Over half of presumed genetic disease cases remain undiagnosed following short-read exome sequencing (SR-ES) or genome sequencing (SR-GS). Long-read GS (LR-GS) shows promise for uncovering etiologies missed by SR genetic testing, particularly structural variants (SVs). However, SV interpretation remains challenging due to limitations in call reliability, population allele frequency estimates, and functional impact prediction. To advance clinical LR-GS implementation, we analyzed the genomes of 19 children with suspected rare genetic conditions and prior negative or inconclusive clinical SR-GS/SR-ES as well as their parents using PacBio HiFi LR-GS. One additional family with limited DNA underwent Illumina SR-GS only, and 11 probands received SR-GS to complement small-variant detection. LR-GS data were processed using phased-assembly and read-based variant-calling pipelines validated in SV-positive control subjects, while SR-GS data were processed with the Illumina DRAGEN pipeline. Variants were prioritized using phenotype-driven approaches. Diagnostic variants (likely pathogenic or pathogenic) were identified in 2/20 (10%) families, while an additional 5/20 (25%) harbored findings of uncertain diagnostic significance, including variants of uncertain significance (VUSs) and variants in genes of uncertain significance (GUSs). All reported variants were detected independently of LR-GS by research SR-GS or by reanalysis of prior clinical SR data. Several LR-GS SV candidates were excluded after population allele frequency filtering, underscoring its importance in clinical SV interpretation. Overall, the observed 10% increase in diagnostic yield was achievable through SR analysis alone, as LR-GS was not required to identify diagnostic variants in this cohort. Functional studies are needed to clarify the clinical relevance of uncertain findings.

To evaluate the associations between parental/family early relational contact in the neonatal intensive care unit (NICU) and the post-discharge childcare quality and behavioural development up to 18-24 months of corrected age (CA). In a longitudinal cohort study (2017-2022), 215 preterm infants were followed. Early relational NICU contact (minutes/day) was measured daily using a 12-item observational checklist. Post-discharge childcare quality was assessed at 18-24 months with the Index of Child Care Environment (ICCE). Behavioural development was assessed using the Bayley Scales of Infant Development III and the Brief Infant-Toddler Social and Emotional Assessment. Multiple regression models examined the associations between these key variables, adjusting for clinical and demographic confounders. The cohort was predominantly male (57.67%), non-Hispanic (74.88%) and White (67.44%), with an average gestational age of 28.3 weeks. At 18-24-month CA, greater early skin-to-skin/soothing contact was linked to better language development (beta = 0.33, p = 0.032), and integrated nurturing contact (characterised by holding combined with verbal interaction) was associated with better language and motor development in female infants (p's < 0.05); strong social support for caregivers was associated with infants' improved cognitive (beta = 0.364, p = 0.018), language (beta = 0.383, p = 0.008) and motor (beta = 0.382, p = 0.015) outcomes. Infants with typical social-emotional competence received higher levels of human stimulation from their caregivers compared with those showing possible competence issues (OR = 1.439, p = 0.020). Greater early NICU contact and higher post-discharge childcare quality are associated with improved developmental outcomes in preterm infants at 18-24 months CA, showing the growing importance of environmental factors in infants' development. Future studies should explore targeted interventions that enhance early bonding and empower parents to support sustained developmental progress.

Objective To identify sex-specific feeding patterns and associations with growth and neurodevelopment in preterm infants during NICU through 2 years of corrected age (CA). Methods A cohort study was conducted with 216 preterm infants (gestational age 28 0/7 to 32 0/7 weeks). Daily feeding regimens, including mother's own milk (MOM), human donor milk, and formula; daily growth; acute and chronic pain/stress were documented during NICU. NICU Network Neurobehavioral Scale (NNNS) (36 to 38 postmenstrual age), and Bayley Scales of Infant and Toddler Development (Bayley) Edition III (1 and 2 years of CA) were measured. Results Between week 9 to 16 after birth, only females showed a positive association between growth z-score and proportion of MOM intake before week 8 (p < 0.05). Sex-differentiated associations between MOM and stress were observed (p < 0.05). MOM proportion was positively correlated with language or cognitive scores at 2 years of CA in females (p = 0.01), this correlation not evident in males. Conclusions We discovered a sex-specific "window of opportunity" for feeding, growth and risk predictors for neurodevelopment up to 2 years of CA. These insights may inform development of tailored feeding regimens, potentially mitigating growth and development differences observed between males and females.

Even with state-of-the-art infection control practices, premature infants can develop life-threatening infections in the neonatal intensive care unit (NICU). The precise sources of most NICU-associated infections frequently remain unknown and, therefore, are difficult to address. In this study, we used a novel microbiome sequencing approach to source-track lethal sepsis-causing Klebsiella, opportunistic pathogens, and commensal bacterial strains colonizing the gut of hospitalized premature infants. An exploratory-methods, case series was at performed Connecticut Children’s Medical Center NICU in 2021. Long-read 16–23 S rRNA gene sequencing was used to analyze fecal samples, mother’s milk, and clinical bacterial isolates derived from a cluster of Klebsiella-infected, and concurrently hospitalized non-infected, premature infants who were simultaneously enrolled in a neonatal microbiome study. Distinct groups of amplicons comprising a unique fingerprint pattern for a given strain were compared among the samples to ascertain relatedness. We confirmed 100

P21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)-a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G>A, p.(D425N), by genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes observed in this individual were global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, failure to thrive, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical phenotypic spectrum.

This study evaluated associations between early neonatal experiences during neonatal intensive care unit (NICU) hospitalization and the development of pain sensitivity, measured by flexion withdrawal reflex (FWR) thresholds, through 18-24 months corrected age in preterm infants. This longitudinal study (2017-2022) in Northeast U.S. specialized level III and IV NICUs monitored preterm infants for approximately 4-6 weeks for NICU pain/stress exposure (using the NICU Infant Stressor Scale - NISS) and analgesic use, assessing FWR thresholds post-discharge at 1, 4, 8-12, and 18-24 months of corrected age. 122 very preterm infants (63.1% male, 82.0% non-Black, 70.5% non-Hispanic) were enrolled, with a mean gestational age of 28.2 ± 2.4 weeks. The mean daily weighted NISS was 90.9 ± 19.9 (acute: 70.7; chronic: 20.2). FWR thresholds declined over time (1.11-0.7 g), suggesting increased population-level sensitivity. However, higher individual cumulative pain/stress exposure at NICU was associated with higher FWR thresholds (lower sensitivity) over time (β = 0.039, p = 0.042). The longitudinal effect of the pain/stress exposures (NISS score) on pain sensitivity (FWR) increased markedly over time in Black females, but was not observed in the Black male, Non-Black female, and Non-Black male subgroups (p < 0.05). Neonatal pain/stress exposure appears to reprogram long-term pain sensitivity development. These findings underscore the critical need for optimized pain management and tailored neuroprotective strategies for all high-risk infants. PERSPECTIVE: The FWR thresholds reflect the maturation of the nociceptive system in preterm infants from birth through 18-24 months of age. Neonatal cumulative pain/stress exposure is associated with altered maturation of FWR thresholds.

Mitochondrial DNA copy number (mtDNAcn) is associated with mitochondrial function contributing various diseases. Understanding of the association between mtDNAcn and infant neurodevelopment, as well as the role of racial disparities, remains unknown. A cohort study was conducted with 55 preterm infants and a single blood sample was collected from each infant during their NICU stay to measure mtDNAcn. NICU Network Neurobehavioral Scale (NNNS) and Bayley Scale of Infant Developmental testing Edition III was assessed during NICU until 2 years of corrected age (CA). Linear regression models were performed to investigate the relationship between the clinical characteristics, neurobehavioral outcomes and mtDNAcn. Majority of our subjects were male, White, non-Hispanic, had C-section, and without preterm premature rupture of membrane (PPROM). Increased mtDNAcn was found associated with younger birth gestational age (GA), nonoptimal neurodevelopment. However, the opposite associations between mtDNAcn and neurodevelopmental outcomes were observed between Black and White infants up to 1 year of CA. Adverse early life experience, together with increased mtDNAcn in White infants, and decreased mtDNAcn in Black infants may be considered as significant positive predictors of poor early life neurodevelopmental outcomes in infants.

SUMMARYP21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelicPAK2variants cause Knobloch syndrome type 2 (KNO2)—a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novelde novoheterozygous missense variant inPAK2, NM_002577.4:c.1273G>A, p.(D425N), by whole genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes include global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, FTT, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging byin silicoanalysis. Previous clinical genetic testing did not report this variant due to unknown relevance ofPAK2variants at the time of testing, highlighting the importance of reanalysis. Our findings also substantiate the candidacy ofPAK2variants in KNO2 and expand the KNO2 clinical spectrum.

OBJECTIVES/GOALS: Early life pain/stress impacts infants’ neurodevelopmental outcomes. Mitochondrial dysfunction may interface between infants’ stress and neurodevelopment. The study aims to investigate the associations between pain/stress, proteins associated with mitochondrial dysfunction, and neurobehavioral responses in preterm infants. METHODS/STUDY POPULATION: A prospective cohort study was conducted with 33 preterm infants enrolled between September 2017 and July 2022 at two affiliated NICUs in Hartford and Farmington, CT. Daily pain/stress experienced during NICU was documented. At 36-38 weeks post-menstrual age (PMA), neurobehavioral outcomes were evaluated using the NICU Network Neurobehavioral Scale (NNNS) and buccal swabs for Mass spectrometry-based proteomics analysis. Lasso statistical methods were conducted to study the association between protein abundance and infants’ NNNS summary scores. Multiple linear regression and Gene Ontology (GO) enrichment analyses were performed to examine how clinical characteristics and neurodevelopmental outcomes may be associated with protein levels and underlying molecular pathways. RESULTS/ANTICIPATED RESULTS: During NICU hospitalization, preterm premature rupture of membrane (PPROM) was negatively associated with neurobehavioral outcomes. The protein functions, including leptin receptor binding activity, glutathione disulfide oxidoreductase activity, and response to oxidative stress, lipid metabolism, phosphate, and proton transmembrane transporter activity, were negatively associated with neurobehavioral outcomes. In contrast, cytoskeletal regulation, epithelial barrier, and protection function were found to be positively associated with neurodevelopmental outcomes. In addition, mitochondrial dysfunction-related proteins (SPRR2A, PAIP1, S100A3, MT-CO2, PiC, GLRX, PHB2, and BNIPL-2, ABLIM1, UNC45A, Keratins, MUC1, and CYB5B) were found to be associated with neurobehavioral outcomes. DISCUSSION/SIGNIFICANCE: Mitochondrial dysfunction-related proteins were observed to be associated with early life pain/stress and neurodevelopmental outcomes in infants. Buccal proteins could be used to predict potential neurobehavioral outcomes. In addition, individualized skin integrity protection should be provided to preterm infants during their NICU stay.

INTRODUCTION:Preterm infants experience tremendous early life pain/stress during their neonatal intensive care unit (NICU) hospitalization, which impacts their neurodevelopmental outcomes. Mitochondrial function/dysfunction may interface between perinatal stress events and neurodevelopment. Nevertheless, the specific proteins or pathways linking mitochondrial functions to pain-induced neurodevelopmental outcomes in infants remain unidentified. Our study aims to investigate the associations among pain/stress, proteins associated with mitochondrial function/dysfunction, and neurobehavioral responses in preterm infants. METHODS:We conducted a prospective cohort study, enrolling 33 preterm infants between September 2017 and July 2022 at two affiliated NICUs located in Hartford and Farmington, CT. NICU Network Neurobehavioral Scale (NNNS) datasets were evaluated to explore potential association with neurobehavioral outcomes. The daily pain/stress experienced by infant's during their NICU stay was documented. At 36-38 weeks post-menstrual age (PMA), neurobehavioral outcomes were evaluated using the NNNS and buccal swabs were collected for further analysis. Mass spectrometry-based proteomics was conducted on epithelial cells obtained from buccal swabs to evaluate protein expression level. Lasso statistical methods were conducted to study the association between protein abundance and infants' NNNS summary scores. Multiple linear regression and Gene Ontology (GO) enrichment analyses were performed to examine how clinical characteristics and neurodevelopmental outcomes may be associated with protein levels and underlying molecular pathways. RESULTS:During NICU hospitalization, preterm premature rupture of membrane (PPROM) was negatively associated with neurobehavioral outcomes. The protein functions including leptin receptor binding activity, glutathione disulfide oxidoreductase activity and response to oxidative stress, lipid metabolism, and phosphate and proton transmembrane transporter activity were negatively associated with neurobehavioral outcomes; in contrast, cytoskeletal regulation, epithelial barrier, and protection function were found to be associated with the optimal neurodevelopmental outcomes. In addition, mitochondrial function-associated proteins including SPRR2A, PAIP1, S100A3, MT-CO2, PiC, GLRX, PHB2, and BNIPL-2 demonstrated positive association with favorable neurodevelopmental outcomes, while proteins of ABLIM1, UNC45A, keratins, MUC1, and CYB5B showed positive association with adverse neurodevelopmental outcomes. CONCLUSION:Mitochondrial function-related proteins were observed to be associated with early life pain/stress and neurodevelopmental outcomes in infants. Large-scale studies with longitudinal datasets are warranted. Buccal proteins could be used to predict potential neurobehavioral outcomes.

P21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)-a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G>A, p.(D425N), by whole genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes include global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, FTT, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings also substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical spectrum.

Intestinal colonization with Klebsiella has been linked to necrotizing enterocolitis (NEC), but methods of analysis usually failed to discriminate Klebsiella species or strains. A novel ~ 2500-base amplicon (StrainID) that spans the 16S and 23S rRNA genes was used to generate amplicon sequence variant (ASV) fingerprints for Klebsiella oxytoca and Klebsiella pneumoniae species complexes (KoSC and KpSC, respectively) and co-occurring fecal bacterial strains from 10 preterm infants with NEC and 20 matched controls. Complementary approaches were used to identify cytotoxin-producing isolates of KoSC. Klebsiella species colonized most preterm infants, were more prevalent in NEC subjects versus controls, and replaced Escherichia in NEC subjects. Single KoSC or KpSC ASV fingerprinted strains dominated the gut microbiota, suggesting exclusionary Klebsiella competition for luminal resources. Enterococcus faecalis was co-dominant with KoSC but present infrequently with KpSC. Cytotoxin-producing KoSC members were identified in most NEC subjects and were less frequent in controls. Few Klebsiella strains were shared between subjects. We conclude that inter-species Klebsiella competition, within an environment of KoSC and E. faecalis cooperation, appears to be an important factor for the development of NEC. Preterm infants seem to acquire Klebsiella primarily through routes other than patient-to-patient transmission.

IntroductionBlack African American (B/AA) women have a 2-fold to 3-fold elevated risk compared with non-Hispanic White (W) women for preterm birth. Further, preterm birth is the leading cause of mortality among B/AA infants, and among survivors, preterm infant adverse health outcomes occur disproportionately in B/AA infants. Racial inequities in maternal and infant health continue to pose a public health crisis despite the discovery >100 years ago. The purpose of this study was to expand on reported preterm infant outcome disparities. A life-course approach, accumulation of lifelong stress, including discrimination, may explain social factors causing preterm birth rate and outcome inequities in B/AA mothers.MethodsAnthropometric measures and clinical treatment information for 197 consented participants were milled from electronic health records across 4 years. The Neonatal Infant Stressor Scale was used to tally acute and chronic painful/stressful procedures. Neurobehavioral differences were investigated using the Neonatal Intensive Care Unit (NICU) Network Neurobehavioral Scale.ResultsB/AA mothers gave birth to preterm infants earlier than W mothers. NICU hospitalization stays were extended more than 2 weeks for the significantly smaller B/AA preterm infants in comparison to the age-matched W preterm infants. A higher number of chronic lifesaving procedures with demonstrated altered stress response patterns were recorded for B/AA preterm infants.DiscussionThis cross-sectional analysis of preterm birth rates and preterm infant developmental and neurodevelopmental outcomes are presented in the context of NICU stress and pain, with attendant implications for infant mortality and future health disparities. Preterm birth rate and outcome inequities further support the need to develop interventions and policies that will reduce the impact of discrimination and improve social determinants of health for Black, Indigenous, and other People of Color.

Asthma is associated with significant morbidity. The gut microbiome has been shown to effect asthma development and exacerbation. In this study, we tested an oral supplement containing Boswellia serrata (Indian frankincense) tree resin on allergic pulmonary inflammation and gut microbiome. An OVA based allergic airway model was used and mice were orally gavaged 100 mg/kg of Boswellia serrata as a supplementation throughout asthma sensitization and challenge. Treated mice showed significant weight loss, lower total lung leukocytes, eosinophil and Th2 cytokines, improved histology scoring and reduced reactivity to methacholine challenge. Asthmatic mice without Boswellia serrata supplementation showed a decrease in overall bacterial diversity, while treated mice were protected against loss. Boswellia serrata treated mice had a significant increase in Bifidobacterium, which was identified as Bifidobacterium pseudolongum. Oral administration of B. pseudolongum also reduced airway inflammation, suggesting Boswellia serrata may work as an anti-asthma agent via increases in B. pseudolongum from prebiotic influences.

Objective:Preterm infants are subjected to numerous painful procedures during their neonatal intensive care unit (NICU) hospitalization. Despite advancements in pain alleviation, nurses remain challenged to provide timely and effective pain management for preterm infants. Greater understanding of the lived experience of nurses caring for preterm infants in pain could provide novel insights to improve pain management for this vulnerable population. The aim of this meta-ethnography was to synthesize and interpret qualitative findings of nurses' experiences of taking care of preterm infants in pain. Methods:An extensive literature search in PubMed, CINAHL, PsycINFO, Scopus, BIOSIS and ProQuest Dissertation and Theses Database was conducted, including studies within the past 10 years. Two nursing researchers conducted data extraction and analysis independently. Inclusion criteria were applied to search for qualitative studies of nurse participants who worked in the NICU taking care of preterm infants. Studies published in a language other than English, articles that did not include qualitative data and qualitative data that could not be extracted from the findings or did not discuss nurses' experiences were excluded. Critical Appraisal Skills Programme was used for literature quality evaluation. Results:Eight studies remained after further screening according to inclusion and exclusion criteria. These eight studies were conducted from 2013 to 2018 and totally enrolled 205 nurses from Iran, Canada, the United States, Finland, Sweden, Switzerland, and Australia. Five themes emerged on the nurses' perspectives of taking care of preterm infants in pain: 1) They sense the neonatal pain; 2) Adverse consequences of unrelieved pain; 3) Barriers of managing pain; 4) Concerns of available approaches for pain relief; 5) Failure to work with parents. Conclusions:This meta-ethnography identified nurses' understanding of pain in preterm infants that can be assessed, and they acknowledged that unrelieved pain could cause developmental deficits in infants. The barriers are lack of training and support on pain assessment and intervention in preterm infants. Optimizing workload and environment, developing age-specified pain assessment and intervention, receiving emotional support and training, and building up a rapport with parents are urgent needs for nurses to provide better care to infants having pain.

Pharmacological activation of the xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding their physiological functions on the gut microbiome. In this study, we discovered bivalent hormetic functions of PXR/CAR modulating the richness of the gut microbiome using genetically engineered mice. The absence of PXR or CAR increased microbial richness, and absence of both receptors synergistically increased microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria Helicobacteraceae and Helicobacter. Deficiency in both PXR and CAR increased the relative abundance of Lactobacillus, which has bile salt hydrolase activity, corresponding to decreased primary taurine-conjugated bile acids (BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. The basal effect of PXR/CAR on the gut microbiome was distinct from pharmacological and toxicological activation of these receptors. Common PXR/CAR-targeted bacteria were identified, the majority of which were suppressed by these receptors. hPXR-TG mice had a distinct microbial profile as compared to wild-type mice. This study is the first to unveil the basal functions of PXR and CAR on the gut microbiome.

Early life stress is commonly experienced by infants, especially preterm infants, and may impact their neurodevelopmental outcomes in their early and later lives. Mitochondrial function/dysfunction may play an important role underlying the linkage of prenatal and postnatal stress and neurodevelopmental outcomes in infants. This review aimed to provide insights on the relationship between early life stress and neurodevelopment and the mechanisms of mitochondrial function/dysfunction that contribute to the neuropathology of stress. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement was used to develop this systematic review. PubMed, Scopus, PsycINFO, and Biosis databases were searched for primary research articles published between 2010 and 2021 that examined the relationships among mitochondrial function/dysfunction, infant stress, and neurodevelopment. Thirty studies were identified. There is evidence to support that mitochondrial function/dysfunction mediates the relationship between prenatal and postnatal stress and neurodevelopmental outcomes in infants. Maternal transgenerational transmission of mitochondrial bioenergetic patterns influenced prenatal stress induced neurodevelopmental outcomes and behavioral changes in infants. Multiple functionally relevant mitochondrial proteins, genes, and polymorphisms were associated with stress exposure. This is the first review of the role that mitochondrial function/dysfunction plays in the association between stress and neurodevelopmental outcomes in full-term and preterm infants. Although multiple limitations were found based on the lack of data on the influence of biological sex, and due to invasive sampling, and lack of longitudinal data, many genes and proteins associated with mitochondrial function/dysfunction were found to influence neurodevelopmental outcomes in the early life of infants.

The human gut harbors a complex community of microbes, including several species and strains that could be commensals or pathogens depending on context. The specific environmental conditions under which a resident microbe changes its relationship with a host and adopts pathogenic behaviors, in many cases, remain poorly understood.

Background The gut microbiome is an important determinant of health and disease in preterm infants. Objectives The objective of this article was to share our current protocol for other neonatal intensive care units to potentially expand their existing protocols, aiming to characterize the relationship between the intestinal microbiome and health outcomes in preterm infants. Methods This prospective, longitudinal study planned to recruit 160 preterm infants born <32 weeks gestational age or weighing <1,500 g and admitted to one of two Level III/IV neonatal intensive care units. During the neonatal intensive care unit period, the primary measures included events of early life pain/stress, gut microbiome, host genetic variations, and neurobehavioral assessment. During follow-up visits, gut microbiome; pain sensitivity; and medical, growth, and developmental outcomes at 4, 8–12, and 18–24 months corrected age were measured. Discussion As of February 14, 2020, 214 preterm infants have been recruited. We hypothesize that infants who experience greater levels of pain/stress will have altered gut microbiome, including potential adverse outcomes such as necrotizing enterocolitis and host genetic variations, feeding intolerance, and/or neurodevelopmental impairments. These will differ from the intestinal microbiome of preterm infants who do not develop these adverse outcomes. To test this hypothesis, we will determine how alterations in the intestinal microbiome affect the risk of developing necrotizing enterocolitis, feeding intolerance, and neurodevelopmental impairments in preterm infants. In addition, we will examine the interaction between the intestinal microbiome and host genetics in the regulation of intestinal health and neurodevelopmental outcomes.

Achieving strain-level resolution is a major obstacle for source tracking and temporal studies of microbiomes. In this study, we describe a novel deep-sequencing approach that provides species- and strain-level resolution of the neonatal microbiome.