Neonatal Microbiome: Early Colonization, Preterm Dysbiosis, and Clinical Implications

Jin Kyu Kim

doi:10.5385/nm.2025.32.2.55

Neonatal Med > Volume 32(2); 2025 > Article

Kim: Neonatal Microbiome: Early Colonization, Preterm Dysbiosis, and Clinical Implications

Review Article

Neonatal Medicine 2025;32(2):55-69.

Published online: November 30, 2025

DOI: https://doi.org/10.5385/nm.2025.32.2.55

Neonatal Microbiome: Early Colonization, Preterm Dysbiosis, and Clinical Implications

Jin Kyu Kim, MD, PhD^1,²

¹Department of Pediatrics, Jeonbuk National University Medical School, Jeonju, Korea

²Research Institute of Clinical Medicine of Jeonbuk National University-Biomedical Research Institute of Jeonbuk National University Hospital, Jeonju, Korea

Correspondence to: Jin Kyu Kim, MD, PhD Department of Pediatrics, Jeonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju 54907, Korea
Tel: +82-63-250-1460 Fax: +82-63-250-1464 E-mail: kyunim99@gmail.com

Received May 26, 2025 Revised October 22, 2025 Accepted November 5, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The neonatal microbiome constitutes a dynamic and rapidly evolving ecosystem that exerts profound effects on immune, metabolic, and neurodevelopmental processes during early life. This review synthesizes current evidence on the establishment, maturation, and functional roles of the neonatal microbiome, emphasizing the differences between preterm and term infants and their implications for neonatal diseases. Microbial colonization commences at birth and is shaped predominantly by maternal transmission, mode of delivery, feeding practices, and antibiotic exposure. The early assembly of oral and gut microbiota provides the foundation for immune education, intestinal barrier integrity, and metabolic homeostasis; however, disruptions during this critical developmental window are associated with adverse outcomes. Preterm infants exhibit delayed and dysbiotic microbial development, characterized by reduced diversity, enrichment of Proteobacteria, and depletion of short-chain fatty acid-producing taxa. These alterations are strongly implicated in the pathogenesis of necrotizing enterocolitis, sepsis, bronchopulmonary dysplasia, and neurodevelopmental impairment, predominantly through mechanisms involving exaggerated lipopolysaccharide—Toll-like receptor 4—nuclear factor kappa B signaling, impaired barrier function, and metabolic deficiencies. Advances in germ-free and gnotobiotic models, combined with multi-omics approaches, have elucidated key host–microbe interactions and mechanistic pathways underpinning neonatal health. However, major challenges persist in establishing causality, standardizing analytical methods, and translating mechanistic findings into effective clinical interventions. Understanding the neonatal microbiome as a central regulator of early-life programming holds promise for the development of microbiome-targeted preventive and therapeutic strategies in neonatal and pediatric medicine.

Keywords: Dysbiosis; Infant, newborn; Microbiota; Multiomics

INTRODUCTION

The neonatal gastrointestinal tract begins to acquire its microbiome within hours of birth, initiating a complex and dynamic process of microbial colonization [1]. This process has been aptly likened to painting on a blank canvas—starting with an initial sketch derived from the maternal microbiota and evolving into a diverse, individualized composition shaped by multiple environmental factors [2]. Early colonization of the neonatal microbiome extends far beyond simple gut occupancy; it plays a critical role in immune development, modulation of nutrient absorption and metabolism, and the establishment of defenses against pathogenic invasion [3,4].

The perinatal period represents a critical developmental window during which the neonatal microbiome undergoes rapid establishment and maturation. Multiple factors—including mode of delivery, maternal diet, antibiotic exposure, and feeding practices—interact in complex ways to shape microbial diversity and influence long-term health trajectories [2,3]. The neonatal microbiome has emerged as a key determinant of infant health, with implications for immune maturation, metabolic programming, neurodevelopment, and behavioral outcomes [5,6]. In particular, preterm neonates exhibit distinct patterns of microbial colonization and succession compared with term neonates, a disparity associated with increased susceptibility to necrotizing enterocolitis (NEC), late-onset sepsis (LOS), bronchopulmonary dysplasia (BPD), feeding intolerance (FI), and neurodevelopmental impairment [7-9]. Given these extensive implications, a comprehensive understanding of the neonatal microbiome is essential for developing strategies that promote infant health and prevent diseases [10,11]. Contemporary research integrates diverse methodological approaches —including advanced animal models and integrative multiomics technologies—to elucidate the complex host–microbiome interactions that occur in early life [12-14].

The aim of this review is to provide a comprehensive synthesis of current evidence on the neonatal microbiome, elucidate the mechanistic pathways through which it influences immune, metabolic, and neurodevelopmental processes, and explore how these insights may inform future preventive and therapeutic strategies.

INITIAL COLONIZATION AND DEVELOPMENT OF THE NEONATAL MICROBIOME

At birth, neonates are exposed to a diverse array of maternal microbes that initiate the foundational stages of microbiome development. Although the intrauterine environment has traditionally been considered sterile, recent studies have identified microbial signals in the placenta and meconium; however, evidence supporting viable in utero colonization remains highly debated and may be confounded by contamination bias [15,16]. Despite this controversy, the maternal microbiome— particularly through vertical transmission during vaginal delivery and immediate postnatal contact—remains the predominant source of early neonatal microbiota. Within these nascent microbial ecosystems, the oral–gastrointestinal axis serves as a critical site for initial colonization. The oral cavity functions as a primary entry point for microbial exposure and plays a central role in seeding microbial communities throughout the oral and gastrointestinal tracts. The developing gut microbiota subsequently contributes to immune system maturation, nutrient metabolism, microbial metabolite production, and protection against pathogenic invasion [4,17].

1. Oral microbiome

The neonatal oral microbiome is established immediately upon exposure to the external environment at birth. Its initial composition is strongly influenced by the mode of delivery: vaginally delivered neonates acquire microbes predominantly from the maternal vaginal and cervical microbiota, whereas those born via cesarean section more commonly inherit the maternal skin flora. Transmission of maternal oral microbes has also been reported [18,19]. Early oral colonizers typically include aerobic and facultative anaerobic genera, including Streptococcus and Staphylococcus. The neonatal oral microbiome evolves rapidly, particularly during the first few days of life and remains dynamic thereafter, shaped by a range of host and environmental factors. Among these, feeding mode is considered one of the most influential determinants [20]. Mother’s own milk (MOM) contains immunologically active components, such as secretory immunoglobulin A (sIgA), lactoferrin, and human milk oligosaccharides (HMOs), and may also include commensal bacteria, including Bifidobacterium. Together, these factors support microbial diversity and stability within the oral and gastrointestinal environments [21]. Conse quently, breastfed infants tend to harbor a greater abundance of beneficial bacterial taxa than those who are formula-fed [22].

Initial oral colonization typically begins with pioneer species such as Streptococcus salivarius, which exhibit strong adherence to oral epithelial cells. These early colonizers modulate the local microenvironment and facilitate the attachment of secondary bacterial species through mechanisms such as co-aggregation and co-adhesion, thereby enhancing microbial diversity [23]. In preterm infants, the composition of the early oral microbiome may differ substantially owing to variations in clinical management and environmental exposures, although it generally converges toward that of term infants within the first few months of life [24]. Around 6 months of age, tooth eruption introduces new hard surfaces that support colonization by additional bacterial species, markedly increasing the complexity of the oral microbiome. By approximately 4 years of age, as dentition and dietary patterns stabilize, the oral microbiota reaches a mature state that closely resembles that of adults [3].

2. Gut microbiome

The neonatal gut initially exists in a relatively aerobic state compared with the adult gut. Early microbial colonization is dominated by facultative anaerobes, such as Staphylococcus and Enterococcus, which consume oxygen and progressively create an anaerobic environment. This transition subsequently allows the expansion of strict anaerobes, including Bifidobacterium, members of the class Clostridia (phylum Firmicutes), and taxa belonging to the phylum Bacteroidetes [25,26].

Several physiological characteristics distinguish the neonatal gut from that of adults, including higher oxygen levels, lower microbial diversity, immature epithelial structures, and an underdeveloped immune repertoire. These conditions collectively shape the selection and succession of colonizing microbes. Initial maternal inoculation and perinatal factors continue to influence microbiome assembly during early infancy. As dietary complexity and environmental exposures increase, microbial diversity expands, typically reaching an adult-like configuration between 3 and 5 years of age [27,28]. Feeding method remains a key determinant of microbial composition: breastfed infants exhibit greater enrichment of Bifidobacterium, which plays a critical role in promoting gut health [29,30]. Gut colonization establishes the foundation for stable host–microbiota symbiosis, and successful early microbial establishment is essential for the maturation of immune, metabolic, and neurodevelopmental functions. Conversely, disruptions during this critical developmental window owing to perinatal exposure may increase susceptibility to immunological or metabolic disorders later in life. Beyond its role in digestion and immune regulation, the gut microbiome also contributes to the production of neuroactive compounds that influence behavior and cognition [27,31].

3. Oral–gut microbiome axis

The neonatal oral cavity serves as a primary route for microbial entry and subsequent colonization of the gastrointestinal tract. Although the oral and gut microbiomes develop under distinct environmental conditions, they remain closely interconnected through microbial migration and interactions, forming what is referred to as the oral–gut microbiome axis [32]. A central mechanism underlying this axis is microbial translocation: microorganisms inhabiting the oral cavity or oropharynx may be swallowed and subsequently establish residence in the gut. Additionally, certain oral pathogens may disseminate hematogenously and establish niches within the gut mucosa [33,34]. Several factors modulate the development and interaction of the oral–gut axis, among which breastfeeding exerts a particularly significant effect. Breast milk provides both microbial taxa and HMOs, which selectively support the growth of beneficial bacteria in both the oral and gut ecosystems [35].

Emerging evidence indicates that certain resident oral bacteria are implicated not only in oral pathologies but also in gastrointestinal disorders [36]. The migration and potential pathogenicity of these oral-derived microbes underscore the clinical significance of the oral–gut microbiome axis. Recognition of this bidirectional relationship highlight the value of implementing lactation support and oral care practices that facilitate beneficial microbial seeding. A more comprehensive understanding of this interaction may contribute to the prevention and management of diseases in neonates and infants [36].

ROLE OF THE MICROBIOME IN NEONATAL HEALTH AND DISEASE

Microbial colonization of the gut and oral cavity during early life represents far more than passive coexistence; it is a dynamic and reciprocal interaction in which the microbiome acts as a central regulator of immune and metabolic system development. The neonatal microbiome plays a pivotal role in shaping physiological development and long-term health outcomes [37]. Its primary functions can be broadly categorized as described below.

1. Gut barrier integrity and pathogen defense

The microbiome plays a crucial role in maintaining intestinal barrier integrity, thereby preventing the translocation of harmful substances, pathogenic bacteria, and microbial endotoxins— such as lipopolysaccharide (LPS)—across the epithelium into systemic circulation [38]. By occupying ecological niches, competing for nutrients, and producing antimicrobial compounds, commensal microbes establish colonization resistance that effectively limits the establishment of potentially pathogenic invaders [39]. Additionally, commensal bacteria stimulate the development and maturation of the intestinal mucus layer by promoting goblet cell activity and mucin secretion. In parallel, they induce the expression of tight junction proteins in intestinal epithelial cells, thereby strengthening intercellular adhesion and reducing paracellular permeability. In addition, they produce short-chain fatty acids (SCFAs), which enhance barrier integrity, inhibit pathogen growth, and modulate immune responses by promoting sIgA production and the release of anti-inflammatory cytokines. These effects collectively reinforce the gut barrier and provide effective protection against infections [40].

2. Immune development and education

The early-life microbiome is essential for healthy maturation and education of the neonatal immune system, enabling it to distinguish between self-antigens, innocuous antigens (food-derived or commensal), and harmful pathogens. Proper immunological programming during this period is essential for preventing immune-mediated disorders such as allergies and autoimmune diseases [41,42].

Gut microbes interact with pattern recognition receptors expressed by intestinal epithelial and resident immune cells to promote the development of gut-associated lymphoid tissue [43]. Furthermore, the microbiome indirectly promotes the maturation of microfold cells within Peyer’s patches, primarily through immune-mediated signaling pathways rather than direct induction. Microbial colonization also induces the differentiation and maturation of key immune cell subsets, including group 3 innate lymphoid cells—which are supported but not exclusively induced by microbial signals—as well as by γδ T cells and regulatory T cells (Tregs), which play a central role in establishing immune tolerance [27].

The neonatal microbiome also stimulates the production of sIgA, which not only restricts pathogen invasion but also promotes the containment of commensal microbes and the establishment of mucosal immune tolerance, thereby contributing to microbial homeostasis. In addition, specific microbial metabolites, particularly SCFAs such as acetate, propionate, and butyrate, modulate immune responses by promoting Treg cell differentiation through histone deacetylase inhibition and supporting the development of oral tolerance to dietary and commensal antigens [44].

3. Metabolic and nutritional support

The gut microbiome exerts a significant influence on host metabolism and nutritional status. Certain bacterial species synthesize essential vitamins such as vitamin K and biotin, which cannot be produced endogenously. Moreover, these microbes ferment indigestible dietary fibers into SCFAs, which serve as major energy sources for the colonic epithelial cells and modulate metabolic functions in distant organs, including the liver and brain. Collectively, the microbiome enhances overall nutrient utilization and absorption efficiency [45].

4. Systemic influence and organ health

Beyond the gut, the microbiome modulates various systemic pathways, including the gut–brain and gut–lung axis, endocrine signaling, and neurodevelopment. It has also been implicated in shaping neonatal neurocognitive development. A well-developed gut microbiome supports not only gastrointestinal health but also the function of distant organs such as the brain, liver, and lungs, underscoring its role as a central regulatory system in human health [46-48]. Disruptions in early microbiome assembly owing to antibiotic exposure, altered feeding practices, or infections can increase the risk of longterm health conditions. Conversely, vertical transmission of the maternal microbiota exerts beneficial effects on immune maturation and microbial colonization during this critical developmental window [37,49].

DIFFERENCES IN MICROBIOME DEVELOPMENT BETWEEN PRETERM AND TERM INFANTS

Compared with term infants, preterm neonates exhibit marked differences in microbiome establishment and maturation. These disparities arise not only from physiological immaturity but also from extrinsic factors, such as the sterile environment of neonatal intensive care units (NICUs), early and broadspectrum antibiotic exposure, reliance on total parenteral nutrition, and delayed or restricted breastfeeding—all of which collectively alter early microbial colonization [50]. In practice, preterm infants are more frequently delivered via cesarean section and are less likely to receive early and sustained mother’s milk, further limiting the vertical transmission of beneficial microbes. Term infants typically achieve rapid microbial stabilization dominated by health-associated taxa within the first few weeks of life; however, preterm infants often exhibit delayed maturation accompanied by prolonged dysbiosis. This is characterized by reduced α-diversity, delayed and sometimes insufficient colonization by Bifidobacterium and Bacteroides, and relative overgrowth of Proteobacteria [51,52]. In contrast, the gut microbiota of term infants is enriched with Lactobacillus, Bifidobacterium, and Bacteroides, with increased diversity and enhanced capacity for SCFA biosynthesis [53].

Longitudinal studies have revealed that, although the microbiota of preterm infants gradually matures and becomes increasingly similar to that of term infants over time, significant differences in microbial diversity and composition often persist for years. Even at 3.5 years of age, preterm children exhibit lower Shannon diversity and distinct microbial community structures compared with their term counterparts [54]. These early-life disparities are associated with increased risk of complications such as NEC, LOS, BPD, and impaired postnatal growth. Moreover, alterations in early microbial assembly may have long-term effects, predisposing to allergic, autoimmune, and metabolic disorders later in life [55].

NEONATAL DISEASES AND THE MICROBIOME

The major neonatal diseases discussed below, along with their key mechanistic underpinnings and clinical implications, are summarized in Table 1. Although the causal direction of these associations remains under investigation, converging evidence from cohort studies, gnotobiotic transfer models, and interventional trials suggests that dysbiosis—characterized by reduced microbial diversity, increased abundance of Proteobacteria, and depletion of SCFA-producing taxa—may act as an upstream contributor rather than solely a secondary response in certain contexts [56,57]. During the neonatal period, the establishment of a balanced microbiome is shaped by perinatal exposures and is closely associated with both short-term morbidity and long-term metabolic and immune outcomes [28]. Disruptions during this critical developmental window can impede normal immune education and epithelial and metabolic maturation, thereby increasing susceptibility to immunemediated and metabolic disorders later in life [58]. A recurrent mechanistic pathway underlies many neonatal diseases: microbiome dysbiosis leads to SCFA depletion, which results in compromised epithelial barrier integrity and increased inflammatory signaling. SCFAs such as butyrate, acetate, and propionate are essential for maintaining mucosal health, modulating immune responses, and supporting metabolic maturation. Their deficiency promotes disease development across multiple organ systems, with clinical manifestations varying according to tissue type and the nature of microbial disruptions (Figure 1) [59]. Large-scale epidemiological studies further demonstrate that cesarean delivery is associated with reduced microbial diversity, microbial community imbalance, and an increased risk of downstream conditions such as asthma, allergic diseases, obesity, and autoimmunity [60]. The initial colonization patterns established during the neonatal period have lasting effects on health, underscoring the importance of early-life exposures and interventions in shaping lifelong health trajectories. Accordingly, NICU practices that emphasize human milk feeding and antibiotic stewardship are biologically well-founded and have demonstrated benefits for specific outcomes such as NEC prevention; however, their efficacy for others, including BPD and neurodevelopmental outcomes, remains uncertain [61].

Emerging evidence underscores the importance of antibiotic stewardship programs (ASPs) in NICU settings, where they help minimize microbiota disruption, limit the emergence of resistant and pathogenic organisms, and reduce the risk of invasive infections such as sepsis [62]. Implementation of ASPs effectively limits unnecessary antibiotic exposure, one of the most significant modifiable risk factors for NEC and sepsis in preterm infants. Moreover, the maternal microbiome, encompassing vaginal, gastrointestinal, and skin flora, together with peripartum antibiotic exposure, directly influences the initial microbiota composition acquired by neonates. Notably, perinatal antibiotic use has been associated with microbiome disruption and an increased risk of early-onset sepsis [63]. When clinically appropriate, rationalizing antibiotic use during pregnancy may enhance neonatal microbial colonization and improve infection-related outcomes [64]. Collectively, these findings highlight the central role of the neonatal microbiome in disease pathogenesis, the shared mechanistic link between dysbiosis and SCFA depletion, and the importance of targeted interventions and antibiotic stewardship in optimizing neonatal health.

1. Necrotizing enterocolitis

Dysbiotic alterations in the gut microbiome have been shown to precede the onset of NEC by several days, as demonstrated in large twin cohort studies. Affected twins typically exhibit a marked decrease in microbial diversity accompanied by an overexpression of Proteobacteria—particularly Klebsiella pneumoniae, Escherichia coli, and Clostridium species—and a concomitant loss of protective Bifidobacterium and other SCFA-producing bacteria [65-67]. These compositional alterations create a pro-inflammatory milieu that primes the immature gut for aberrant immune activation. A key mechanism involves Toll-like receptor 4 (TLR4) signaling in enterocytes. TLR4, the receptor for gram-negative bacterial LPS, is overexpressed in the premature intestine and further upregulated during NEC. Activation of TLR4 by LPS triggers nuclear factor kappa B (NF-κB)-mediated inflammatory cascades, leading to enterocyte apoptosis and impaired mucosal repair. This pathway-level vulnerability in the premature gut is well characterized and provides strong biological plausibility for microbe-to-host causation [68]. Concomitantly, reduced levels of SCFA, particularly butyrate and acetate, and altered bile acid profiles are consistently observed in preterm infants at risk for NEC. SCFA depletion compromises epithelial barrier integrity and amplifies the Proteobacteria–LPS–TLR4 axis, thereby promoting early mucosal injury [68-70]. Accordingly, SCFA and bile acid profiles have emerged as promising early biomarkers and potential targets for prevention [69,71]. As mucosal injury progresses, microbe–inflammation interactions are likely to become bidirectional [72]. Despite the strong biological plausibility and temporality of these associations, it is noteworthy that not all infants with dysbiosis develop NEC, and the specificity and consistency of the dysbiosis–NEC link vary across studies. These observations highlight the multifactorial nature of NEC pathogenesis, in which microbial, host, and environmental factors interact to shape disease risk.

MOM provides robust protection against NEC, primarily because it contains immunoglobulins, growth factors, and bioactive components such as HMOs, which promote the growth of beneficial Bifidobacterium species while suppressing the abundance of potential pathogenic microbes [73,74]. In the absence of MOM, pasteurized donor human milk (DHM) represents the next best alternative, conferring protection through many of the same bioactive mechanisms [75]. HMOs, the principal prebiotic component of both MOM and DHM, drive unique beneficial shifts in gut microbial composition, supporting a microbiota less susceptible to NEC than that observed with formula feeding [73]. In addition, recent randomized controlled trials, meta-analyses, and the latest Cochrane review provide compelling evidence that supplementation with specific probiotic strains reduces the incidence of NEC and mortality, although the certainty of benefit varies according to strain and trial quality [75,76].

2. Late-onset sepsis in preterm infants

The role of the gut microbiome in sepsis development, particularly in preterm infants, has gained considerable attention. Compared with term infants, preterm neonates exhibit reduced microbial diversity and a lower abundance of anaerobes, factors that may increase their susceptibility to infections and dysregulated inflammatory responses [77]. Overgrowth of pathogenic gut bacteria can predispose these infants to systemic infections such as sepsis. Distinct microbial signatures have been observed between septic and non-septic preterm infants, with the former showing elevated levels of potential pathogens, including E. coli, K. pneumoniae, and Staphylococcus species. Similarly, reduced microbial diversity has been reported in preterm infants affected by LOS and pneumonia [7,78]. Diseasespecific mechanisms underlie the heightened risk of sepsis in preterm infants. In those with dysbiosis, compromised gut barrier facilitates the translocation of pathogenic bacteria and their products into the bloodstream, triggering systemic infection and inflammation [79]. Recent longitudinal studies further indicate that specific microbial signatures—particularly reduced abundance of Bifidobacterium and enrichment of Escherichia-Shigella—may facilitate the identification of highrisk subgroups among extremely low-birth-weight infants. Antibiotic exposure has been independently associated with a dramatic reduction in Bifidobacterium populations (up to 250-fold reduction), an effect that persists even after adjustment for confounders [80]. The loss of protective commensals further impairs barrier integrity and immune regulation, thereby increasing the risk of invasive infections. Therefore, the interplay between dysbiosis, SCFA depletion, and barrier dysfunction provides a mechanistic framework for understanding the increased susceptibility of preterm infants to sepsis.

3. Bronchopulmonary dysplasia: the gut–lung axis

BPD in preterm infants is increasingly conceptualized through the framework of the gut–lung axis, a bidirectional communication pathway that connects the intestinal and pulmonary systems via microbial, metabolic, and immune mechanisms. Disruptions in the gut microbiota, characterized by reduced microbial diversity and overgrowth of Proteobacteria, can compromise intestinal barrier integrity, allowing the translocation of bacterial products and metabolites into the circulation and ultimately to the lungs. This process can trigger systemic and pulmonary inflammation, exacerbate lung injury, and promote the development and progression of BPD [81]. Observational evidence suggests that greater exposure to MOM or DHM is associated with a lower risk of BPD, although evidence from randomized trials remains limited [82]. Mechanistically, gut dysbiosis in preterm infants increases intestinal permeability, facilitating the migration of immune cells and inflammatory mediators from the gut to the lungs. Experimental models further demonstrate that perinatal disruption of intestinal commensals—for instance, through antibiotic exposure— exacerbates BPD severity, leading to increased lung fibrosis, vascular remodeling, and alveolar inflammation. Collectively, depletion of SCFAs and increased intestinal permeability provide a plausible mechanistic link between gut dysbiosis and pulmonary inflammation in preterm infants [83]. Recent studies have identified the migration of immune cells—such as innate lymphoid cells and dendritic cells—between the gut and lungs as an important contributor to mucosal immune regulation and BPD pathogenesis [81]. Despite these insights, clinical evidence supporting microbiome-targeted interventions for BPD remains limited. Meta-analyses have shown no significant preventive effect of probiotics against BPD, and findings on prebiotics and SCFA supplementation remain preliminary. Furthermore, substantial heterogeneity across study populations, interventions, and outcomes continues to limit definitive conclusions [84,85]. Future large-scale, standardized cohort studies and interventional trials are essential to elucidate the therapeutic potential of gut microbiome modulation in the prevention and management of BPD.

4. Neurodevelopmental outcomes: the gut–brain axis

Preterm infants are at an elevated risk of neurodevelopmental impairment, and recent evidence implicates the gut–brain axis as a key modulator of early brain development in this vulnerable population. The gut–brain axis represents a bidirectional communication network integrating the gut microbiota, immune system, neural pathways, and developing brain [86]. Mechanistic insights from animal and human studies have identified key pathways mediating this connection. In preterm infants, gut dysbiosis is associated with increased intestinal permeability and pro-inflammatory cytokine release, which can cross the immature blood–brain barrier and affect white matter development and neuronal growth [87]. Microbial metabolites— particularly SCFAs, such as butyrate and acetate, produced by commensal bacteria such as Bifidobacterium and Bacteroides— play a central role in maintaining neuroimmune homeostasis. SCFAs promote neurogenesis, modulate microglial activation, and preserve blood–brain barrier integrity; their deficiency, observed in dysbiosis, correlates with reduced neuroprotective signaling and increased susceptibility to adverse neurodevelopmental outcomes [87]. Clinical evidence increasingly links early-life microbiome characteristics to neurodevelopmental outcomes in preterm infants. A lower abundance of healthassociated microbial taxa, as well as alterations in both static and dynamic microbiome features during the first month of life, correlate with poorer neurodevelopmental metrics, including reduced head circumference growth and lower neurobehavioral test scores at discharge and early childhood follow-up. For example, Oliphant et al. [88] found that preterm infants with poor head growth during their NICU stay exhibited consistently lower levels of Bacteroidetes and Lachnospiraceae. Similarly, Sun et al. [89] linked alterations in Clostridiales, Lactobacillales, and Enterobacterales order-level abundances with higher neonatal stress/abstinence scores and atypical behavioral responses. Disruption of the gut–brain axis has also been implicated in later neurodevelopmental difficulties; however, although observational signals exist, the causal links to specific diagnoses such as cerebral palsy and autism spectrum disorder—as well as broader cognitive and developmental impairments—remain uncertain [90]. Despite strong biological plausibility, large randomized control trials—including multistrain probiotic interventions (ProPrems) and donor milk vs. preterm formula comparisons (DoMINO)—have not consistently demonstrated improvements in primary neurodevelopmental endpoints at 18 to 24 months [91,92].

5. Feeding intolerance

FI is a common complication among preterm infants, characterized by delayed advancement of enteral feeding, gastrointestinal dysmotility, and prolonged hospitalization. Emerging evidence links FI to distinct microbial alterations, including Proteobacteria enrichment and reduced Firmicutes abundance [93]. Mechanistically, FI appears to result from impaired gut barrier integrity and mucosal inflammation, driven by the overgrowth of opportunistic pathogens and a lack of protective commensals. This imbalance may increase intestinal permeability, disrupt motility, perpetuate feeding difficulties, and increase the risk of subsequent complications, such as NEC. Notably, microbial disruptions associated with FI often persist beyond the period of clinical recovery, suggesting long-term effects on gut ecosystem development. Data from twin and NICU-based cohort studies suggest partially shared microbial trajectories among infants with FI; however, notable intra-pair and intra-unit differences underscore the complex interplay between host and environmental factors [94]. Understanding FI through the framework of microbiome dynamics may enable the development of function-informed diagnostics and targeted interventions to reestablish microbial balance.

METHODOLOGICAL APPROACHES IN NEONATAL MICROBIOME RESEARCH

Research on the neonatal microbiome is essential for elucidating the complex host–microbiota interactions that underpin early-life health and disease prevention. Various advanced experimental and analytical techniques have been developed and widely applied to investigate these interactions.

1. Germ-free and gnotobiotic animal models

Animal studies have contributed significantly to elucidating the structural and functional roles of the microbiome in neonatal gastrointestinal and immune system development. Models with anatomical and physiological characteristics comparable to those of humans can provide valuable insights into how gut microbial communities shape immune maturation and disease pathogenesis. In particular, germ-free (GF) and gnotobiotic models are essential tools for establishing causal relationships between specific microbial configurations and host physiology.

GF animals are raised under sterile conditions from birth and remain completely free of microbial exposure. These models are invaluable for assessing how the absence of microbiota affects host development and disease susceptibility [95]. Gnotobiotic animals—created by colonizing GF hosts with defined microbial consortia, ranging from single strains or synthetic communities— enable controlled, causal investigations of how the microbiota modulates key host functions such as immunity, metabolism, and inflammation [96]. These models provide a controlled experimental setting to evaluate how early-life exposures affect microbial colonization patterns and immune system development. For instance, gnotobiotic animals inoculated with beneficial microbes have been used to delineate their effects on intestinal barrier integrity, immune cell differentiation, and systemic inflammatory responses.

More recently, gnotobiotic models using mice, rats, and piglets with physiological features analogous to those of human neonates—have advanced the translational relevance of microbiome studies. These models closely replicate key features of the human neonatal gut architecture, immune development, and feeding responses, thereby providing an essential preclinical platform for exploring microbiome-targeted therapies and personalized probiotic interventions in early life [97].

2. Omics-based analytical techniques

The advent of high-throughput sequencing technologies has revolutionized microbiome research, enabling large-scale genomic and functional analysis of microbial communities. Omics-based approaches—including comprehensive molecular profiling methods such as genomics, transcriptomics, proteomics, and metabolomics—have substantially advanced neonatal microbiome research. These tools facilitate an integrated system-level understanding of the role of the microbiome in health and disease [5].

Metagenomics involves the direct sequencing of microbial DNA within a sample to characterize both its taxonomic composition and functional potential. Recent advances in singlecell and genome-resolved approaches, such as single-amplified genomes, Hi-C–assisted binning, and microfluidics-based partitioning, enable higher-resolution genome reconstruction for individual cells and low-abundance taxa [98].

Transcriptomics examines RNA transcripts to identify genes actively expressed under specific conditions, thereby offering insights into microbial activity and gene expression dynamics. Proteomics complements this by characterizing the complete set of proteins expressed in the microbiome. Given that proteins represent the functional execution of genetic information, proteomic analyses provide information on the biochemical functions currently performed by the microbial community [99].

Metabolomics identifies and quantifies the small molecules produced or modified by the microbiome, including SCFAs, vitamins, bile acids, amino acids, lipids, and other bioactive molecules that mediate host–microbe chemical interactions. Volatile organic compounds, a distinct subset of metabolomic signatures, are increasingly investigated as potential noninvasive diagnostic biomarkers for neonatal diseases such as NEC [100].

Multi-omics integration combines metagenomic, transcriptomic, proteomic, and metabolomic datasets to provide a comprehensive understanding of microbiome–host interactions. By correlating gut microbial composition with metabolic profiles and host immune mediators such as cytokines, this approach enables the elucidation of complex molecular mechanisms that connect microbiota dynamics to neonatal immune maturation and maternal–fetal signaling [13,101].

This review highlights the critical role of the neonatal microbiome in shaping infant health and disease outcomes. The establishment of the early-life microbiome constitutes a foundational element that influences the development of the immune system, metabolic processes, and neurodevelopment. Among these systems, healthy development of the gut microbiome is particularly important. Dysbiosis during the neonatal period is strongly associated with the pathogenesis of severe conditions such as NEC and LOS in preterm infants [5]. However, several aspects remain subjects of ongoing debate and investigation.

1) Mechanistic studies

In-depth investigation of the molecular and cellular mechanisms underlying host–microbe interactions is essential for uncovering the biological basis of microbiome-mediated effects. Although many studies have identified associations between dysbiosis and diseases, establishing causality and delineating the precise mechanistic pathways involved remain major challenges.

2) Longitudinal studies

Long-term cohort studies are essential to track microbiome developmental trajectories and their sustained associations with pediatric and adult health outcomes. Ongoing debates regarding the influence of perinatal interventions, such as antibiotic exposure, probiotic supplementation, and delivery mode, on the microbiome and subsequent health, highlight the need for robust, multicenter, and diverse cohort designs.

3) Integration of multi-omics technologies

The application of integrative omics approaches, including metagenomics, metabolomics, transcriptomics, and proteomics, offers the potential for a comprehensive system-level understanding of the microbiome–host interface. However, realizing this potential requires overcoming significant technical and methodological challenges such as sample collection, data integration, and biological interpretation.

CONCLUSION

In summary, the neonatal microbiome represents more than a biological phenomenon—it functions as a central regulator of early-life developmental programming. Continued advances in this field hold the potential to transform neonatal and pediatric medicine by enabling the development of personalized, microbiome-based therapeutic and preventive strategies. These innovations may represent a paradigm shift in clinical practice; however, their implementation requires careful consideration of ongoing controversies, rigorous research, and consensus within the field.

ARTICLE INFORMATION

Ethical statement

None

Conflicts of interest

Jin Kyu Kim is an editorial board member of the journal, but he was not involved in the peer reviewer selection, evaluation, or decision process of this article.

Author contributions

Conception or design: JKK.

Acquisition, analysis, or interpretation of data: JKK.

Drafting the work or revising: JKK.

Final approval of the manuscript: JKK.

Funding

None

Acknowledgments

None

Figure 1.

Factors influencing neonatal microbiome development and pathways linking early dysbiosis to adverse outcomes. Schematic representation of perinatal factors influencing neonatal microbiome development and key pathways linking early dysbiosis to adverse outcomes. Perinatal factors, including delivery mode, gestational age, feeding mode, antibiotic exposure, and environmental factors, shape initial microbial colonization and maturation of the neonatal microbiome. Breast milk, appropriate antibiotic stewardship, and probiotic supplementation may promote eubiosis, which is characterized by balanced microbial communities and optimal immune and metabolic functions. In contrast, dysbiosis, marked by reduced microbial diversity (decreased Bifidobacterium, increased Proteobacteria), impaired metabolic activity (lower short-chain fatty acid [SCFA] production, barrier dysfunction), and heightened innate immune activation (increased Toll-like receptor 4 [TLR4] → nuclear factor kappa B [NF-κB] signaling), is associated with an increased risk of necrotizing enterocolitis (NEC), late-onset sepsis (LOS), and bronchopulmonary dysplasia (BPD). Dysbiosis may also affect the gut–brain and gut–lung axes through compromised immune programming, impaired barrier integrity, and altered metabolic support.

Table 1.

Microbiome-Disease Links in Neonates

Condition	Key mechanistic link	clinical implication	Limitations	References
Necrotizing enterocolitis (NEC)	Immature gut with high TLR4 signaling; dysbiosis with Proteobacteria↑& Bifidobacterium↓ → barrier failure, necroptosis; Paneth-cell dysfunction; SCFA↓, bile-acid/amino-acid pathway shifts; antibiotic pressure signatures preceding NEC	Human milk feeding reduces NEC risk; antibiotic stewardship; probiotics: possible NEC benefit but strainspecific & safety/regulatory concerns; HMOs are promising but not yet standard	Heterogeneity (strains/dose/timing, feed ing policies), potential publication bias; limited ELBW data; biomarker standardization needed	[5,61,68,74,75,92]
Late-onset neonatalsepsis (LOS)	Gut colonization → bloodstream; dysbiosis with diversity↓, Enterobacte riaceae/Enterococcus↑; antibiotic-driven pathogen dominance; MOM/HMOs promote Bifidobacterium & colonization resistance	Antibiotic stewardship (limit empiric duration); MOM early & adequate; probiotics: little/no effect on LOS; lactoferrin not beneficial	Multiple LOS pathways (gut, catheter, skin); definitions vary; older cohorts vs. modern NICU; probiotic safety/quality/regulation issues	[7,61,78,79,80]
Bronchopulmonary dysplasia (BPD)	Gut–lung axis: dysbiosis & low SCFA → systemic inf lammation impairs alveolarization; airway microbiome: lower diversit y, ↑Ureaplasma/Sta phylococcus/Enterobacteriaceae; exposures (ventilation/oxygen/antibiotics) interact with microbiome	MOM diet associated w ith lower BPD risk (dose–response signals); minimize broad/long empiric anti-biotics; probiotics not effective for BPD prevention	Association ≠ causation; sampling/site/method heterogeneity; few trials targeting BPD as primary endpoint; evolving NICU practices limit generalizability	[62,81-83,85]
Neurodevelopmental outcomes	Gut–brain axis via immune activation (microglial priming), BBB stability, SCFA & tryptophan–serotonin/kynurenine pathways; early infection/antibiotics linked to later NDI	MOM prioritized (biologic plausibility & observational links), but RCTs show limited/neutral effects on primary neurodevelopmental endpoints; probiotics not indicated for neurodevelopment gains	Predominantly observational data; varied sampling/omics pipelines; diverse developmental tools/timepoints; strain-specificity & timing issues	[21,59,86,87,91]
Feeding intolerance (FI)	Dysbiosis → mucosal inflammation/barrier dysfunction & ENS–motility disruption; SCFA support mucus/tight junction integrity & motility; early/prolonged antibiotics → delayed FEF	MOM programs show FI improvement signals; standardize feeding protocols; probiotics: mixed—some RCTs suggest ↓FI & faster FEF, newer meta-analysis shows uncertain effects; cautious use given safety	FI definitions are non-standard & subjective; small single-center trials; probiotic strain/dose/start vary; limited HMO RCTs	[49,60,76,93,94]

Abbreviations: TLR4, Toll-like receptor 4; SCFA, short-chain fatty acid; HMO, human milk oligosaccharide; ELBW, extremely low birth weight; MOM, mother’s own milk; NICU, neonatal intensive care unit; BPD, bronchopulmonary dysplasia; BBB, blood–brain barrier; NDI, neurodevelopmental Impairment; RCT, randomized controlled trial; ENS, enteric nervous system; FEF, full enteral feeding.

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