Multi-kingdom ecological drivers of microbiota assembly in preterm infants.

The gut microbiota of preterm infants develops predictably1-7, with pioneer species colonizing the gut after birth, followed by an ordered succession of microorganisms. The gut microbiota is vital to the health of preterm infants8,9, but the forces that shape these predictable dynamics of microbiome assembly are unknown. The environment, the host and interactions between microorganisms all potentially shape the dynamics of the microbiota, but in such a complex ecosystem, identifying the specific role of any individual factor is challenging10-14. Here we use multi-kingdom absolute abundance quantification, ecological modelling and experimental validation to address this challenge. We quantify the absolute dynamics of bacteria, fungi and archaea in a longitudinal cohort of 178 preterm infants. We uncover microbial blooms and extinctions, and show that there is an inverse correlation between bacterial and fungal loads in the infant gut. We infer computationally and demonstrate experimentally in vitro and in vivo that predictable assembly dynamics may be driven by directed, context-dependent interactions between specific microorganisms. Mirroring the dynamics of macroscopic ecosystems15-17, a late-arriving member of the microbiome, Klebsiella, exploits the pioneer microorganism, Staphylococcus, to gain a foothold within the gut. Notably, we find that interactions between different kingdoms can influence assembly, with a single fungal species-Candida albicans-inhibiting multiple dominant genera of gut bacteria. Our work reveals the centrality of simple microbe-microbe interactions in shaping host-associated microbiota, which is critical both for our understanding of microbiota ecology and for targeted microbiota interventions.

Proteomics-Based Mapping of Bronchopulmonary Dysplasia-Associated Changes in Noninvasively Accessible Oral Secretions.

OBJECTIVE: To determine if oral secretions (OS) can be used as a noninvasively collected body fluid, in lieu of tracheal aspirates (TA), to track respiratory status and predict bronchopulmonary dysplasia (BPD) development in infants born <32 weeks. STUDY DESIGN: This was a retrospective, single center cohort study that included data and convenience samples from week-of-life (WoL) 3 from 2 independent preterm infant cohorts. Using previously banked samples, we applied our sample-sparing, high-throughput proteomics technology to compare OS and TA proteomes in infants born <32 weeks admitted to the Neonatal Intensive Care Unit (NICU) (Cohort 1; n = 23 infants). In a separate similar cohort, we mapped the BPD-associated changes in the OS proteome (Cohort 2; n = 17 infants including 8 with BPD). RESULTS: In samples collected during the first month of life, we identified 607 proteins unique to OS, 327 proteins unique to TA, and 687 overlapping proteins belonging to pathways involved in immune effector processes, neutrophil degranulation, leukocyte mediated immunity, and metabolic processes. Furthermore, we identified 37 OS proteins that showed significantly differential abundance between BPD cases and controls: 13 were associated with metabolic and immune dysregulation, 10 of which (eg, SERPINC1, CSTA, BPI) have been linked to BPD or other prematurity-related lung disease based on blood or TA investigations, but not OS. CONCLUSIONS: OS are a noninvasive, easily accessible alternative to TA and amenable to high-throughput proteomic analysis in preterm newborns. OS samples hold promise to yield actionable biomarkers of BPD development, particularly for prospective categorization and timely tailored treatment of at-risk infants with novel therapies.

Fecal Elastase in Preterm Infants to Predict Growth Outcomes.

OBJECTIVES: Preterm infants are born functionally pancreatic insufficient with decreased pancreatic production of lipase and proteases. Developmental pancreatic insufficiency (PI) may contribute to reduced nutrient absorption and growth failure. We sought to determine longitudinal fecal elastase (ELA1) levels in a cohort of preterm infants and whether levels are associated with growth outcomes. METHODS: Prospective observational study of 30 infants 24-34 weeks gestational age and birth weight ≤1250 g fed the exclusive human milk diet, consisting of human milk with human milk-based fortifier. ELA1 was quantified by ELISA during the first 2 weeks of life [Early; 7.5 ± 1.8 days of life (DOL)] and after attainment of full, fortified feedings (Late; 63.6 ± 24.1 DOL). RESULTS: Early ELA1 levels were 192.2 ± 96.4 µg/g, and Late ELA1 levels were 268.0 ± 80.3 µg/g, 39.4% higher (P = 0.01). Infants with early PI (ELA1 < 200 µg/g) were more likely male and of lower gestational age, weight, length, and head circumference at birth. These variables, but not PI status, independently predicted somatic growth. CONCLUSIONS: Fecal ELA1 in preterm infants fed exclusive human milk diet increases with postnatal age. Although pancreatic function in preterm infants may serve as a biological contributor to early postnatal growth failure, additional studies using fecal ELA1 as a predictive biomarker for growth failure are needed in larger cohorts.

Acetaminophen and Xenobiotic Metabolites in Human Milk and the Development of Bronchopulmonary Dysplasia and Retinopathy of Prematurity in a Cohort of Extremely Preterm Infants.

This retrospective cohort study sought to identify the association between certain xenobiotic metabolites in maternal breast milk and the diagnoses of bronchopulmonary dysplasia and retinopathy of prematurity in extremely preterm infants. Several acetaminophen metabolites were associated with a 3- to 6-fold increased odds of these disorders, and metabolites of certain food products, benzoate, and caffeine were associated with decreased odds.

Systemic Inflammation in the First 2 Weeks after Birth as a Determinant of Physical Growth Outcomes in Hospitalized Infants with Extremely Low Gestational Age.

OBJECTIVE: To examine associations of systemic inflammation with growth outcomes at neonatal intensive care unit discharge or transfer among infants with extremely low gestational ages. STUDY DESIGN: We studied 850 infants at born at 23-27 weeks of gestation. We defined inflammatory protein elevation as the highest quartile of C-reactive protein (CRP), Interleukin (IL)-6, tumor necrosis factor-∝, or IL-8 on postnatal days 1, 7, and 14. We compared z-scores of weight, length, and head circumference at neonatal intensive care unit discharge or transfer between infants with vs without inflammatory protein elevation, adjusting in linear regression for birth size z-score, sex, gestational age, diet, comorbidities, medications, and length of hospitalization. RESULTS: The mean gestational age was 25 weeks (range, 23-27 weeks) and birth weight z-score 0.14 (range, -2.73 to 3.28). Infants with a high CRP on day 7 had lower weights at discharge or transfer (-0.17 z-score; 95% CI, -0.27 to -0.06) than infants without CRP elevation, with similar results on day 14. Infants with CRP elevation on day 14 were also shorter (-0.21 length z-scores; 95% CI, -0.38 to -0.04), and had smaller head circumferences (-0.18 z-scores; 95% CI, -0.33 to -0.04) at discharge or transfer. IL-6 elevation on day 14 was associated with lower weight (-0.12; 95% CI, -0.22 to -0.02); IL-6 elevation on day 7 was associated with shorter length (-0.27; 95% CI, -0.43 to -0.12). Tumor necrosis factor-∝ and IL-8 elevation on day 14 were associated with a lower weight at discharge or transfer. CONCLUSIONS: Postnatal systemic inflammation may contribute to impaired nutrient accretion during a critical period in development in infants with extremely low gestational ages.

Parenteral lipid emulsions induce unique ileal fatty acid and metabolomic profiles but do not increase the risk of necrotizing enterocolitis in preterm pigs.

Necrotizing enterocolitis (NEC) is a manifestation of maladaptive intestinal responses in preterm infants centrally medicated by unattenuated inflammation. Early in the postnatal period, preterm infants develop a deficit in arachidonic and docosahexaenoic acid, both potent regulators of inflammation. We hypothesized that the fatty acid composition of parenteral lipid emulsions uniquely induces blood and intestinal fatty acid profiles which, in turn, modifies the risk of NEC development. Forty-two preterm pigs were randomized to receive one of three lipid emulsions containing 100% soybean oil (SO), 15% fish oil (MO15), or 100% fish oil (FO100) with enteral feedings over an 8-day protocol. Blood and distal ileum tissue were collected for fatty acid analysis. The distal ileum underwent histologic, proteomic, and metabolomic analyses. Eight pigs [3/14 SO (21%), 3/14 MO15 (21%), and 2/14 FO100 (14%)] developed NEC. No differences in NEC risk were evident between groups despite differences in induced fatty acid profiles in blood and ileal tissue. Metabolomic analysis of NEC versus no NEC tissue revealed differences in tryptophan metabolism and arachidonic acid-containing glycerophospholipids. Proteomic analysis demonstrated no differences by lipid group; however, 15 proteins differentiated NEC versus no NEC in the domains of tissue injury, glucose uptake, and chemokine signaling. Exposure to parenteral lipid emulsions induces unique intestinal fatty acid and metabolomic profiles; however, these profiles are not linked to a difference in NEC development. Metabolomic and proteomic analyses of NEC versus no NEC intestinal tissue provide mechanistic insights into the pathogenesis of NEC in preterm infants.NEW & NOTEWORTHY Exposure to parenteral lipid emulsions induces unique intestinal fatty acid and metabolomic profiles; however, these profiles are not linked to a difference in NEC risk in preterm pigs. Metabolomic and proteomic analyses provide mechanistic insights into NEC pathogenesis. Compared with healthy ileal tissue, metabolites in tryptophan metabolism and arachidonic acid-containing glycerophospholipids are increased in NEC tissue. Proteomic analysis differentiates NEC versus no NEC in the domains of tissue injury, glucose uptake, and chemokine signaling.

Developmental Accretion of Docosahexaenoic Acid Is Independent of Fatty Acid Transporter Expression in Brain and Lung Tissues of C57BL/6 and Fat1 Mice.

BACKGROUND: Developmental expression of fatty acid transporters and their role in polyunsaturated fatty acid concentrations in the postnatal period have not been evaluated. OBJECTIVE: We hypothesized that transporter expression is developmentally regulated, tissue-specific, and that expression can modulate fatty acid accretion independently of diet. METHODS: Brain and lung transporter expression were quantified in C57BL/6 wild-type (WT) and Fat1 mice. Pups were dam-fed until day 21. Dams were fed AIN-76A 10% corn oil to represent a typical North American/European diet. After weaning, mice were fed the same diet as dams. Gene expression of Fatp1, Fatp4, Fabp5, and Fat/cd36 was quantified by quantitative reverse transcriptase-polymerase chain reaction. Fatty acid concentrations were measured by GC-MS. RESULTS: Brain docosahexaenoic acid (DHA) concentrations increased from day 3 to day 28 in both genotypes, with higher concentrations at days 3 and 14 in Fat1 than in WT mice [median (IQR)]: 10.7 (10.6-11.2) mol% compared with 6.6 (6.4-7.2) mol% and 12.5 (12.4-12.9) mol% compared with 8.9 (8.7-9.1) mol%, respectively; P < 0.05). During DHA accrual, transporter expression decreased. Fold changes in brain Fatp4, Fabp5, and Fat/cd36 were inversely correlated with fold changes in brain DHA concentrations in Fat1 relative to WT mice (ρ = -0.85, -0.75, and -0.78, respectively; P ≤ 0.001). Lung DHA concentrations were unchanged across the 3 time points for both genotypes. Despite unchanging DHA concentrations, there was increased expression of Fatp1 at days 14 and 28 (5-fold), Fatp4 at day 14 (2.3-fold), and Fabp5 at day 14 (3.8-fold) relative to day 3 in Fat1 mice. In WT mice, Fatp1 increased almost 5-fold at day 28 relative to day 3. There was no correlation between lung transporters and DHA concentrations in Fat1 relative to WT mice. CONCLUSIONS: Development of fatty acid transporter expression in C57BL/6 WT and Fat1 mice is genotype and tissue specific. Further, postnatal accretion of brain DHA appears independent of transporter status, with tissue concentrations representing dietary contributions.

Effect of polyunsaturated fatty acids on postnatal ileum development using the fat-1 transgenic mouse model.

BACKGROUND: Long-chain polyunsaturated fatty acids (LCPUFAs) play a critical role in neonatal health. We hypothesized that LCPUFAs play an essential role in priming postnatal gut development. We studied the effect of LCPUFAs on postnatal gut development using fat-1 transgenic mice, which are capable of converting n-6 to n-3 LCPUFAs, and wild-type (WT) C57BL/6 mice. METHODS: Distal ileum sections were collected from fat-1 and WT mice on days 3, 14, and 28. Fatty acid analyses, histology, RT-qPCR and intestinal permeability were performed. RESULTS: Fat-1 mice, relative to WT mice, showed increased n-3 LCPUFAs levels (α-linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid, p < 0.05) and decreased arachidonic acid levels (p < 0.05) in the ileum. Preweaning fat-1 mice, compared to WT, showed >50% reduced muc2, Tff3, TLR9, and Camp expression (p < 0.05), markers of the innate immune response. There was a >two-fold increased expression of Fzd5 and EphB2, markers of cell differentiation (p < 0.05), and Fabp2 and 6, regulators of fatty acid transport and metabolism (p < 0.05). Despite reduced expression of tight junction genes, intestinal permeability in fat-1 was comparable to WT mice. CONCLUSIONS: Our data support the hypothesis that fatty acid profiles early in development modulate intestinal gene expression in formative domains, such as cell differentiation, tight junctions, other innate host defenses, and lipid metabolism.

Early Serum Gut Hormone Concentrations Associated With Time to Full Enteral Feedings in Preterm Infants.

OBJECTIVES: The primary objective of this study was to evaluate early postnatal serum gut hormone concentrations in preterm infants as predictors of time to full enteral feedings. The secondary objective was to identify infant characteristics and nutritional factors that modulate serum gut hormone concentrations and time to full enteral feedings. METHODS: Sixty-four preterm infants less than 30 weeks of gestation were included in this retrospective cohort study. Serum gut hormone concentrations at postnatal days 0 and 7 were measured using enzyme-linked immunosorbent assays. Linear regression and mediation analyses were performed. RESULTS: Median (interquartile range) serum concentrations of glucose-dependent insulinotropic peptide (GIP) and peptide YY (PYY) on postnatal day 7 were 31.3 pg/mL (18.2, 52.3) and 1181.7 pg/mL (859.0, 1650.2), respectively. GIP and PYY concentrations on day 7 were associated with days to full enteral feedings after adjustment for confounders (ß = -1.1, P = 0.03; and ß = -0.002, P = 0.02, respectively). Nutritional intake was correlated with serum concentrations of GIP and PYY on postnatal day 7 and time to full enteral feedings. Mediation analysis revealed that the effect of serum gut hormone concentrations on time to full enteral feedings was not fully explained by nutritional intake. Intrauterine growth restriction, mechanical ventilation on postnatal day 7, and patent ductus arteriosus treated with indomethacin were associated with longer time to full enteral feedings. CONCLUSIONS: Serum concentrations of GIP and PYY on postnatal 7 are independently associated with time to full enteral feedings. The link between serum gut hormone concentrations and time to full enteral feedings is not fully mediated by nutritional factors, suggesting an independent mechanism underlying the influence of gut hormones on feeding tolerance and time to full enteral feedings.

A prospective analysis of intake and composition of mother's own milk in preterm newborns less than 32 weeks' gestational age.

Background Mothers of preterm (PT) infants have difficulty providing adequate quantities of human milk (HM) for their babies during their hospital stay. The macronutrient content in HM changes over time, varying across and within individual mothers. The research aim of the strudy was to describe the intake of mothers' own milk (MOM) and its composition according to gestational (GA) and postnatal age (PNA) in infants born <32 weeks' GA and to correlate them with neonatal weight, length and morbidities. Methods A prospective observational study of 176 premature infants in a unit without a donor milk bank was conducted. Daily milk intake was recorded. HM macronutrients were determined by mid-infrared spectrophotometric analysis at 7, 15 and 30 days after delivery and monthly until hospital discharge. Results Intake of MOM increased during the first 2 weeks after birth and decreased steadily thereafter. Protein concentration varied inversely with PNA. Carbohydrate and lipid concentrations increased over the first few days and remained stable thereafter. A fall in weight percentiles from birth to 60 days was found. No correlation was found between total protein and calorie intakes at 3 and 15 days of life and growth velocity (GV) between 15 and 30 days, even when broken down into parenteral nutrition (PN), formula and MOM. Conclusion To improve MOM feeding in PT newborns, intensive support strategies at the prenatal stage along entire hospitalization income should be encouraged. New protocols for fortification of HM should be implemented to optimize postnatal weight gain while preserving the health benefits of HM.

Practical guide to exocrine pancreatic insufficiency - Breaking the myths.

BACKGROUND: Exocrine pancreatic insufficiency (EPI) is characterized by a deficiency of exocrine pancreatic enzymes, resulting in malabsorption. Numerous conditions account for the etiology of EPI, with the most common being diseases of the pancreatic parenchyma including chronic pancreatitis, cystic fibrosis, and a history of extensive necrotizing acute pancreatitis. Treatment for EPI includes dietary management, lifestyle changes (i.e., decrease in alcohol consumption and smoking cessation), and pancreatic enzyme replacement therapy. DISCUSSION: Many diagnostic tests are available to diagnose EPI, however, the criteria of choice remain unclear and the causes for a false-positive test are not yet understood. Despite multiple studies on the treatment of EPI using exogenous pancreatic enzymes, there remains confusion amongst medical practitioners with regard to the best approach to diagnose EPI, as well as dosing and administration of pancreatic enzymes. Appropriate use of diagnostics and treatment approaches using pancreatic enzymes in EPI is essential for patients. This opinion piece aims to address the existing myths, remove the current confusion, and function as a practical guide to the diagnosis and treatment of EPI.

Defining the diagnostic value of hyperlipasemia for acute pancreatitis in the critically ill.

BACKGROUND/OBJECTIVES: Hyperlipasemia is frequently encountered in patients in the intensive care unit (ICU). The degree to which it should be valued in making the diagnosis of acute pancreatitis (AP) in critically ill patients remains uncertain. We sought to determine the diagnostic accuracy of hyperlipasemia and the optimal lipase cutoff for diagnosing AP in critically ill patients. METHODS: Four hundred and seventeen ICU patients with hyperlipasemia, defined as lipase greater than three times the upper limit of normal from 2009 to 2012 were retrospectively identified. A diagnosis of AP was confirmed by the additional presence of either characteristic abdominal pain or cross-sectional imaging. RESULTS: The overall positive predictive value (PPV) of hyperlipasemia was 38.1%. Median initial lipase levels were 1164 IU/L in patients with AP and 284.5 IU/L in patients without AP (p < 0.001). The optimal diagnostic lipase cutoff of 532 IU/L correlated with a sensitivity, specificity, negative predictive value and PPV of 77.4%, 78.0%, 84.9%, and 67.0% respectively. The most common primary diagnoses in non-AP patients with elevated lipase included shock, cardiac arrest and malignancy. CONCLUSIONS: Physicians should maintain caution when interpreting hyperlipasemia in the critically ill due its relatively low PPV. However, a greater lipase cutoff improves its diagnostic value in AP and helps to reduce unnecessary imaging in these patients.

Association of the gut microbiota mobilome with hospital location and birth weight in preterm infants.

BackgroundThe preterm infant gut microbiota is vulnerable to different biotic and abiotic factors. Although the development of this microbiota has been extensively studied, the mobilome-i.e. the mobile genetic elements (MGEs) in the gut microbiota-has not been considered. Therefore, the aim of this study was to investigate the association of the mobilome with birth weight and hospital location in the preterm infant gut microbiota.MethodsThe data set consists of fecal samples from 62 preterm infants with and without necrotizing enterocolitis (NEC) from three different hospitals. We analyzed the gut microbiome by using 16S rRNA amplicon sequencing, shot-gun metagenome sequencing, and quantitative PCR. Predictive models and other data analyses were performed using MATLAB and QIIME.ResultSThe microbiota composition was significantly different between NEC-positive and NEC-negative infants and significantly different between hospitals. An operational taxanomic unit (OTU) showed strong positive and negative correlation with NEC and birth weight, respectively, whereas none showed significance for mode of delivery. Metagenome analyses revealed high levels of conjugative plasmids with MGEs and virulence genes. Results from quantitative PCR showed that the plasmid signature genes were significantly different between hospitals and in NEC-positive infants.ConclusionOur results point toward an association of the mobilome with hospital location in preterm infants.

Factors Determining Optimal Fatty Acid Absorption in Preterm Infants.

OBJECTIVES: The aim of the present study was to quantify absorption coefficients of specific fatty acids in preterm infants as a function of diet, formula or breast milk (BM), and postnatal age; to identify the fatty acid structural characteristics that determine optimal fatty acid absorption. METHODS: Fatty acids from dietary and fecal samples were extracted and quantified by gas chromatography-mass spectroscopy. Fatty acid absorption coefficients (FA-CFAs) were calculated by comparing the total amount of fatty acids supplied by the diet to the amount quantified in the total fecal output during a 3-day period. RESULTS: A total of 18 infants (BM 8, formula 10) were studied at 2 weeks of age, and 20 infants (BM 10, formula 10) were studied at 6 weeks of age. FA-CFAs decreased with increasing carbon length in formula-fed infants at 2 and 6 weeks. Results were similar but less in magnitude in BM-fed infants at 2 weeks with no difference at 6 weeks. CONCLUSIONS: Preterm infants fed formula demonstrated lower FA-CFAs as a function of increasing carbon length. This is consistent with limited pancreatic lipase production and with lipase being present in BM but not in formula. The fact that this pattern was seen in BM-fed infants at 2 weeks but not 6 weeks of age suggests that intestinal immaturity may also play a role in impaired fatty acid absorption. These data highlight principles that need to be considered to optimize delivery and absorption of dietary long-chain polyunsaturated fatty acids in preterm infants.

Comparison of Nasal Potential Difference and Intestinal Current Measurements as Surrogate Markers for CFTR Function.

OBJECTIVES: Nasal potential difference (NPD) measurement is part of the diagnostic criteria for cystic fibrosis (CF) and now used routinely as an endpoint in clinical trials of correcting the basic defect in CF. Intestinal current measurement (ICM), measured ex vivo on a rectal biopsy, has been used to study cystic fibrosis transmembrane conductance regulator (CFTR) function but has not been compared to NPD in the same subject in adults and children. The aim of the study is to evaluate the potential usefulness of ICM as a marker of CFTR function for treatment studies compared NPD in patients with CF and in healthy control subjects. METHODS: ICM and NPD were performed on healthy controls and patients with CF. The healthy adults were individuals undergoing routine screening colonoscopy at the Beth Israel Deaconess Medical Center. The healthy children were undergoing colonoscopy for suspicion of inflammation in Hadassah Hebrew University Medical Center. The CF adults were recruited from Boston Children's Hospital CF Center and CF Center Worcester Mass, the children with CF from Hadassah CF Center. RESULTS: ICM measurements in healthy control subjects (n = 16) demonstrated a mean (±SE) carbachol response of 16.0 (2.2) µA/cm, histamine response of 13.2 (2.1) µA/cm and a forskolin response of 6.3 (2.0) µA/cm. Basal NPD of -15.9 (1.9) and response to Cl free + isoproterenol of -13.8 (2.0). These responses were inverted in CF subjects (n = 12) for ICM parameters with carbachol response of -3.0 (0.5) µA/cm, histamine -1.0 (0.8) µA/cm and a forskolin response of 0.5 (0.3) and also for NPD parameters; basal NPD of -42.2 (4.3) and response to Cl free + isoproterenol of 4.3 (0.7). Pearson correlation test showed the comparability of ICM and NPD in assessing CFTR function. CONCLUSIONS: ICM is equivalent to NPD in the ability to distinguish patients with CF from controls and could be used as surrogate markers of CFTR activity in treatment protocols.

A digital twin of the infant microbiome to predict neurodevelopmental deficits.

Despite the recognized gut-brain axis link, natural variations in microbial profiles between patients hinder definition of normal abundance ranges, confounding the impact of dysbiosis on infant neurodevelopment. We infer a digital twin of the infant microbiome, forecasting ecosystem trajectories from a few initial observations. Using 16S ribosomal RNA profiles from 88 preterm infants (398 fecal samples and 32,942 abundance estimates for 91 microbial classes), the model (Q-net) predicts abundance dynamics with R2 = 0.69. Contrasting the fit to Q-nets of typical versus suboptimal development, we can reliably estimate individual deficit risk (Mδ) and identify infants achieving poor future head circumference growth with ≈76% area under the receiver operator characteristic curve, 95% ± 1.8% positive predictive value at 98% specificity at 30 weeks postmenstrual age. We find that early transplantation might mitigate risk for ≈45.2% of the cohort, with potentially negative effects from incorrect supplementation. Q-nets are generative artificial intelligence models for ecosystem dynamics, with broad potential applications.