Synergistic regulation of gut microbiota and metabolites by breast milk and probiotics in the prevention of neonatal necrotizing enterocolitis: mechanisms and prospects

Introduction

Necrotizing enterocolitis (NEC) is a critical intestinal emergency that predominantly affects neonates, especially those born prematurely. While advances in perinatal medicine and neonatal intensive care have significantly improved the survival rates of preterm infants, this progress has been accompanied by a rise in the incidence of NEC, with a global prevalence of approximately 7.0% among infants with extremely low birth weight (ELBW). Challenges in early diagnosis and limited treatment options—primarily supportive care and surgical intervention (1)—contribute to a persistently high mortality rate of 20–30% (2,3). Survivors often face significant long-term complications, such as neurodevelopmental delay and short bowel syndrome, which impose substantial economic and caregiving burdens on families and society (4). The precise etiology and pathogenesis of NEC remain incompletely understood; it is generally posited to be associated with multiple factors, such as intestinal barrier immaturity in preterm infants, gut microbiota dysbiosis, inappropriate feeding practices, and abnormal inflammatory responses (5,6).

Recent studies indicate that breastfeeding is an effective preventive measure against NEC (7). Breast milk is rich in active components, including immunoglobulins, human milk oligosaccharides (HMOs), and lactoferrin, which confer protective benefits by facilitating the maturation of the intestinal barrier, modulating the immune system, and influencing the gut microbiota. Concurrently, probiotics have emerged as a promising microecological strategy for preventing NEC, exerting protective effects by competitively inhibiting pathogenic bacteria, enhancing the intestinal epithelial barrier function, and modulating immune responses. A key area of current scientific inquiry focuses on how breastfeeding and probiotics synergistically shape the development of a healthy “gut microbiota” and regulate the production of its “metabolites”, thereby establishing a physiological defense mechanism against NEC.

This review focuses on the synergistic axis of probiotics-breast milk-gut microbiota-host to systematically elucidate the molecular mechanisms underlying the interaction between breast milk and probiotics. By coordinately regulating intestinal microecology and host physiological functions, these factors contribute to the prevention of NEC. We further identify critical unresolved questions and controversies in the current literature, propose future research directions, and provide a theoretical basis for refining clinical preventive strategies against NEC.

Breast milk and probiotics as synergistic upstream ecological modulators

Breast milk serves as the foundational early-life exposure that shapes the neonatal gut ecosystem (8). It supplies both substrates that select for beneficial microbes and molecular signals that prime host immune development. Among these substrates, HMOs are preferentially metabolized by Bifidobacteria, a key beneficial gut bacterium (9). This selective utilization fosters the proliferation of Bifidobacteria and enhances the production of short-chain fatty acid (SCFA), which support intestinal epithelial integrity and immune regulation. Furthermore, bioactive components such as lactoferrin and lysozyme in breast milk help create a permissive niche for beneficial microbes. Lactoferrin restricts the growth of iron-dependent pathogens and can tilt the microbial community towards beneficial taxa, enhancing the colonization of probiotics like Bifidobacterium and Lactobacillus (10,11). These antimicrobial proteins, together with HMOs, support the establishment of a healthy microbiota, crucial for reducing the risk of NEC.

Breast milk components shape gut microbiota and host immunity

Breast milk serves as both the foundational exposure that shapes the neonatal gut ecosystem and the optimal standard for infant health, offering superior nutrition and multifaceted support. Firstly, bioactive components like antibodies and lactoferrin offer direct immune protection (12,13). Secondly, breastfeeding significantly diminishes the risk of numerous infectious diseases, including respiratory infections, gastrointestinal infections, and NEC (14-16). Furthermore, it promotes neurodevelopment and long-term cognitive function (17,18). Breast milk also plays a crucial role in establishing a healthy gut microbiome. Components including HMOs prevent NEC primarily by promoting the growth and colonization of beneficial bacteria (Figure 1) (19-21).

Figure 1 Key bioactive components in breast milk that regulate infant gut microbiota. (I) HMOs promote the growth of bifidobacteria and enhance SCFA production. When added to formula or probiotic products, HMOs help shape a gut microbiota structure resembling that of breastfed infants. Maternal probiotic intake may further modulate HMOs composition in breast milk. (II) Lactoferrin suppresses pathogenic bacteria while stimulating probiotic proliferation. It also exhibits anti-inflammatory activity, increases intestinal SCFA levels, promotes Treg production, and enhances the expression of tight junction proteins and INAVA, thereby strengthening the intestinal barrier. (III) Immunoglobulins provide passive immunity by reducing GBS colonization. Through an “immune coating” mechanism, they facilitate the transfer of maternal gut bacteria to the infant, promoting beneficial colonization, and support dendritic cell activation for specific immune responses. (IV) Lysozyme, a key antibacterial protein, directly lyses bacteria by hydrolyzing peptidoglycan in bacterial cell walls. Expressed mainly in intestinal immune cells, it selectively supports the growth of beneficial gut bacteria. Created with Biorender. Siyi Chen. [2025] (https://BioRender.com/7v7ji74). HMOs, human milk oligosaccharides; Lr DSM 17938, Lactobacillus reuteri DSM 17938; SCFA, short-chain fatty acid; sIgA, secretory IgA; TLRs, Toll-like receptors; Treg, regulatory T cell; ZO-1, zonula occludens-1.

HMOs

HMOs are critical constituents of breast milk, playing a significant role in promoting brain development (22,23). As essential prebiotics, HMOs modulate the gut microbiota to enhance infant health. Although infants cannot digest them, HMOs reach the colon and serve as a selective substrate to foster the proliferation of probiotics, especially bifidobacteria (24,25). Many probiotics possess specialized mechanisms to metabolize HMOs (26). Clinical studies demonstrate that HMOs supplementation in formula-fed infants results in a gut microbiota composition akin to that of breastfed infants (27). Notably, adding 2'-fucosyllactose (2'-FL) significantly elevates the abundance of bifidobacteria with relevant metabolic genes, aligning their proportion more closely with breastfed infants (28,29). Research demonstrates a synergistic relationship between HMOs and probiotics in enhancing gut health. Adding HMOs to formula containing Limosilactobacillus reuteri DSM 17938 (Lr DSM 17938) shifted infant gut microbiota toward a breastfed-like profile, underscoring a synergistic effect on probiotic colonization (30). In vitro studies further corroborate that co-culturing Bifidobacterium longum subsp. infantis with specific HMOs mixtures significantly enhances SCFAs production compared to either alone, suggesting amplified beneficial metabolic outputs (31). Additionally, maternal probiotic supplementation may modify the composition of HMOs in breast milk, affecting the concentrations of specific compounds such as 3-fucosyllactose and 3'-sialyllactose, therapy influencing the amount of HMOs ingested by the infant (32). In conclusion, specific fucosylated and sialylated HMOs reduce pathogen adherence, blunt epithelial Toll-like receptor 4 (TLR4) signaling, and promote epithelial maturation. In experimental NEC models, HMO-enriched feeding decreases mucosal injury scores and inflammatory cytokines while increasing tight junction proteins and goblet-cell mucus production. At the ecosystem level, HMOs act as selective carbon sources for HMO-utilizing bifidobacteria, generating acetate and lactate that acidify the lumen and restrict Enterobacteriaceae expansion. HMOs primarily exert prebiotic effects on intestinal microecology. This not only explains their direct benefits in infection resistance and intestinal barrier fortification but may also represent the fundamental pathway for their immunomodulatory and potential neurodevelopmental advantages (22,23).

Lactoferrin

Lactoferrin, a pivotal multifunctional protein found in breast milk, plays a crucial role in promoting gut health. It exerts direct antibacterial effects by binding to iron, which inhibits the growth and biofilm formation of various pathogens, including Listeria, Salmonella, and Escherichia coli (33-35). This mechanism also provides a competitive advantage to beneficial microbes, such as Bifidobacterium and Lactobacillus, by limiting iron availability to harmful bacteria (20,33). Mechanistically, lactoferrin enhances gut health through multiple pathways. It increases the abundance of beneficial genera, including Eubacterium xylanophilum group, Tuzzerella, and Oscillibacter, while reducing the presence of harmful bacteria like Desulfovibrio, Erysipelatoclostridium, Bacteroides, and Alistipes (36). Lactoferrin also plays a key role in restoring antibiotic-induced dysbiosis (37). Additionally, lactoferrin strengthens both the physical and immune barriers of the gut. It upregulates tight junction proteins, such as zonula occludens-1 (ZO-1) and INAVA, which enhance epithelial cell integrity and prevent pathogen invasion (35). On the immune front, lactoferrin modulates inflammation by promoting anti-inflammatory cytokines (e.g., IL-10, TGF-β) and reducing pro-inflammatory factors (e.g., TNF-α, IL-6), partly through the modulation of Toll-like receptors (TLRs) like TLR2, TLR8, and TLR9 (37,38). Lactoferrin also promotes the production of SCFAs such as butyrate, propionate, and acetate, which nourish intestinal epithelial cells and contribute to systemic anti-inflammatory effects by enhancing regulatory T cell (Treg) production (39). Furthermore, lactoferrin’s influence on the microbiota-gut-brain axis has been linked to improvements in cognitive function (38).

In conclusion, lactoferrin supports gut health by exerting antibacterial, microbiota-modulating, barrier-strengthening, and immunoregulatory effects, thus helping maintain homeostasis and counteracting dysbiosis. Animal studies have demonstrated that lactoferrin supplementation preserves tight junctions and reduces mucosal inflammation (TNF-α, IL-6), while increasing IL-10, reflecting reduced macrophage-driven inflammation. Clinical studies evaluating bovine lactoferrin supplementation (with or without probiotics) have shown mixed results in preventing sepsis and NEC, emphasizing the need for standardized dosing and product characterization.

Immunoglobulin

Secretory IgA (sIgA) is the principal antibody in intestinal mucosal immunity and a critical component of breast milk, conferring passive immunity to neonates (40). sIgA fulfills several protective functions within the infant gut. Primarily, sIgA provides direct immune defense by acting as the initial barrier against pathogens. For instance, sIgA binds to the surface of group B Streptococcus (GBS), thereby preventing its adherence to the mucosa and significantly diminishing its colonization (41). Animal studies substantiate that maternally derived antibodies in breast milk prevent intestinal pathogen infections in neonates (42). Additionally, sIgA precisely modulates gut microbiota to maintain homeostasis. A core mechanism is “immune coating”, wherein the mother’s gut microbiota, coated with sIgA, is transferred via breast milk, facilitating the colonization of beneficial bacteria such as lactobacilli and bifidobacteria (43,44). Research indicates a positive correlation between breast sIgA content and infant gut probiotic abundance (45), and this beneficial modulation of the gut microecology effectively reduces the risk of NEC. Concurrently, sIgA also inhibits the adhesion of bacteria to the intestinal epithelium, thereby mitigating excessive Th17 responses and inflammation (46). Furthermore, sIgA bridges the innate and adaptive immunity. Bacteria coated with sIgA can be transported via M cells, thereby activating dendritic cells and inducing specific immune responses that enhance the infant’s immune defenses (47-49). Consequently, exclusively breastfed infants exhibit elevated sIgA levels (50), while probiotic-supplemented infant formula has also been shown to help maintain stable sIgA levels in infants (51). In conclusion, sIgA shapes early immunity by limiting epithelial contact with pro-inflammatory taxa, reducing excessive Th17 skewing, and facilitating tolerogenic antigen sampling via M cells and dendritic cells. In NEC-relevant settings, higher luminal sIgA coating is associated with a microbiota richer in bifidobacteria/lactobacilli and lower inflammatory cytokine expression in the mucosa.

Lysozyme

Lysozyme is a crucial antibacterial protein in breast milk, with concentration varying from approximately 0.36–0.37 g/L in colostrum to 0.83–0.89 g/L in late mature milk (52), aligning with developmental immune needs. Its primary function is to lyse bacterial cells directly by hydrolyzing peptidoglycan in cell walls, primarily targeting gram-positive bacteria (53). Beyond direct antibacterial effects, lysozyme regulates the gut microecosystem by selectively promoting infant-associated Bifidobacterium (B. bifidum, B. breve, B. longum) and inhibiting animal-derived strains like B. animalis, exhibiting unique lineage specificity (54,55). Lysozyme supplementation increases lactic acid bacteria in the gut, reduces Escherichia coli, and promotes the production of beneficial gut metabolites such as L-glutamine, thereby supporting intestinal development and modulating the expression of inflammatory cytokines (56-58). In NEC pathology, lysozyme expression is concentrated in intestinal immune cells, particularly macrophages and neutrophils (59). High lysozyme levels may play a protective role in local inflammatory responses. However, during severe systemic inflammation, lysozyme may shift to promoting inflammation, thereby impairing gut barrier function. Notably, lysozyme acts synergistically with other immune proteins such as sIgA (60), collectively regulating the neonatal immune response and enhancing resistance to gastrointestinal and respiratory infections. Lysozyme not only helps infants establish an initial line of immune defense but also contributes to the long-term development of their immune systems. In addition to direct bacteriolysis, lysozyme can reshape microbial community function by selecting for infant-adapted bifidobacterial lineages and altering amino-acid and glutamine-related metabolite pools. These metabolite shifts may influence epithelial proliferation and redox balance, providing a plausible route by which lysozyme affects barrier repair during inflammatory stress.

Key microorganisms and probiotic strains in NEC

The human gastrointestinal tract harbors a highly diverse community of microorganisms, which exerts pivotal roles in the early postnatal development of neonates (61). A healthy gut microbiota is essential for barrier function, immune regulation, and nutrient metabolism (62). In neonates, the colonization and succession of gut microbiota constitute a dynamic, multi-stage process influenced by various factors, such as delivery mode, feeding method, antibiotic use, and environmental conditions, all of which contribute to the maturation of gut barrier function and the development of the immune system (63-65). Typically, detectable bacteria begin to colonize the gut within 16 hours after birth. Anaerobic or facultative anaerobic bacteria rapidly adapt to the infant gut environment and proliferate forming the earliest colonizing communities. Over time, and influenced by breastfeeding or formula feeding, bifidobacteria gradually become the predominant group, particularly in breastfed infants. These bacteria utilize HMOs in as a nutrient source, therapy producing health-promoting SCFAs, such as acetate and lactate. Consequently, the colonization of the neonatal gut microbiota not only affects its composition but also influences the gut’s metabolic functions. However, the gastrointestinal tract of preterm infants often exhibits significant developmental immaturity, characterized by slowed gut motility, impaired nutrient utilization, underdeveloped Paneth cells, and abnormalities in innate immune system (66,67). These physiological deficiencies collectively impair the ability of preterm infants to regulate the gut microbiota, increasing susceptibility to dysbiosis (68,69). Growing evidence links such microbial imbalance to NEC (70,71). In patients with NEC, gut microbial diversity is reduced, with Firmicutes and Proteobacteria sequentially becoming the dominant phyla prior to disease onset, while Bifidobacterium levels decline 2–3 weeks before symptoms onset (72-74).

Research indicates that probiotics can significantly alter the gut microbiota of preterm infants (75). By the time preterm infants receiving probiotic supplementation reach full-term equivalence, their gut microbiota closely resembles that of full-term infants of the same age (76). Orally administered probiotics, particularly those containing Bifidobacterium and Lactobacillus, have been shown to increase beneficial populations while inhibiting the growth of potentially pathogenic bacteria (77,78). The administration of probiotics not only suppresses the growth of pathogenic and opportunistic bacteria (79) but also reduces feeding intolerance in preterm infants, which is crucial for the nutritional support of the nervous system and subsequent growth and development (80). Moreover, a systematic review and meta-analysis of 70 clinical studies (involving 8,319 cases and 9,283 controls) demonstrated that probiotics significantly reduce both the incidence of NEC and related mortality (81), with the effect being particularly notable in preterm infants (82). These findings are supported by additional studies. For instance, an assessment of probiotic efficacy in reducing severe NEC across neonatal intensive care units (NICUs) in New Zealand reported a 38% decrease in stage 2 or higher NEC, most evident in preterm infants. The combination of Lactobacillus and Bifidobacterium yielded the most favorable outcomes (83). Based on recent evidence from network meta-analyses and subgroup-based systematic reviews, multi-strain probiotic regimens in preterm infants (especially very low birth weight (VLBW) and/or ≤34 weeks’ gestation) are associated with more consistent reductions in NEC (Bell stage ≥ II) and all-cause mortality; in contrast, the evidence for single-strain regimens is more variable across outcomes or does not always reach statistical significance (84,85). Importantly, the protective or pathogenic influence of probiotics on NEC depends on the specific strain used (86); even different strains within the same species may exert distinct effects. Clinical evidence highlights that selecting appropriate probiotic strains is essential for effective NEC prevention (87). Beyond protection (88-92), the gut microbiota and specific metabolites may serve as potential biomarkers for the early diagnosis of NEC, thereby aiding in the prediction of disease onset (70,93,94). Collectively, these studies highlight the significant role of probiotics in preventing and treating NEC, and below we summarize representative organisms and strains.

Lactobacillus

Lactobacillus plays a crucial role in preventing NEC through multiple mechanisms, including modulating intestinal inflammation, enhancing gut barrier function, and regulating immune responses. It is one of the earliest identified probiotics and remains widely used in clinical applications (95,96). The abundance of Lactobacillus is typically lower in high-risk preterm infants compared to breastfed controls (97). After supplementation, Lactobacillus colonization is often associated with increased lactate and acetate production, as well as a reduction in inflammatory markers. Lactobacillus strains, such as Lactobacillus rhamnosus GG (LGG) and Lactobacillus reuteri, are among the most studied, showing potential in reducing NEC incidence, particularly Lactobacillus reuteri, which is associated with enhanced feeding tolerance, growth, and prevention of NEC in preterm infants (98-101).

Mechanistically, Lactobacillus strains exert their protective effects by regulating several molecular pathways. These mechanisms include competition with Enterobacteriaceae for adhesion sites and nutrients, the production of antimicrobial metabolites like reuterin, and enhancement of epithelial restitution and tight junction expression (102,103). Additionally, Lactobacillus modulates immune responses by dampening TLR4-NF-κB signaling and promoting Treg programs (104-106). For example, Lactobacillus reuteri enhances intestinal barrier function and reduces inflammation via its biofilm state, which is more effective and durable in preventing NEC than its planktonic state (107). Furthermore, L. reuteri has been shown to mitigate NEC-induced neuroinflammation and improve cognitive function through modulation of the gut-brain axis (108). Some strains of Lactobacillus also regulate oxidative stress and improve intestinal permeability. LGG, for instance, mitigates TLR-mediated intestinal damage by upregulating inhibitory factors like SIGIRR and A20, suppressing excessive TLR4 activation (105,106). Lactobacillus acidophilus has been shown to reduce NEC incidence by inhibiting intestinal epithelial apoptosis and reducing pro-inflammatory cytokines like TNF-α and IL-6 (109,110). Similarly, Lactobacillus gasseri maintains intestinal epithelial integrity by producing acetate, which activates GPR41/43 receptors (111).

In conclusion, Lactobacillus exerts protective effects against NEC by targeting specific molecular pathways, such as TLR-related signaling, G protein-coupled receptor pathways, and pathways associated with lipid peroxidation and inflammation (112,113). It also produces metabolites like reuterin, histamine, N-carbamoylglutamate, SCFAs, and tryptophan derivatives that contribute to its protective role. The cellular targets of Lactobacillus include intestinal epithelial cells, where it reduces apoptosis, enhances intestinal permeability, and maintains epithelial integrity. Lactobacillus also acts on immune cells, regulating T-cell subsets, enhancing dendritic cell tolerance, and reducing pro-inflammatory cytokines. Notably, these protective effects are strain-specific, with distinct molecular targets and cellular effector sites among different Lactobacillus strains (98,105,106,114).

Bifidobacterium

Bifidobacterium plays a significant role in the prevention of NEC by fortifying the intestinal barrier, modulating immune responses, and improving gut microbiota composition. Research has demonstrated that decreased levels of Bifidobacterium, particularly weeks before the clinical onset of NEC, correlate with an increase in Proteobacteria and elevated luminal lipopolysaccharide (LPS) levels (115,116). Reconstitution with Bifidobacterium has been shown to enhance acetate and indole-3-lactic acid (ILA) levels, improve mucin layer function, and reduce epithelial IL-8 responses via aryl hydrocarbon receptor (AHR)/NF-κB modulation (89,117). Furthermore, Bifidobacterium is commonly administered alongside other probiotics, such as Lactobacillus rhamnosus, which significantly lowers NEC incidence (118). Studies also suggest that strains such as Bifidobacterium animalis subsp. lactis can serve as an early marker for NEC detection in infants (93). Mechanistically, Bifidobacterium exerts its protective effects through several molecular pathways. It strengthens the intestinal barrier by decreasing permeability and increasing the expression of tight junction proteins, such as ZO-1, claudin-1, and occluding (119). For example, B. breve AHC3 has been shown to enhance intestinal barrier function and alleviate NEC pathology (120). Additionally, Bifidobacterium modulates immune responses by regulating TLR signaling. B. breve M-16V, for instance, inhibits TLR4 expression and enhances TLR2 expression, leading to reduced pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6 (121). Secretions from Bifidobacterium infantis also regulate the NF-κB pathway to reduce IL-8 expression, thus alleviating intestinal inflammation (89). Moreover, B. longum and its secretions can inhibit IL-1β-induced inflammatory responses via TLR-4 and decrease IL-6 production, while its secreted ILA modulates the AHR transcription factor to further reduce immune responses (117,122).

In summary, the protective effects of Bifidobacterium, particularly B. breve, B. longum, and B. lactis, are attributed to their regulation of specific molecular pathways, including TLR-related signaling, the NF-κB pathway, and the AHR pathway. These mechanisms collectively enhance intestinal barrier integrity, modulate immune responses, and reduce inflammation, thereby lowering the incidence and severity of NEC. However, the optimal strain combinations and dosages require further validation through randomized controlled trials (RCTs).

Saccharomyces

Invasive Candida species are uncommon as primary triggers but can complicate NEC with severe barrier disruption and sepsis, particularly in ELBW infants and after broad-spectrum antibiotics. Conversely, S. boulardii may confer protection in some studies through competitive exclusion and immunomodulation; however, the risk-benefit balance is uncertain in immunologically fragile neonates. Saccharomyces exhibits a complex dual role in the development of NEC. On one hand, Candida species are recognized as significant pathogens. Although NEC directly attributable to Candida is relatively uncommon, its occurrence is associated with severe consequences. Clinical studies have demonstrated that C. albicans directly impairs the intestinal epithelial barrier, independently induces NEC, and leads to cecal perforation with high mortality rate in preterm infants (123,124). An autopsy report revealed that the prevalence of intestinal Candida infection in NEC specimens complicated by intestinal perforation was approximately 7.5% (124). Furthermore, Candida infection is closely linked to NEC progression; a clinical study documented that up to 58% of ELBW infants developed systemic infection with Candida parapsilosis during NEC progression (125). This organism forms biofilms, adheres to intestinal epithelium, exhibits strong drug resistance, and disseminates into the bloodstream, ultimately resulting in intestinal perforation, sepsis, and multiple organ failure. Conversely, Saccharomyces boulardii is regarded as a promising probiotic that may help reduce NEC incidence (126). However, in vulnerable preterm infants with immature immune systems, the use of probiotics, including Saccharomyces boulardii, requires caution due to the potential increased risk of Candida infections. A meta-analysis highlighted that although a compound preparation containing S. boulardii can expedite full enteral feeding and reduce overall NEC risk and mortality, an increased incidence of Candida infection was also observed (127). Therefore, for the primary objective of preventing NEC and mortality, current evidence more robustly supports the use of multi-strain probiotic formulations, and the use of S. boulardii alone is not recommended (128). In conclusion, yeast species play a dual role in NEC. Future research is urgently required to investigate how to effectively balance the benefits of probiotics against the potential risk of Candida infection in this vulnerable preterm population.

Mechanistically, the opposing roles of yeast in NEC may be attributed to distinct molecular pathways and cellular effects. Pathogenic Candida species disrupt tight junction proteins, trigger excessive inflammatory responses, and induce apoptosis in intestinal epithelial cells, thereby compromising barrier integrity. In contrast, probiotic Saccharomyces boulardii may modulate immune signaling pathways, enhance epithelial repair, and maintain intestinal homeostasis. However, the underlying molecular crosstalk between yeast, epithelial cells, and immune cells in the immature gut remains incompletely defined. Further exploration of these specific pathways will facilitate the safe and targeted application of yeast‑based probiotics in preterm infants at risk of NEC.

Enterococcus and Streptococcus

Enterococcus and Streptococcus show time-dependent and strain-dependent dynamics. Early overgrowth of pro-inflammatory Enterococcus (e.g., E. faecalis) and certain Streptococcus species can coincide with heightened TLR2/TLR4 signaling and barrier injury, while other strains (e.g., E. faecium in some models; S. thermophilus as part of multi-strain products) may support recovery via butyrate-associated pathways and competitive exclusion. Enterococcus is an early colonizer of the neonatal gut (129) and may significantly influence NEC onset and recovery, with different subspecies exerting contrasting effects. Clinical studies using 16S rRNA sequencing identify Enterococcus as a major differential genus in the gut microbiota of neonates with NEC, suggesting its potential as an early diagnostic biomarker. Its abundance fluctuations may reflect dysbiosis and disease progression (130). Studies in NICUs in China (131) have revealed that Enterococcus, predominantly E. faecalis, is the dominant intestinal genus during NEC progression. Several clinical and animal studies have demonstrated that E. faecalis may contribute to the early inflammatory response of NEC. It proliferates excessively in the intestines of preterm infants with NEC, exacerbating inflammation in intestinal epithelial cells and disrupting the intestinal barrier by activating the TLR2/TLR4 signaling pathways (132,133). Recent clinical research observed significant changes in Enterococcus abundance in neonates with NEC (134). Certain NEC infants exhibited an increase in Enterococcus faecium abundance during the recovery phase, suggesting this strain may contribute to restoring intestinal homeostasis by regulating microbial community structure. A multi-omics study in a neonatal rabbit NEC model found that Enterococcus faecium exerts protective effects by competitively inhibiting intestinal pathogens, regulating butyrate metabolism, and promoting barrier repair (135). Enterococcus durans, a commensal increased by breastfeeding in some animal models, is linked to enhanced intestinal mucosal integrity, potentially protecting against NEC through metabolites like SCFAs (136). Thus, Enterococcus demonstrates a dual role with some strains facilitating recovery and others contributing to early inflammation. Collectively, the dual role of Enterococcus in NEC is mediated via divergent signaling pathways and cellular responses. Pathogenic strains such as E. faecalis promote intestinal inflammation and barrier disruption by activating TLR2/TLR4 signaling in epithelial cells, while protective strains including E. faecium and E. durans maintain mucosal integrity via regulating microbial balance, butyrate metabolism, and epithelial repair. These strain-specific molecular mechanisms highlight the potential of tailored Enterococcus‑based interventions for NEC prevention and treatment.

Streptococcus similarly exhibits a dual role in NEC. On one hand, many studies indicate certain species may contribute to the pathogenesis of NEC. Clinical investigations have revealed a significant increase in Streptococcus abundance in the gut microbiota of neonates with NEC, particularly in early stages (137). A study of preterm infants in China found Enterococcus, Streptococcus, and Peptoclostridium dominating the gut microbiota during NEC progression (131). The persistent colonization of Streptococcus, from meconium to pre-onset stool samples, suggesting its potential pathogenic role (138). Certain streptococci, such as Streptococcus salivarius, are significantly elevated in children with NEC and are regarded as early predictive biomarkers (70). Furthermore, infection with pathogenic strains, such as GBS, may directly precipitate or exacerbate NEC (139). Conversely, specific probiotic streptococcal strains have demonstrated protective potential. Some Streptococcus species is associated with protective gut microbiota against NEC (140,141). Additionally, both animal and clinical studies have shown that multi-strain probiotic formulations containing Streptococcus thermophilus can effectively reduce NEC incidence (126,142,143). In conclusion, both Enterococcus and Streptococcus exhibit complex dual roles in NEC. For Enterococci, E. faecalis may promote disease, whereas E. faecium could support recovery. Among Streptococcus, certain species are harmful when overabundant, but probiotics like S. thermophilus offer therapeutic benefits.

In summary, probiotics exert preventive and protective effects against NEC, as they can reduce the incidence of NEC and remodel the intestinal microbiota. Notably, their effects are strain-dependent, and Table 1 summarizes the mechanisms of action of different species and their respective strains. Future studies must delineate these divergent, strain-specific impacts to inform clinical strategies.

Evidence from RCTs of breastfeeding plus probiotics

Because the standard of care in many NICUs is to prioritize mother’s own milk (or donor human milk) as the primary enteral feed, most probiotic RCTs were effectively conducted on a background of predominant human-milk feeding. These trials therefore provide pragmatic evidence for the combined strategy of “breast milk + probiotics” (Table 2). However, heterogeneity in probiotic strain(s), dose, initiation timing, feeding composition (mother’s milk vs. donor milk vs. formula), and baseline NEC risk contributes to mixed results across studies.

Overall, multi-strain Bifidobacterium/Lactobacillus-based products administered after enteral feeds are started have shown the most consistent reduction in Bell stage II or higher NEC in several trials [e.g., Lin et al. (147), Bin-Nun et al. (148), and ProPrems/Jacobs et al. (149)], with no clear reduction in late-onset sepsis or mortality in some large multicenter studies. In contrast, other rigorously designed studies reported no significant benefit on NEC, sepsis, or death overall (152,153,155). Importantly, subgroup analyses and cohort re-analyses suggest that the protective effect may be stronger when human milk is the predominant feed (150), supporting a biologically plausible synergy via substrate availability (e.g., HMOs) and antimicrobial/immune factors in milk.

Microbial metabolites as key proximal effectors

The metabolites produced by the gut microbiota play a significant role in preventing NEC. Microbiota-derived metabolites such as SCFAs (acetate, propionate, butyrate) enhance tight junction expression, support epithelial energy metabolism, and regulate inflammation, which are critical in maintaining gut integrity. HMOs and probiotic strains help co-regulate these metabolite pools. Breast milk provides the essential substrates for the growth of beneficial microbiota that produce these metabolites, which in turn promote intestinal barrier function and modulate immune responses. Additionally, tryptophan-derived indoles, produced by probiotics such as Bifidobacterium and Lactobacillus, activate the AHR signaling pathway, which further strengthens the epithelial barrier and modulates immune responses, contributing to a balanced immune system and reduced inflammation.

Synergistic regulation of intestinal metabolites by breast milk and probiotics: the core mediating role in NEC prevention

While taxonomic signatures of NEC are informative, metabolites represent a mechanistically actionable “common currency” linking diet, microbes, and host responses. Breast milk supplies substrates (HMOs, lactoferrin-bound iron dynamics, antimicrobial peptides) and signaling molecules that steer microbial metabolism, whereas probiotics introduce metabolic capacities (HMO utilization, SCFA production, tryptophan catabolism, redox control) that reshape the luminal and mucosal metabolite landscape. Below we summarize metabolite classes with the strongest mechanistic connections to epithelial integrity and immune homeostasis in NEC.

SCFAs: acetate, propionate, butyrate

SCFAs are produced via fermentation of HMOs and other carbohydrates (156). Acetate and butyrate can enhance tight junction expression, support epithelial energy metabolism, and suppress inflammation via GPR41/43 signaling and histone deacetylase (HDAC) inhibition, promoting Treg differentiation and limiting NF-κB activation (157-160). However, in the extremely immature gut, excessive or imbalanced SCFA exposure may be harmful (161); thus, the protective window likely depends on concentration, site, and developmental stage. This duality remains a key controversy that warrants standardized metabolomics-linked trials.

Tryptophan-derived indoles and aryl hydrocarbon receptor signaling

Indole derivatives (e.g., ILA) produced by Bifidobacterium and some Lactobacillus strains activate AHR-dependent transcriptional programs in epithelial and immune cells (162). AHR signaling can reinforce barrier function, promote IL-22-mediated mucosal defense, and reduce epithelial chemokine responses (e.g., IL-8) during inflammatory challenge (117,163). Because both breast milk and probiotic strains can shift tryptophan availability and microbial catabolic routes, this axis is a plausible mechanistic “hub” for synergy.

Bile acid (BA) intermediates and nuclear receptor signaling

Although disrupted BA homeostasis is implicated in NEC, its role in NEC pathogenesis remains unclear (164). Microbial bile-acid transformation influences epithelial and innate immune tone through farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5) (165). In preterm infants, bile-acid pools are developmentally constrained and can be perturbed by antibiotics and dysbiosis (166,167). Whether breast milk and probiotics jointly normalize bile-acid profiles to protect against NEC remains incompletely defined, representing a major open question for multi-omics studies.

Other key metabolites: lactate, tricarboxylic acid cycle (TCA) intermediates, and redox molecules

Additional metabolite candidates include lactate (cross-feeding substrate for butyrate producers), succinate and other TCA intermediates reported as early biomarkers, polyamines that support epithelial proliferation, and redox-active compounds that modulate oxidative stress and lipid peroxidation (168-170). Future work should move beyond association to causality by integrating targeted metabolite supplementation, receptor knockouts, and gnotobiotic models.

Conclusions

NEC is a critical condition predominantly affecting preterm infants, with a rising incidence (171). Despite the availability of current treatments, which include supportive care and surgical interventions, the high mortality rate and long-term health complications associated with NEC underscore the urgent need for effective preventive measures. Existing research suggests that breastfeeding and probiotic interventions are crucial in NEC prevention. In recent 5 years, accumulating evidence has consistently supported the beneficial potential of breastfeeding and probiotic interventions in preventing neonatal NEC, while also highlighting critical unresolved mechanisms and controversial research gaps. Multiple systematic reviews, RCTs, and meta-analyses have confirmed that human milk, including maternal and donor milk, significantly reduces the risk of NEC in very preterm and VLBW infants compared with formula (172), possibly by supplying immunomodulatory factors, promoting intestinal barrier maturation, and optimizing gut microbiota colonization; human milk-derived fortifiers may further enhance this protective effect (173), although the key bioactive components (such as immunoglobulins, HMOs, and extracellular vesicles) and their precise mechanisms in the immature gastrointestinal tract remain unclear (174). For probiotics, recent systematic reviews and network meta-analyses indicate that multi-strain combinations (e.g., Bifidobacterium, Lactobacillus, and Enterococcus) confer more consistent benefits in reducing NEC incidence and all-cause mortality than single-strain supplements, which show greater heterogeneity (175). However, substantial variations in strain selection, dosage, timing, and duration, along with insufficient high-quality large-sample trials in very preterm or VLBW infants, limit the strength of evidence, and the safety profile regarding sepsis or other severe infections remains inconclusive. Furthermore, the synergistic effects between human milk and probiotics, the application strategies of prebiotics or synbiotics combined with human milk, and the mechanisms by which microbiome interventions regulate host immune development and intestinal barrier function represent hot but highly debated areas (176). In summary, although human milk and probiotic interventions are consistently shown to reduce NEC risk and improve short-term outcomes, important questions remain unanswered regarding their exact biological mechanisms, optimal clinical combinations and timing, safety evaluation, and applicability in extremely preterm subgroups.

Looking forward, an in-depth, mechanism-oriented synthesis is urgently needed because current evidence is fragmented across disciplines and often reports endpoints (NEC incidence) without resolving upstream causal chains, such as which microbial taxa and metabolite shifts are true drivers of NEC onset, and which are downstream consequences of inflammation, antibiotics, or feeding interruption. By organizing breastfeeding and probiotics along a shared microbiota-metabolite-barrier-immunity mainline, this review aims to support rational synbiotic design, improve trial comparability, and accelerate translation toward safer, personalized NEC prevention strategies. Future work combining longitudinal multi-omics, functional assays, and careful clinical stratification will be key to identify actionable biomarkers and timing windows for intervention.

Acknowledgments

None.

Footnote

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-913/prf

Funding: The work was supported by funds from Top Medical Expert Team of Wuxi Taihu Talent Program (grant No. DJTD202106), Medical Key Discipline Program of Wuxi Health Commission (grant No. ZDXK2021007), Top Talent Support Program for Young and Middle-aged People of Wuxi Health Committee (grant No. BJ2020089), Major Program of Wuxi Health Commission (grant No. Z202109), General Program of Wuxi Health Commission (grant Nos. M202003 and M202208), Wuxi Science and Technology Development Fund (grant Nos. N20202003, N20192039, and Y20222001), the Natural Science Foundation of Jiangsu Province, China (grant No. BK20221086), the National Natural Science Foundation of China (grant No. 82504804), the Jiangsu Funding Program for Excellent Postdoctoral Talent (grant No. 2022ZB484), “Taihu Light” Science and Technology Project-Basic Research (grant No. K20241003, Wuxi), and Wuxi Youth Science and Technology Talent Support Project (grant No. TJXD-2025-215).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-913/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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