Azithromycin therapy for prevention of chronic lung disease of prematurity (AZTEC): a multicentre, double-blind, randomised, placebo-controlled trial.

BACKGROUND: Systematic reviews have reported conflicting evidence on whether macrolide antibiotics reduce rates of chronic lung disease of prematurity (CLD) in at-risk preterm infants born at less than 30 weeks' gestation, including in those colonised with pulmonary Ureaplasma spp. Since an adequately powered trial has been lacking, we aimed to assess if the macrolide azithromycin improved survival without the development of physiologically defined moderate or severe CLD in preterm infants. METHODS: AZTEC was a multicentre, double-blind, randomised, placebo-controlled trial conducted in 28 tertiary neonatal intensive care units in the UK. Infants were eligible if they were born at less than 30 weeks' gestation and had received at least 2 h of either non-invasive (continuous positive airway pressure or humidified high flow nasal cannula therapy) or invasive respiratory support (via endotracheal tube) within 72 h of birth. Eligible infants were randomly allocated in a 1:1 ratio using random permuted blocks of four to receive either intravenous azithromycin at 20 mg/kg per day for 3 days followed by 10 mg/kg for 7 days, or to placebo. Allocation was stratified by centre and gestational age at birth (<28 weeks vs ≥28 weeks). Azithromycin and placebo vials were encased in tamper-evident custom cardboard cartons to ensure masking for clinicians, parents, and the research team. The primary outcome was survival without development of physiologically defined moderate or severe CLD at 36 weeks' postmenstrual age. Outcomes and safety were analysed on an intention-to-treat basis (all randomly allocated infants, regardless of any post-randomisation events). The study was registered with ISRCRN (11650227) and is closed. FINDINGS: Infants were recruited between Oct 9, 2019, and March 22, 2022. 799 (53·1%) of 1505 eligible infants underwent random allocation; three infants were withdrawn, including consent to use their data, leaving 796 infants for analysis. Survival without moderate or severe CLD occurred in 166 (42%) of 394 infants in the intervention group and 179 (45%) of 402 in the placebo group (three-level adjusted OR [aOR] 0·84, 95% CI 0·55-1·29, p=0·43). Pulmonary Ureaplasma spp colonisation did not influence treatment effect. Overall, seven serious adverse events were reported for the azithromycin group (five graded as severe, two as moderate), and six serious adverse events were reported in the placebo group (two severe, two moderate, and two mild), as assessed by the local principal investigators. INTERPRETATION: Since prophylactic use of azithromycin did not improve survival without development of physiologically-defined CLD, regardless of Ureaplasma spp colonisation, it cannot be recommended in clinical practice. FUNDING: UK National Institute for Health and Care Research.

The core phageome and its interrelationship with preterm human milk lipids.

Phages and lipids in human milk (HM) may benefit preterm infant health by preventing gastrointestinal pathobiont overgrowth and microbiome modulation. Lipid association may promote vertical transmission of phages to the infant. Despite this, interrelationships between lipids and phages are poorly characterized in preterm HM. Shotgun metagenomics and untargeted lipidomics of phage and lipid profiles from 99 preterm HM samples reveals that phages are abundant and prevalent from the first week and throughout the first 100 days of lactation. Phage-host richness of preterm HM increases longitudinally. Core phage communities characterized by Staphylococcus- and Propionibacterium-infecting phages are significantly correlated with long-chain fatty acid abundances over lactational age. We report here a phage-lipid interaction in preterm HM, highlighting the potential importance of phage carriage in preterm HM. These results reveal possible strategies for phage carriage in HM and their importance in early-life microbiota development.

Age of onset of necrotising enterocolitis (NEC) and focal intestinal perforation (FIP) in very preterm and low birthweight infants: a systematic review.

OBJECTIVE: Review of age of onset of necrotising enterocolitis (NEC) and focal intestinal perforation (FIP) in very preterm (≤32 weeks) and/or very low birthweight (VLBW, ≤1500 g) infants. DESIGN: Preregistered review undertaken according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses in July 2021 and updated October 2021. DATA SOURCES: MEDLINE/ PubMed, Embase, CINAHL and Cochrane Central Register of Controlled Trials. ELIGIBILITY: Eligible studies reported age of onset of NEC and/or FIP in randomised controlled trials of >200 or observational studies of >500 infants. DATA EXTRACTION AND SYNTHESIS: Titles/abstracts were screened; eligible articles underwent data extraction. Age of onset as day of life (DOL) and/or corrected gestational age (CGA) were extracted alongside study information, such as NEC definition, included population, intervention, location and dates studied. Weighted means were used to compare onset by birth gestation, study type, NEC definition, trial intervention, location and dates studied. Comparison was done by Mann-Whitney U test or one-way analysis of variance. RESULTS: Of the 747 screened studies 188 were eligible. Removal of duplicates, studies without onset data and ineligible populations left 10 RCTs and 14 observational studies contributing 51 NEC cohorts; 49 reported onset DOL and 14 CGA. 2984 cases of NEC had average DOL onset of 16.7 (15.5 in RCTs, 16.9 in observational studies), and CGA onset of 30.1 weeks. Gestation did not impact DOL onset. No other demographic feature impacted NEC onset. Few studies included data on FIP. CONCLUSIONS: Average onset of NEC in exclusively very preterm/very low birthweight infants is in the third week of life and unlike in cohorts including more mature or heavier infants is not impacted by birth gestation.

How frequent is routine use of probiotics in UK neonatal units?

OBJECTIVE: There is a lack of UK guidance regarding routine use of probiotics in preterm infants to prevent necrotising enterocolitis, late-onset sepsis and death. As practices can vary, we aimed to determine the current usage of probiotics within neonatal units in the UK. DESIGN AND SETTING: Using NeoTRIPS, a trainee-led neonatal research network, an online survey was disseminated to neonatal units of all service levels within England, Scotland, Northern Ireland and Wales in 2022. Trainees were requested to complete one survey per unit regarding routine probiotic administration. RESULTS: 161 of 188 (86%) neonatal units responded to the survey. 70 of 161 (44%) respondents routinely give probiotics to preterm infants. 45 of 70 (64%) use the probiotic product Lactobacillus acidophilus NCFM/Bifidobacterium bifidum Bb-06/B. infantis Bi-26 (Labinic™). 57 of 70 (81%) start probiotics in infants ≤32 weeks' gestation. 33 of 70 (47%) had microbiology departments that were aware of the use of probiotics and 64 of 70 (91%) had a guideline available. Commencing enteral feeds was a prerequisite to starting probiotics in 62 of 70 (89%) units. The majority would stop probiotics if enteral feeds were withheld (59 of 70; 84%) or if the infant was being treated for necrotising enterocolitis (69 of 70; 99%). 24 of 91 (26%) units that did not use probiotics at the time of the survey were planning to introduce them within the next 12 months. CONCLUSIONS: More than 40% of all UK neonatal units that responded are now routinely administering probiotics, with variability in the product used. With increased probiotic usage in recent years, there is a need to establish whether this translates to improved clinical outcomes.

Particular genomic and virulence traits associated with preterm infant-derived toxigenic Clostridium perfringens strains.

Clostridium perfringens is an anaerobic toxin-producing bacterium associated with intestinal diseases, particularly in neonatal humans and animals. Infant gut microbiome studies have recently indicated a link between C. perfringens and the preterm infant disease necrotizing enterocolitis (NEC), with specific NEC cases associated with overabundant C. perfringens termed C. perfringens-associated NEC (CPA-NEC). In the present study, we carried out whole-genome sequencing of 272 C. perfringens isolates from 70 infants across 5 hospitals in the United Kingdom. In this retrospective analysis, we performed in-depth genomic analyses (virulence profiling, strain tracking and plasmid analysis) and experimentally characterized pathogenic traits of 31 strains, including 4 from CPA-NEC patients. We found that the gene encoding toxin perfringolysin O, pfoA, was largely deficient in a human-derived hypovirulent lineage, as well as certain colonization factors, in contrast to typical pfoA-encoding virulent lineages. We determined that infant-associated pfoA+ strains caused significantly more cellular damage than pfoA- strains in vitro, and further confirmed this virulence trait in vivo using an oral-challenge C57BL/6 murine model. These findings suggest both the importance of pfoA+ C. perfringens as a gut pathogen in preterm infants and areas for further investigation, including potential intervention and therapeutic strategies.

Impact of Transfer for Surgical Management of Preterm Necrotising Enterocolitis or Focal Intestinal Perforation.

OBJECTIVE: To compare outcomes after surgically managed necrotising enterocolitis (NEC) and focal intestinal perforation (FIP) in infants <32 weeks requiring transfer to or presenting in a single surgical centre. DESIGN: Retrospective review of transferred and inborn NEC or FIP, from January 2013 to December 2020. PATIENTS: 107 transfers with possible NEC or FIP contributed 92 cases (final diagnoses NEC (75) and FIP (17)); 113 inborn cases: NEC (84) and FIP (29). RESULTS: In infants with a final diagnosis of NEC, medical management after transfer was as common as when inborn (41% TC vs 54% p = 0.12). Unadjusted all-cause mortality was lower in inborn NEC (19% vs 27%) and FIP (10% vs 29%). In infants undergoing surgery unadjusted mortality attributable to NEC or FIP was lower if inborn (21% vs 41% NEC, 7% vs 24% FIP). In regression analysis of surgically treated infants, being transferred was associated with increased all-cause mortality (OR 2.55 (1.03-6.79)) and mortality attributable to NEC or FIP (OR 4.89 (1.80-14.97)). CONCLUSIONS: These data require replication, but if confirmed, suggest that focusing care for infants at highest risk of developing NEC or FIP in a NICU with on-site surgical expertise may improve outcomes.

Effect of an Exclusive Human Milk Diet on the Gut Microbiome in Preterm Infants: A Randomized Clinical Trial.

Importance: The effect of using an exclusive human milk diet compared with one that uses bovine products in preterm infants is uncertain, but some studies demonstrate lower rates of key neonatal morbidities. A potential mediating pathway is the gut microbiome. Objective: To determine the effect of an exclusive human milk diet on gut bacterial richness, diversity, and proportions of specific taxa in preterm infants from enrollment to 34 weeks' postmenstrual age. Design, Setting, and Participants: In this randomized clinical trial conducted at 4 neonatal intensive care units in the United Kingdom from 2017 to 2020, microbiome analyses were blind to group. Infants less than 30 weeks' gestation who had only received own mother's milk were recruited before 72 hours of age. Statistical analysis was performed from July 2019 to September 2021. Interventions: Exclusive human milk diet using pasteurized human milk for any shortfall in mother's own milk supply and human milk-derived fortifiers (intervention) compared with bovine formula and bovine-derived fortifier (control) until 34 weeks' postmenstrual age. Fortifier commenced less than 48 hours of tolerating 150 mL/kg per day. Main Outcomes and Measures: Gut microbiome profile including alpha and beta diversity, and presence of specific bacterial taxa. Results: Of 126 preterm infants enrolled in the study, 63 were randomized to control (median [IQR] gestation: 27.0 weeks [26.0-28.1 weeks]; median [IQR] birthweight: 910 g [704-1054 g]; 32 [51%] male) and 63 were randomized to intervention (median [IQR] gestation: 27.1 weeks [25.7-28.1 weeks]; median [IQR] birthweight: 930 g [733-1095 g]; 38 [60%] male); 472 stool samples from 116 infants were analyzed. There were no differences in bacterial richness or Shannon diversity over time, or at 34 weeks between trial groups. The exclusive human milk diet group had reduced relative abundance of Lactobacillus after adjustment for confounders (coefficient estimate, 0.056; P = .03), but not after false discovery rate adjustment. There were no differences in time to full feeds, necrotizing enterocolitis, or other key neonatal morbidities. Conclusions and Relevance: In this randomized clinical trial in preterm infants using human milk-derived formula and/or fortifier to enable an exclusive human milk diet, there were no effects on overall measures of gut bacterial diversity but there were effects on specific bacterial taxa previously associated with human milk receipt. These findings suggest that the clinical impact of human milk-derived products is not modulated via microbiomic mechanisms. Trial Registration: ISRCTN trial registry identifier: ISRCTN16799022.

Exploring Human Milk, Nutrition, Growth, and Breastfeeding Rates at Discharge(HUMMINGBIRD Study): a protocol for a pilot randomised controlled trial.

INTRODUCTION: Mother's own breast milk (MOM) is the optimal nutrition for preterm infants as it reduces the incidence of key neonatal morbidities and improves long-term outcomes. However, MOM shortfall is common and either preterm formula or pasteurised donor human milk (DHM) may be used, although practice varies widely. Limited data suggest that the use of DHM may impact maternal beliefs and behaviours and therefore breastfeeding rates. The aim of this pilot study is to determine if longer duration of DHM exposure increases breastfeeding rates, and if a randomised controlled trial (RCT) design is feasible. METHODS AND ANALYSIS: The Human Milk, Nutrition, Growth, and Breastfeeding Rates at Discharge (HUMMINGBIRD) Study is a feasibility and pilot, non-blinded RCT with a contemporaneous qualitative evaluation. Babies born less than 33 weeks' gestation or with birth weight <1500 g whose mothers intend to provide MOM are randomly assigned to either control (DHM used to make up shortfall until full feeds and preterm formula thereafter) or intervention (DHM used for shortfall until 36 weeks' corrected age or discharge if sooner). The primary outcome is breast feeding at discharge. Secondary outcomes include growth, neonatal morbidities, length of stay, breastfeeding self-efficacy and postnatal depression using validated questionnaires. Qualitative interviews using a topic guide will explore perceptions around use of DHM and analysed using thematic analysis. ETHICS APPROVAL AND DISSEMINATION: Nottingham 2 Research Ethics Committee granted approval (IRAS Project ID 281071) and recruitment commenced on 7 June 2021. Results will be disseminated in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ISRCTN57339063.

Mechanisms affecting the gut of preterm infants in enteral feeding trials: a nested cohort within a randomised controlled trial of lactoferrin.

OBJECTIVE: To determine the impact of supplemental bovine lactoferrin on the gut microbiome and metabolome of preterm infants. DESIGN: Cohort study nested within a randomised controlled trial (RCT). Infants across different trial arms were matched on several clinical variables. Bacteria and metabolite compositions of longitudinal stool and urine samples were analysed to investigate the impact of lactoferrin supplementation. SETTING: Thirteen UK hospitals participating in a RCT of lactoferrin. PATIENTS: 479 infants born <32 weeks' gestation between June 2016 and September 2017. RESULTS: 10 990 stool and 22 341 urine samples were collected. Analyses of gut microbiome (1304 stools, 201 infants), metabolites (171 stools, 83 infants; 225 urines, 90 infants) and volatile organic compounds (314 stools, 117 infants) were performed. Gut microbiome Shannon diversity at 34 weeks corrected age was not significantly different between infants in the lactoferrin (mean=1.24) or placebo (mean=1.06) groups (p=0.11). Lactoferrin receipt explained less than 1% variance in microbiome compositions between groups. Metabolomic analysis identified six discriminative features between trial groups. Hospital site (16%) and postnatal age (6%) explained the greatest variation in microbiome composition. CONCLUSIONS: This multiomic study identified minimal impacts of lactoferrin but much larger impacts of hospital site and postnatal age. This may be due to the specific lactoferrin product used, but more likely supports the findings of the RCT in which this study was nested, which showed no impact of lactoferrin on reducing rates of sepsis. Multisite mechanistic studies nested within RCTs are feasible and help inform trial interpretation and future trial design.

Early infancy growth, body composition and type of feeding in late and moderate preterms.

BACKGROUND: Late and moderate preterm (LMPT) infants are at risk for adverse later life outcomes. We determined the association between feeding method at enrolment and growth and body composition of LMPT infants until 3 months corrected age (3mCA). METHODS: Infants born between 32+0 and 36+6 weeks of gestation (n = 107) were enrolled up to 4 weeks corrected age and stratified according to feeding at enrolment. We performed anthropometric measurements at enrolment, term equivalent age (TEA) and 3mCA, including skinfold measurements and body composition using dual X-ray absorptiometry (DEXA). RESULTS: Feeding method at enrolment was associated with fat mass (FM) (breast 554.9 g, mixed 716.8 g, formula 637.7 g, p = 0.048), lean body mass (LM) (2512 g, 2853 g, 2722 g, respectively, p = 0.009) and lean mass index (LMI) (10.6 kg/m2, 11.6 kg/m2,11.2 kg/m2 respectively, p = 0.008) at TEA, but not 3mCA. Breastfed infants demonstrated greater increase in LM (breast 1707 g, mixed 1536 g, formula 1384 g, p = 0.03) and LMI (1.23 kg/m2, 0.10 kg/m2, 0.52 kg/m2, respectively, p = 0.022) between TEA and 3mCA. CONCLUSIONS: Breastfed LMPT infants have lower FM and greater LM increase and LMI increase up to 3mCA compared to formula or mixed-fed infants. These findings stress the importance of supporting breastfeeding in this population. IMPACT: Infants born late and moderate preterm age who are exclusively breastfed soon after birth gain more lean mass up to 3 months corrected age compared to mixed- or formula-fed infants. Breastfed infants have lower lean and fat mass at term equivalent age compared to mixed- and formula-fed infants. This is the first study exploring this population's growth and body composition in detail at 3 months corrected age. Our results underline the importance of supporting mothers to initiate and continue breastfeeding at least until 3 months corrected age.

Milk-Based Bionutrient Trials to Improve Outcomes in Preterm Infants: Challenges and Opportunities.

OBJECTIVE: Bionutrients (or immunonutrients) are dietary components present in milk, or supplements that could be added to milk diets, that impact health and disease. With few exceptions, most of these are present in human breastmilk and the majority are also present in amniotic fluid. STUDY DESIGN: Bionutrients can be proteins and peptides including enzymes, hormones, immunoglobulins, and growth factors and can also be molecules such as human milk oligosaccharides, amino acids, or lipids such as docosahexaenoic acid. Many of these have ancient origins, are found in other species, and existed before mammalian lactation evolved. Bionutrients may act in diverse ways when administered enterally: they may impact gut bacterial communities or epithelial cell metabolism, or they may pass into the lamina propria where they interact with the gut and systemic immune systems. Clinical trials have often used bovine analogs such as lactoferrin or may use artificially synthesized or recombinant compounds including insulin, bile salt stimulated lipase, or oligosaccharides. RESULTS: Challenges arise because the bioactivity of proteins, such as lactoferrin, may be affected by processing and pasteurization meaning that the impacts of commercial products may differ. The challenge of determining the optimal bioactivity of any single preparation may be even greater in complex compounds such as milk fat globule membrane. It is also possible that bioactivity is affected by the milk matrix, that is, may differ between formula and human milk. CONCLUSION: Finally, it is important to appreciate that nutrients do not function in isolation, and most will not act like drugs, that is, they may take several days or longer to exert an affect. KEY POINTS: · Breastmilk contains high concentrations of bionutrients and provides more than macro- and micronutrients.. · Bionutrients can be proteins (e.g. enzymes, hormones, or immunoglobulins) or molecules (e.g. human milk oligosaccharides or amino acids).. · Bionutrients can be added to milk feeds but high quality trials are needed..

Early antibiotic use and incidence of necrotising enterocolitis in very preterm infants: a protocol for a UK based observational study using routinely recorded data.

INTRODUCTION: Necrotising enterocolitis (NEC) remains a major contributor to preterm mortality and morbidity. Prolonged duration of antibiotic therapy after delivery is associated with later NEC development but recent evidence suggests that absence of antibiotic treatment after delivery may also increase NEC risk. We will explore this controversy using a large pre-existing dataset of preterm infants in the UK. METHODS AND ANALYSIS: This is a retrospective cohort study using data from UK National Neonatal Research Database (NNRD) for infants born 1 January 2012 to 31 December 2020. Eligible infants will be <32 weeks gestation, alive on day 3. Primary outcome is development of severe NEC, compared in infants receiving early antibiotics (days 1-2 after birth) and those not. Subgroup analysis on duration of early antibiotic exposure will also occur. Secondary outcomes are: late onset sepsis, total antibiotic use, predischarge mortality, retinopathy of prematurity, intraventricular haemorrhage, bronchopulmonary dysplasia, focal intestinal perforation and any abdominal surgery. To address competing risks, incidence of death before day 7, 14 and 28 will be analysed. We will perform logistic regression and propensity score matched analyses. Statistical analyses will be guided by NEC risk factors, exposures and outcome presented in a causal diagram. These covariates include but are not limited to gestational age, birth weight, small for gestational age, sex, ethnicity, delivery mode, delivery without labour, Apgar score, early feeding and probiotic use. Sensitivity analyses of alternate NEC definitions, specific antibiotics and time of initiation will occur. ETHICS AND DISSEMINATION: We will use deidentified data from NNRD, which holds permissions for the original data, from which parents can opt out and seek study-specific research ethics approval. The results will help to determine optimal use of early antibiotics for very preterm infants. IMPLICATIONS: This data will help optimise early antibiotic use in preterm infants. TRIAL REGISTRATION NUMBER: ISRCTN55101779.

Strain-specific impacts of probiotics are a significant driver of gut microbiome development in very preterm infants.

The development of the gut microbiome from birth plays important roles in short- and long-term health, but factors influencing preterm gut microbiome development are poorly understood. In the present study, we use metagenomic sequencing to analyse 1,431 longitudinal stool samples from 123 very preterm infants (<32 weeks' gestation) who did not develop intestinal disease or sepsis over a study period of 10 years. During the study period, one cohort had no probiotic exposure whereas two cohorts were given different probiotic products: Infloran (Bifidobacterium bifidum and Lactobacillus acidophilus) or Labinic (B. bifidum, B. longum subsp. infantis and L. acidophilus). Mothers' own milk, breast milk fortifier, antibiotics and probiotics were significantly associated with the gut microbiome, with probiotics being the most significant factor. Probiotics drove microbiome transition into different preterm gut community types (PGCTs), each enriched in a different Bifidobacterium sp. and significantly associated with increased postnatal age. Functional analyses identified stool metabolites associated with PGCTs and, in preterm-derived organoids, sterile faecal supernatants impacted intestinal, organoid monolayer, gene expression in a PGCT-specific manner. The present study identifies specific influencers of gut microbiome development in very preterm infants, some of which overlap with those impacting term infants. The results highlight the importance of strain-specific differences in probiotic products and their impact on host interactions in the preterm gut.

Follow-up study of infants recruited to the randomised, placebo-controlled trial of azithromycin for the prevention of chronic lung disease of prematurity in preterm infants-study protocol for the AZTEC-FU study.

BACKGROUND: Preterm birth, especially at less than 30 weeks' gestation, is significantly associated with respiratory, neurodevelopmental and growth abnormalities. The AZTEC study has recruited 799 infants born at < 30 weeks' gestation to determine if a ten-day intravenous treatment with azithromycin improves survival without development of chronic lung disease of prematurity (CLD) at 36 weeks' post menstrual age (PMA) when compared to placebo. The follow-up studies will compare respiratory, neurodevelopmental and growth outcomes up to 2 years of corrected age between infants who received azithromycin and those who received placebo in the early neonatal period. METHODS: Survivors at 36 weeks' PMA from the main Azithromycin Therapy for Chronic Lung Disease of Prematurity (AZTEC) study with parental consent will continue to be followed up to discharge from the neonatal unit and to 2 years of corrected age. Length of stay, rates of home oxygen, length of supplemental oxygen requirement, hospital admissions, drug usage, respiratory illness, neurodevelopmental disability and death rates will be reported. Data is being collected via parentally completed respiratory and neurodevelopmental questionnaires at 1 and 2 years of corrected age respectively. Additional information is being obtained from various sources including hospital discharge and clinical letters from general practitioners and hospitals as well as from national databases including the National Neonatal Research Database and NHS Digital. DISCUSSION: The AZTEC-FU study will assess mortality and important neonatal morbidities including respiratory, neurodevelopmental and growth outcomes. Important safety data will also be collected, including the incidence of potential consequences of early macrolide use, primarily pyloric stenosis. This study may have implications on future neonatal care. TRIAL REGISTRATION: The study was retrospectively registered on ISRCTN (ISRCTN47442783).

Optimisation and Application of a Novel Method to Identify Bacteriophages in Maternal Milk and Infant Stool Identifies Host-Phage Communities Within Preterm Infant Gut.

Human milk oligosaccharides, proteins, such as lactoferrin, and bacteria represent just some of the bioactive components of mother's breast milk (BM). Bacteriophages (viruses that infect bacteria) are an often-overlooked component of BM that can cause major changes in microbial composition and metabolism. BM bacteriophage composition has been explored in term and healthy infants, suggesting vertical transmission of bacteriophages occurs between mothers and their infants. Several important differences between term and very preterm infants (<30 weeks gestational age) may limit this phenomenon in the latter. To better understand the link between BM bacteriophages and gut microbiomes of very preterm infants in health and disease, standardised protocols are required for isolation and characterisation from BM. In this study, we use isolated nucleic acid content, bacteriophage richness and Shannon diversity to validate several parameters applicable during bacteriophage isolation from precious BM samples. Parameters validated include sample volume required; centrifugal sedimentation of microbes; hydrolysis of milk samples with digestive enzymes; induction of temperate bacteriophages and concentration/purification of isolated bacteriophage particles in donor milk (DM). Our optimised method enables characterisation of bacteriophages from as little as 0.1 mL BM. We identify viral families that were exclusively identified with the inclusion of induction of temperate bacteriophages (Inoviridae) and hydrolysis of milk lipid processes (Iridoviridae and Baculoviridae). Once applied to a small clinical cohort we demonstrate the vertical transmission of bacteriophages from mothers BM to the gut of very preterm infants at the species level. This optimised method will enable future research characterising the bacteriophage composition of BM in very preterm infants to determine their clinical relevance in the development of a healthy preterm infant gut microbiome.

Integrating longitudinal clinical and microbiome data to predict growth faltering in preterm infants.

Preterm birth affects more than 10% of all births worldwide. Such infants are much more prone to Growth Faltering (GF), an issue that has been unsolved despite the implementation of numerous interventions aimed at optimizing preterm infant nutrition. To improve the ability for early prediction of GF risk for preterm infants we collected a comprehensive, large, and unique clinical and microbiome dataset from 3 different sites in the US and the UK. We use and extend machine learning methods for GF prediction from clinical data. We next extend graphical models to integrate time series clinical and microbiome data. A model that integrates clinical and microbiome data improves on the ability to predict GF when compared to models using clinical data only. Information on a small subset of the taxa is enough to help improve model accuracy and to predict interventions that can improve outcome. We show that a hierarchical classifier that only uses a subset of the taxa for a subset of the infants is both the most accurate and cost-effective method for GF prediction. Further analysis of the best classifiers enables the prediction of interventions that can improve outcome.

Development of the gut microbiome in early life.

NEW FINDINGS: What is the topic of this review? The importance of the early life gut microbiome, with a focus on preterm infants and microbially related diseases. Current techniques to study the preterm gut microbiome are appraised, and the potential of recent methodological advancements is discussed. What advances does it highlight? Recent findings in the field achieved by the application of advanced technologies, the applicability of intestinally derived organoid models to study host-microbiome interactions in the preterm gut, and recent developments in enhancing the physiological relevance of such models. Preterm intestinally derived organoids may provide novel insights into the mechanisms underlying preterm disease, as well as diagnosis and treatment opportunities. These models have huge translational potential, offering a step towards precision medicine. ABSTRACT: Accumulating evidence affirms the importance of the gut microbiome in both health and disease. In early life, there exists a critical period in which the composition of gut microbes is particularly malleable and subject to a wide range of influencing factors. Disturbances to microbial communities during this time may be beneficial or detrimental to short and long-term health outcomes. For infants born prematurely, naïve immune systems, immature gastrointestinal tracts and additional clinical needs put this population at high risk of abnormal microbial colonisation, resulting in increased susceptibility to diseases including necrotising enterocolitis (NEC) and late-onset sepsis (LOS). Traditional cell culture methods, gnotobiotic animals, molecular sequencing techniques (16S rRNA gene sequencing and metagenomics) and advanced 'omics' technologies (transcriptomics, proteomics and metabolomics) have been fundamental in exploring the associations between diet, gut microbes, microbial functions and disease. Despite significant investment and ongoing research efforts, prevention and treatment strategies in NEC and LOS remain limited. Recent endeavours have focused on searching for new, more physiologically relevant models to simulate the preterm intestine. Preterm intestinally derived organoids represent a promising in vitro approach in the study of host-microbiome interactions in the preterm infant gut, offering new and exciting possibilities in this field.

Discriminating necrotising enterocolitis and focal intestinal perforation.

Discriminating necrotising enterocolitis (NEC) and focal intestinal perforation (FIP) is important for clinical trials, observational cohorts, quality improvement and aetiological understanding. Literature suggests that timing and key features diagnose and discriminate, and that NEC subclassifications exist. We used a detailed 10-year cohort of NEC and FIP cases in preterm infants born <32 weeks' gestation from a single centre to explore antecedent factors, presentation and potential NEC subclassifications. 785 infants had 144 episodes of NEC and 38 of FIP. FIP presented earlier than NEC, but ranges overlapped, and 30% of NEC presented before day 14. Antecedent events (other than feed volumes) and outcomes did not differ between NEC and FIP. Currently used diagnostic/discriminatory features performed poorly, and subclassification identified few cases except transfusion-associated NEC. Contrary to existing literature, postnatal age at NEC presentation was not dependent on gestational age. Detailed review rather than simple definitions are required to separate NEC from FIP.