Preterm Infant Gut Research Articles

Bifidobacterium regulates premature infant gut metabolites, reducing serum inflammatory factors: a randomised controlled trial.

Analyse the effects of Bifidobacterium BB-12 on intestinal metabolites and serum inflammatory factors in premature infants. 71 premature infants at gestational age of ≤32 weeks were randomly divided into the probiotic (n = 36) and control (n = 35) groups. Faecal and blood samples were collected from the two groups of premature infants at the 2nd and 4th week of life for intestinal metabolite detection and assessment of the level of the serum inflammatory markers TLR4, NF- κ B, IL-1β, and TNF- α. Compared to the control group, the probiotic group contained more amino acids, these elements were enriched on multiple amino acid metabolic pathways, and the probiotic group showed significantly lower levels of the serum inflammatory markers TLR4, NF-κB, IL-1β, and TNF-α. Finally, the probiotic group showed a lower incidence of feeding intolerance. The administration of Bifidobacterium BB-12 is associated with increasing the levels of glutamine, glutamic acid, and kynurenine in the gut of premature infants, and associated with reducing the levels of TLR4 and NF-κB in the serum, further decreasing the secretion of the pro-inflammatory factors IL-1β and TNF-α, and alleviating systemic inflammatory reactions, thereby reducing the incidence of feeding intolerance. 1. The use of Bifidobacterium BB-12 in premature infants can increase the levels of amino acids in the intestine. 2. Increases in Bifidobacterium BB-12 may decrease the serum levels of TLR4, NF-κB, IL-1β, and TNF-α. 3. Kynurenine may improve the prognosis of preterm infants by reducing inflammation. 4. Bifidobacterium BB-12 may improve the feeding tolerance of premature infants, thus reducing the incidence of feeding intolerance.

Clinical sequelae of gut microbiome development and disruption in hospitalized preterm infants

Aberrant preterm infant gut microbiota assembly predisposes to early-life disorders and persistent health problems. Here, we characterize gut microbiome dynamics over the first 3months of life in 236 preterm infants hospitalized in three neonatal intensive care units using shotgun metagenomics of 2,512 stools and metatranscriptomics of 1,381 stools. Strain tracking, taxonomic and functional profiling, and comprehensive clinical metadata identify Enterobacteriaceae, enterococci, and staphylococci as primarily exploiting available niches to populate the gut microbiome. Clostridioides difficile lineages persist between individuals in single centers, and Staphylococcus epidermidis lineages persist within and, unexpectedly, between centers. Collectively, antibiotic and non-antibiotic medications influence gut microbiome composition to greater extents than maternal or baseline variables. Finally, we identify a persistent low-diversity gut microbiome in neonates who develop necrotizing enterocolitis after day of life 40. Overall, we comprehensively describe gut microbiome dynamics in response to medical interventions in preterm, hospitalized neonates.

Linking preterm infant gut microbiota to nasograstric enteral feeding tubes: exploring potential interactions and microbial strain transmission.

Preterm birth is a growing problem worldwide. Staying at a neonatal intensive care unit (NICU) after birth is critical for the survival of preterm infants whose feeding often requires the use of nasogastric enteral feeding tubes (NEFT). These can be colonized by hospital-associated pathobionts that can access the gut of the preterm infants through this route. Since the gut microbiota is the most impactful factor on maturation of the immune system, any disturbance in this may condition their health. Therefore, the aim of this study is to assess the impact of NEFT-associated microbial communities on the establishment of the gut microbiota in preterm infants. A metataxonomic analysis of fecal and NEFT-related samples obtained during the first 2 weeks of life of preterm infants was performed. The potential sharing of strains isolated from the same set of samples of bacterial species involved in NICU's outbreaks, was assessed by Random Amplification of Polymorphic DNA (RAPD) genotyping. In the samples taken 48 h after birth (NEFT-1 and Me/F1), Staphylococcus spp. was the most abundant genera (62% and 14%, respectively) and it was latter displaced to 5.5% and 0.45%, respectively by Enterobacteriaceae. Significant differences in beta diversity were detected in NEFT and fecal samples taken at day 17 after birth (NEFT-3 and F3) (p = 0.003 and p = 0.024, respectively). Significant positive correlations were found between the most relevant genera detected in NEFT-3 and F3. 28% of the patients shared at least one RAPD-PCR profile in fecal and NEFT samples and 11% of the total profiles were found at least once simultaneously in NEFT and fecal samples from the same patient. The results indicate a parallel bacterial colonization of the gut of preterm neonates and the NEFTs used for feeding, potentially involving strain sharing between these niches. Moreover, the same bacterial RAPD profiles were found in neonates hospitalized in different boxes, suggesting a microbial transference within the NICU environment. This study may assist clinical staff in implementing best practices to mitigate the spread of pathogens that could threaten the health of preterm infants.

TENSOR REGRESSION FOR INCOMPLETE OBSERVATIONS WITH APPLICATION TO LONGITUDINAL STUDIES.

Multivariate longitudinal data are frequently encountered in practice such as in our motivating longitudinal microbiome study. It is of general interest to associate such high-dimensional, longitudinal measures with some univariate continuous outcome. However, incomplete observations are common in a regular study design, as not all samples are measured at every time point, giving rise to the so-called blockwise missing values. Such missing structure imposes significant challenges for association analysis and defies many existing methods that require complete samples. In this paper we propose to represent multivariate longitudinal data as a three-way tensor array (i.e., sample-by-feature-by-time) and exploit a parsimonious scalar-on-tensor regression model for association analysis. We develop a regularized covariance-based estimation procedure that effectively leverages all available observations without imputation. The method achieves variable selection and smooth estimation of time-varying effects. The application to the motivating microbiome study reveals interesting links between the preterm infant's gut microbiome dynamics and their neurodevelopment. Additional numerical studies on synthetic data and a longitudinal aging study further demonstrate the efficacy of the proposed method.

Dynamic impact of delivery modes on gut microbiota in preterm infants hospitalized during the initial 4 weeks of life

Preterm infants face a high risk of various complications, and their gut microbiota plays a pivotal role in health. Delivery modes have been reported to affect the development of gut microbiota in term infants, but its impact on preterm infants remains unclear. Here, we collected fecal samples from 30 preterm infants at five-time points within the first four weeks of life. Employing 16 S rRNA sequencing, principal coordinates analysis, the analysis of similarities, and the Wilcoxon rank-sum test, we examined the top dominant phyla and genera, the temporal changes in specific taxa abundance, and their relationship with delivery modes, such as Escherichia-Shigella and Enterococcus based on vaginal delivery and Pluralibacter related to cesarean section. Moreover, we identified particular bacteria, such as Taonella, Patulibacter, and others, whose proportions fluctuated among preterm infants born via different delivery modes at varying time points, as well as the microbiota types and functions. These results indicated the influence of delivery mode on the composition and function of the preterm infant gut microbiota. Importantly, these effects are time-dependent during the early stages of life. These insights shed light on the pivotal role of delivery mode in shaping the gut microbiota of preterm infants and have significant clinical implications for their care and management.

Premature Infant Gut Microbiome relationships with childhood behavioral scales: preliminary insights.

Very Low Birth Weight (VLBW) infants, born weighing less than 1,500 grams, are at risk for both gut dysbiosis and later neuropsychological developmental deficits. Behavioral effects, while related to neurodevelopment, are often more subtle and difficult to measure. The extent of later neurobehavioral consequences associated with such microbial dysbiosis has yet to be determined. We explored associations between the infants' gut microbiome and early childhood behavior at 4 years of age and identified the bacterial taxa through a multivariate analysis by linear models. Parents completed the Child Behavior Checklist (CBCL) focused on different DSM diagnostic categories: affective, anxiety, pervasive developmental, attention deficit/hyperactivity, and oppositional defiant. All the CBCL scores were corrected for gender, delivery method, gestational age, infant birth weight, occurrence of sepsis, and days on antibiotics prior statistical analyses. Canonical correlation analysis (CCA) was performed to determine the relationship between early life gut microbiome and the adjusted CBCL scores. The association of bacterial Amplicon sequence Variants (ASVs) to the CBCL scores were tested with multivariate analysis by linear models (MaAsLin). Nineteen children who were previously born with very low birth weight and studied while hospitalized in the Neonatal Intensive Care Unit (NICU) were included in this study. Statistically significant associations were observed between early life gut bacteria such as Veillonella dispar, Enterococcus, Escherichia coli, and Rumincococcus to later behavior at 4 years. No significant association could be observed with early-life gut microbiome alpha diversity and behavioral measures at 4 years. These preliminary observational data provide insight into the relationships between VLBW gut microbiome dysbiosis and childhood behavior. This study contributes to the literature on gut microbiome analysis by examining various behavioral domains using a standardized tool linked to the Diagnostic and Statistical Manual of Mental Disorders (DSM).

A pilot study of fecal pH and redox as functional markers in the premature infant gut microbiome.

The infant gut microbiome is a crucial factor in health and development. In preterm infants, altered gut microbiome composition and function have been linked to serious neonatal complications such as necrotizing enterocolitis and sepsis, which can lead to long-term disability. Although many studies have described links between microbiome composition and disease risk, there is a need for biomarkers to identify infants at risk of these complications in practice. In this pilot study, we obtained stool samples from preterm infant participants longitudinally during the first postnatal months, and measured pH and redox, as well as SCFA content and microbiome composition by 16S rRNA gene amplicon sequencing. These outcomes were compared to clinical data to better understand the role of pH and redox in infant gut microbiome development and overall health, and to assess the potential utility of pH and redox as biomarkers. We found that infants born earlier or exposed to antibiotics exhibited increased fecal pH, and that redox potential increased with postnatal age. These differences may be linked to changes in SCFA content, which was correlated with pH and increased with age. Microbiome composition was also related to birth weight, age, pH, and redox. Our findings suggest that pH and redox may serve as biomarkers of metabolic state in the preterm infant gut.

Analyzing the Responses of Enteric Bacteria to Neonatal Intensive Care Supplements.

In the neonatal intensive care unit, adequate nutrition requires various enteral products, including human milk and formula. Human milk is typically fortified to meet increased calorie goals, and infants commonly receive vitamin mixes, iron supplements, and less frequently, thickening agents. We examined the growth of 16 commensal microbes and 10 pathobionts found in the premature infant gut and found that formula, freshly pasteurized milk, and donated banked milk generally increased bacterial growth. Fortification of human milk significantly elevated the growth of all microbes. Supplementation with thickeners or NaCl in general did not stimulate additional growth. Vitamin mix promoted the growth of several commensals, while iron promoted growth of pathobionts. These data indicate that pathobionts in the preterm gut have significant growth advantage with preterm formula, fortified donor milk, and supplemented iron and suggest that the choice of milk and supplements may impact the infant gut microbiota.

Strain-resolved metagenomic analysis of the gut as a reservoir for bloodstream infection pathogens among premature infants in Singapore

BackgroundGut dysbiosis contributes to the high risk of bloodstream infection (BSI) among premature infants. Most prior studies of the premature infant gut microbiota were conducted in Western countries and prior to development of current tools for strain-resolved analysis.MethodsWe performed metagenomic sequencing of weekly fecal samples from 75 premature infants at a single hospital in Singapore. We evaluated associations between clinical factors and gut microbiota composition using PERMANOVA and mixed effects linear regression. We used inStrain to perform strain-level analyses evaluating for gut colonization by BSI-causing strains.ResultsMedian (interquartile range) gestation was 27 (25, 29) weeks, and 63% of infants were born via Cesarean section. Antibiotic exposures (PERMANOVA; R2 = 0.017, p = 0.001) and postnatal age (R2 = 0.015, p = 0.001) accounted for the largest amount of variability in gut microbiota composition. Increasing postnatal age was associated with higher relative abundances of several common pathogens (Enterococcus faecalis: p < 0.0001; Escherichia coli: p < 0.0001; Klebsiella aerogenes: p < 0.0001; Klebsiella pneumoniae: p < 0.0001). Antibiotic exposures were generally associated with lower relative abundances of both frequently beneficial bacteria (e.g., Bifidobacterium species) and common enteric pathogens (e.g., Enterobacter, Klebsiella species). We identified strains identical to the blood culture isolate in fecal samples from 12 of 16 (75%) infants who developed BSI, including all infections caused by typical enteric bacteria.ConclusionsAntibiotic exposures were the dominant modifiable factor affecting gut microbiota composition in a large cohort of premature infants from South-East Asia. Strain-resolved analyses indicate that the gut is an important reservoir for organisms causing BSI among premature infants.

Gut pathogen colonization precedes bloodstream infection in the neonatal intensive care unit.

Bacterial bloodstream infections (BSIs) resulting in late-onset sepsis affect up to half of extremely preterm infants and have substantial morbidity and mortality. Bacterial species associated with BSIs in neonatal intensive care units (NICUs) commonly colonize the preterm infant gut microbiome. Accordingly, we hypothesized that the gut microbiome is a reservoir of BSI-causing pathogenic strains that increase in abundance before BSI onset. We analyzed 550 previously published fecal metagenomes from 115 hospitalized neonates and found that recent ampicillin, gentamicin, or vancomycin exposure was associated with increased abundance of Enterobacteriaceae and Enterococcaceae in infant guts. We then performed shotgun metagenomic sequencing on 462 longitudinal fecal samples from 19 preterm infants (cases) with BSI and 37 non-BSI controls, along with whole-genome sequencing of the BSI isolates. Infants with BSI caused by Enterobacteriaceae were more likely than infants with BSI caused by other organisms to have had ampicillin, gentamicin, or vancomycin exposure in the 10 days before BSI. Relative to controls, gut microbiomes of cases had increased relative abundance of the BSI-causing species and clustered by Bray-Curtis dissimilarity according to BSI pathogen. We demonstrated that 11 of 19 (58%) of gut microbiomes before BSI, and 15 of 19 (79%) of gut microbiomes at any time, harbored the BSI isolate with fewer than 20 genomic substitutions. Last, BSI strains from the Enterobacteriaceae and Enterococcaceae families were detected in multiple infants, indicating BSI-strain transmission. Our findings support future studies to evaluate BSI risk prediction strategies based on gut microbiome abundance in hospitalized preterm infants.

The Nonbacterial Microbiome: Fungal and Viral Contributions to the Preterm Infant Gut in Health and Disease.

The intestinal microbiome is frequently implicated in necrotizing enterocolitis (NEC) pathogenesis. While no particular organism has been associated with NEC development, a general reduction in bacterial diversity and increase in pathobiont abundance has been noted preceding disease onset. However, nearly all evaluations of the preterm infant microbiome focus exclusively on the bacterial constituents, completely ignoring any fungi, protozoa, archaea, and viruses present. The abundance, diversity, and function of these nonbacterial microbes within the preterm intestinal ecosystem are largely unknown. Here, we review findings on the role of fungi and viruses, including bacteriophages, in preterm intestinal development and neonatal intestinal inflammation, with potential roles in NEC pathogenesis yet to be determined. In addition, we highlight the importance of host and environmental influences, interkingdom interactions, and the role of human milk in shaping fungal and viral abundance, diversity, and function within the preterm intestinal ecosystem.

Effect of Holder pasteurisation and UV-C irradiation on bacteriophage titres in human milk.

Human milk is the optimal nutrition source for infants and contains a complex mix of bioactive compounds and microorganisms. When unavailable, pasteurised donor milk may be provided, particularly to preterm infants. Holder pasteurisation (HP) is typically implemented in human milk banks to prevent pathogen transmission. Given the impact of heat on milk bioactives, ultraviolet-C irradiation (UV-C) is an alternative being explored and has demonstrated effective bactericidal activity. In addition to bacteria, milk contains viruses including primarily bacteriophages (phages) and which likely influence the developing bacterial microbiome of infants. However, the effect of pasteurisation on human milk phages is unknown. This study assessed the effect of HP and UV-C on titres of exogenous bacteriophages inoculated into human milk. Ten donor human milk samples were tested in parallel with water controls. Milk samples or water controls were inoculated to a final concentration of 1× 104 PFU/mL (±1 log) each of a thermotolerant Escherichia coli phage (T4) and a thermosensitive Staphylococcus aureus phage (BYJ20) and subjected to HP and UV-C treatments. UV-C inactivated both phages within milk and water controls, however, HP was ineffective against the thermotolerant T4 phages. Initial data suggests that UV-C treatment may eliminate phage with potential to affect preterm infant gut colonization. Further studies should extend this to other phages.

A novel approach for combining the metagenome, metaresistome, metareplicome and causal inference to determine the microbes and their antibiotic resistance gene repertoire that contribute to dysbiosis.

The use of whole metagenomic data to infer the relative abundance of all its microbes is well established. The same data can be used to determine the replication rate of all eubacterial taxa with circular chromosomes. Despite their availability, the replication rate profiles (metareplicome) have not been fully exploited in microbiome analyses. Another relatively new approach is the application of causal inferencing to analyse microbiome data that goes beyond correlational studies. A novel scalable pipeline called MeRRCI (Metagenome, metaResistome, and metaReplicome for Causal Inferencing) was developed. MeRRCI combines efficient computation of the metagenome (bacterial relative abundance), metaresistome (antimicrobial gene abundance) and metareplicome (replication rates), and integrates environmental variables (metadata) for causality analysis using Bayesian networks. MeRRCI was applied to an infant gut microbiome data set to investigate the microbial community's response to antibiotics. Our analysis suggests that the current treatment stratagem contributes to preterm infant gut dysbiosis, allowing a proliferation of pathobionts. The study highlights the specific antibacterial resistance genes that may contribute to exponential cell division in the presence of antibiotics for various pathogens, namely Klebsiella pneumoniae, Citrobacter freundii, Staphylococcus epidermidis, Veilonella parvula and Clostridium perfringens. These organisms often contribute to the harmful long-term sequelae seen in these young infants.

Antibiotic resistance and host immune system-induced metal bactericidal control are key factors for microbial persistence in the developing human preterm infant gut microbiome.

The human gut microbiome, which develops and stabilizes during the early stages of infant life, plays an essential role in host health through the production of metabolic resources and the stimulation and training of the immune system. To study colonization and community functional dynamics of the microbiota based on responses to host immune processes during the normal and dysbiotic establishment of the gut, metaproteomics was conducted on 91 fecal samples collected over the first 90 days of life from 17 hospitalized premature infants. Microbial responses to antibiotic administration and host-imposed metal bactericidal control correlated with community assembly and resiliency of microbes in the developing preterm gut. Specifically, proteins related to antibiotic resistance and metal homeostasis mechanisms were predominant in persisting members in the infant gut environment over the first several weeks of life. Overall, this metaproteomics study provides a unique approach to examine the temporal expansion and resilience of microbial colonization, as it allows simultaneous examination of both host and microbial metabolic activities. Understanding the interplay between host and microbes may elucidate the microbiome's potential immunomodulatory roles relevant to necrotizing enterocolitis and other dysbiotic conditions in preterm infants.

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

ObjectiveTo determine the impact of supplemental bovine lactoferrin on the gut microbiome and metabolome of preterm infants.DesignCohort study nested within a randomised controlled trial (RCT). Infants across different trial arms...

Source of human milk (mother or donor) is more important than fortifier type (human or bovine) in shaping the preterm infant microbiome

SummaryMilk fortifiers help meet the nutritional needs of preterm infants receiving their mother’s own milk (MOM) or donor human milk. We conducted a randomized clinical trial (NCT03214822) in 30 very low birth weight premature neonates comparing bovine-derived human milk fortifier (BHMF) versus human-derived fortifier (H2MF). We found that fortifier type does not affect the overall microbiome, although H2MF infants were less often colonized by an unclassified member of Clostridiales Family XI. Secondary analyses show that MOM intake is strongly associated with weight gain and microbiota composition, including Bifidobacterium, Veillonella, and Propionibacterium enrichment. Finally, we show that while oxidative stress (urinary F2-isoprostanes) is not affected by fortifier type or MOM intake, fecal calprotectin is higher in H2MF infants and lower in those consuming more MOM. Overall, the source of human milk (mother versus donor) appears more important than the type of milk fortifier (human versus bovine) in shaping preterm infant gut microbiota.

Antibiotic exposure prevents acquisition of beneficial metabolic functions in the preterm infant gut microbiome

BackgroundAberrations in the preterm microbiome following antibiotic therapy have been reported in previous studies. The objective of this study was to probe potential underlying mechanisms between this observation and susceptibility to adverse prematurity-related outcomes.ResultsMetagenomic shotgun sequencing was performed on 133 stool and 253 skin samples collected at 1 and 3 weeks of age from 68 infants born at <36 weeks postmenstrual age and birth weight <2000 g. After accounting for gestational age and maternal antibiotics, the distribution of organisms in all samples and the corresponding metabolic pathway abundance were compared between infants exposed to postnatal antibiotics and antibiotics-naïve infants.In antibiotic-naïve infants, gestational and postnatal age imparted similar trajectories on maturation of the microbial community and associated metabolic functional capacity, with postnatal age exerting greater contribution. Antibiotic exposure was associated with reversal in maturation trajectory from the first week to the third week of age (p< 0.001). Butyrate-producing genera, including Clostridium and Blautia, were significantly more abundant in antibiotic-naïve neonates at 3 weeks postnatal age. Correspondingly, metabolic pathways required for short-chain fatty acid synthesis were significantly increased in antibiotic-naïve infants, but not in antibiotic-exposed neonates, at 3 weeks after birth.ConclusionsEarly brief antibiotic exposure markedly disrupts developmental trajectory of the neonatal microbiome and its corresponding functional capacity. Our findings may provide a mechanistic explanation for the known associations between antibiotic use and adverse outcomes in preterm infants.459naA43GFKEq_9gJQNK48Video

#20 Bloodstream Infection is Associated with Prior Gut Microbiome Colonization of the Same Strain in the Neonatal Intensive Care Unit

Abstract Background Infants hospitalized in neonatal intensive care units (NICUs) have high incidence of bacterial bloodstream infections (BSIs) leading to up to 15% mortality and substantial morbidity. Common etiologies of BSI in the NICU are pathogens also capable of colonizing the preterm infant gut. However, differentiating susceptible neonates from those in the same environment remains challenging. We hypothesized that the same strains causing BSI increase in abundance in the gut of affected neonates prior to bacteremia and increased above controls in the same environment. Method We performed shotgun metagenomic sequencing on 462 fecal samples from 19 infants with BSI and 37 controls in the same NICUs paired for gestational age, days of life, antibiotic exposure, delivery mode, and sex. We whole genome sequenced the BSI isolate from 19 cases (6 Staphylococcus aureus, 3 Streptococcus agalactiae, 3 Serratia marcescens, 2 Enterococcus faecalis, 2 Klebsiella pneumoniae, 2 Escherichia coli, and 1 Enterobacter cloacae). We utilized MetaPhLan3 to infer taxonomy and InStrain to track the whole genome sequence of the BSI pathogen within the fecal metagenomes longitudinally. We performed generalized linear mixed effect models with MaAsLin2 to determine significant associations between the microbiome and metadata with subject as a random effect. Results We found no differences in gut microbiome diversity or richness between cases and controls relative to bacteremia day of life. However, infants who experience a BSI possess significantly higher median relative abundances of the causative species in their gut relative to paired controls in the 2 weeks prior to bacteremia (Figure 1A, median 21.4% IQR 0.33-52 vs. 0.4% IQR 0.23-10.5, p&amp;lt;0.05 Wilcoxon). 10/19 infants produced at least 1 stool before bacteremia containing an organism with 0-1 genomic substitutions from the BSI isolate, establishing isogenicity between gut residing and invasive pathogen. The coverage (abundance) of the BSI isolate increased in the stool of affected neonates in the 10 days before infection to an average maximum of 0.48/1.0 (Figure 1B, linear mixed effect model p&amp;lt;0.05). We observed the strongest associations between high gut microbiome abundance of E. faecalis, Klebsiella spp., E. coli, and S. marcescens in the gut microbiome of cases whose BSI was caused by that organism compared to all other samples (p&amp;lt;0.01). Conclusion We show high relative abundance of the same strain in stool prior to bacteremia in hospitalized neonates. The relative abundance of the causative species is higher in cases versus paired controls. These data suggest the potential of gut microbial surveillance and management to identify blooms of gut pathogens to limit the incidence of bacteremia.

Individuality of the Extremely Premature Infant Gut Microbiota Is Driven by Ecological Drift.

ABSTRACTThe initial contact between humans and their colonizing gut microbiota after birth is thought to have expansive and long-lasting consequences for physiology and health. Premature infants are at high risk of suffering from lifelong impairments, due in part to aberrant development of gut microbiota that can contribute to early-life infections and inflammation. Despite their importance to health, the ecological assembly and succession processes governing gut microbiome composition in premature infants remained incompletely understood. Here, we quantified these ecological processes in a spatiotemporally resolved 16S rRNA gene amplicon sequencing data set of 60 extremely premature neonates using an established mathematical framework. We found that gut colonization during the first months of life is predominantly stochastic, whereby interindividual diversification of microbiota is driven by ecological drift. Dispersal limitations are initially small but have increasing influence at later stages of succession. Furthermore, we find similar trends in a cohort of 32 healthy term-born infants. These results suggest that the uniqueness of individual gut microbiota of extremely premature infants is largely due to stochastic assembly.IMPORTANCE Our knowledge concerning the initial gut microbiome assembly in human neonates is limited, and scientific progression in this interdisciplinary field is hindered due to the individuality in composition of gut microbiota. Our study addresses the ecological processes that result in the observed individuality of microbes in the gastrointestinal tract between extremely premature and term-born infants. We find that initial assembly is mainly driven by neutral ecological processes. Interestingly, while this progression is predominantly random, limitations to the dispersal of microbiota between infants become increasingly important with age and are concomitant features of gut microbiome stability. This indicates that while we cannot predict gut microbiota assembly due to its random nature, we can expect the establishment of certain ecological features that are highly relevant for neonatal health.

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.