus and Bifidobacterium). At 2 and 4 years of age, the microbiota of these VLBW infants became similar to the mothers' microbiota. The number of microbial taxa shared between the infant or toddler and the mother varied, with least the overlap between infants and mothers. Overall, there was a significant association between the diversity and structure of the microbial community and infant weight and length gain in an at-risk childhood population.

Dichotomous development of the gut microbiome in preterm infants.

BACKGROUND: Preterm infants are at risk of developing intestinal dysbiosis with an increased proportion of Gammaproteobacteria. In this study, we sought the clinical determinants of the relative abundance of feces-associated Gammaproteobacteria in very low birth weight (VLBW) infants. Fecal microbiome was characterized at ≤ 2 weeks and during the 3rd and 4th weeks after birth, by 16S rRNA amplicon sequencing. Maternal and infant clinical characteristics were extracted from electronic medical records. Data were analyzed by linear mixed modeling and linear regression. RESULTS: Clinical data and fecal microbiome profiles of 45 VLBW infants (gestational age 27.9 ± 2.2 weeks; birth weight 1126 ± 208 g) were studied. Three stool samples were analyzed for each infant at mean postnatal ages of 9.9 ± 3, 20.7 ± 4.1, and 29.4 ± 4.9 days. The average relative abundance of Gammaproteobacteria was 42.5% (0-90%) at ≤ 2 weeks, 69.7% (29.9-86.9%) in the 3rd, and 75.5% (54.5-86%) in the 4th week (p < 0.001). Hierarchical and K-means clustering identified two distinct subgroups: cluster 1 started with comparatively low abundance that increased with time, whereas cluster 2 began with a greater abundance at ≤ 2 weeks (p < 0.001) that decreased over time. Both groups resembled each other by the 3rd week. Single variants of Klebsiella and Staphylococcus described variance in community structure between clusters and were shared between all infants, suggesting a common, hospital-derived source. Fecal Gammaproteobacteria was positively associated with vaginal delivery and antenatal steroids. CONCLUSIONS: We detected a dichotomy in gut microbiome assembly in preterm infants: some preterm infants started with low relative gammaproteobacterial abundance in stool that increased as a function of postnatal age, whereas others began with and maintained high abundance. Vaginal birth and antenatal steroids were identified as predictors of Gammaproteobacteria abundance in the early (≤ 2 weeks) and later (3rd and 4th weeks) stool samples, respectively. These findings are important in understanding the development of the gut microbiome in premature infants.

Cytokines and growth factors in the developing intestine and during necrotizing enterocolitis.

Cytokines and growth factors play diverse roles in the uninflamed fetal/neonatal intestinal mucosa and in the development of inflammatory bowel injury during necrotizing enterocolitis (NEC). During gestational development and the early neonatal period, the fetal/premature intestine is exposed to high levels of many "inflammatory" cytokines and growth factors, first via swallowed amniotic fluid in utero and then, after birth, in colostrum and mother's milk. This article reviews the dual, seemingly counter-intuitive roles of cytokines, where these agents play a "trophic" role and promote maturation of the uninflamed mucosa, but can also cause inflammation and promote intestinal injury during NEC.