Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disorder predominantly affecting preterm neonates, marked by inflammation and necrosis of the intestinal wall. Despite substantial advancements in neonatal intensive care, NEC continues to impose a significant burden on neonatal health, with high rates of morbidity, mortality, and long-term sequelae, including intestinal strictures, short bowel syndrome, and neurodevelopmental impairment. The multifactorial etiology of NEC underscores the complex interplay of environmental, host, and microbial factors, yet the precise mechanisms driving its pathogenesis remain incompletely understood.

Central to the development of NEC is the neonatal gut microbiome, a dynamic and essential ecosystem that influences immune maturation and gut homeostasis. In preterm infants, the gut microbiome undergoes aberrant colonization, often dominated by opportunistic pathogens due to factors such as immaturity, antibiotic use, and suboptimal feeding practices. This microbial dysbiosis—characterized by reduced diversity and an imbalance favoring pathogenic over commensal species—has been consistently implicated in the development of NEC. However, the interaction between microbial dysbiosis and clinical risk factors, such as chorioamnionitis, mechanical ventilation, and formula feeding, remains poorly delineated.

This prospective, multicenter cohort study aims to unravel the intricate relationship between microbial dysbiosis and clinical risk factors in the pathogenesis and outcomes of NEC. By combining comprehensive clinical data with advanced microbial profiling, this research seeks to identify specific microbial and clinical biomarkers predictive of NEC risk. Understanding these interactions is critical to advancing preventive and therapeutic strategies, including microbiome-targeted interventions and optimized neonatal care protocols.

This study builds upon existing literature by exploring not only the microbial shifts preceding NEC onset but also the synergistic effects of clinical interventions and perinatal risk factors on gut microbiome dynamics. Ultimately, the findings aim to provide actionable insights to mitigate the burden of NEC and improve the survival and long-term health outcomes of preterm infants.

Methods

Study Design and Participants:

A prospective, multicenter cohort study was conducted across four tertiary neonatal intensive care units (NICUs) over a 24-month period. The study included preterm infants born at ≤32 weeks gestational age who were admitted to the NICUs within 24 hours of birth. Infants with major congenital anomalies, those with a history of antibiotic or probiotic therapy prior to admission, and those with severe infections at birth were excluded.

Clinical Data Collection:

Clinical data were collected through structured questionnaires, including maternal and infant characteristics, antenatal and postnatal risk factors, and clinical outcomes. Antenatal factors, such as maternal diabetes, hypertension, chorioamnionitis, and use of antenatal corticosteroids, were recorded. Postnatal factors, including feeding practices (breastfeeding vs. formula), antibiotic use, and duration of mechanical ventilation, were also documented. The primary outcome was the development of NEC, diagnosed clinically and confirmed radiographically. Secondary outcomes included mortality, need for surgery, and long-term gastrointestinal complications.

Microbial Analysis:

Stool samples were collected from enrolled infants at two time points: on the day of NICU admission and weekly thereafter for the first month of life. Microbial DNA was extracted from stool samples and subjected to 16S rRNA gene sequencing to analyze the composition of the gut microbiome. Diversity indices (alpha and beta diversity) were calculated to assess microbial richness and composition, while specific taxa associated with NEC were identified using bioinformatics tools.

Statistical Analysis:

Descriptive statistics were used to summarize the clinical and microbiome data. Univariate and multivariate logistic regression analyses were performed to identify clinical risk factors associated with the development of NEC. Correlations between microbial diversity and the clinical outcomes of NEC were examined using Spearman’s rank correlation. A p-value of <0.05 was considered statistically significant.

Results

Demographic and Clinical Characteristics:

A total of 450 preterm infants were enrolled in the study, of which 50 (11.1%) developed NEC during their hospital stay. The mean gestational age of the cohort was 28.5 weeks (SD 2.3 weeks), and the mean birth weight was 1,050 grams (SD 350 grams). The primary clinical risk factors for NEC included low birth weight, mechanical ventilation, and early antibiotic use. Infants who developed NEC had a higher incidence of chorioamnionitis and lower rates of breastfeeding compared to those who did not develop NEC.

Table 1: Clinical Characteristics of Preterm Infants with and without NEC

The bar graph represents the clinical characteristics of preterm infants with and without necrotizing enterocolitis (NEC). The graph visually compares the differences across various characteristics, such as gestational age, birth weight, chorioamnionitis incidence, mechanical ventilation, antibiotic use, and exclusive breastfeeding rates between the two groups. ​

Microbial Dysbiosis and NEC:

Stool microbial analysis revealed significant differences in the microbiome composition between infants who developed NEC and those who did not. Preterm infants with NEC had lower microbial diversity (both alpha and beta diversity) at the time of admission compared to controls. The microbiomes of NEC infants were dominated by potential pathogenic species, including Enterobacteriaceae and Clostridia, while beneficial microbes such as Bifidobacteria and Lactobacillus were significantly reduced.

Table 2: Microbial Diversity and Composition in Infants with and without NEC

Multivariate analysis revealed that low microbial diversity at admission was independently associated with an increased risk of NEC (OR 3.5, 95% CI 1.8-6.9, p<0.001). Specific microbial taxa, including Clostridium species, were significantly enriched in infants who developed NEC, while Bifidobacterium was inversely associated with NEC development.

The graph depicts the microbial diversity and composition in infants with and without NEC (Necrotizing Enterocolitis). The data for both groups (NEC (+) and NEC (-) is presented for various microbial indicators.

Table 3: Microbial Taxa Associated with NEC Development

The graph illustrates the microbial taxa associated with NEC development, showing odds ratios with 95% confidence intervals and corresponding p-values.

Clinical Risk Factors and Microbial Interactions in the Development of NEC:

This table summarizes the clinical risk factors and their impact on the microbiome in relation to the development of NEC. It highlights how both antibiotic use (whether broad-spectrum or narrow-spectrum) and feeding practices (formula vs. exclusive breastfeeding) are crucial in shaping the microbial environment, with significant implications for the pathogenesis of NEC.

Discussion

The findings of this study underscore the complex interplay between microbial dysbiosis and clinical risk factors in the pathogenesis of NEC in preterm infants. Our data show that microbial diversity is significantly reduced in infants who develop NEC, and the presence of specific pathogenic microorganisms is strongly associated with the onset of the disease. These results support the hypothesis that the gut microbiome plays a pivotal role in the development of NEC.

Necrotizing enterocolitis (NEC) remains one of the most severe and complex gastrointestinal diseases affecting preterm infants. Despite advances in neonatal care, it continues to contribute significantly to morbidity, mortality, and long-term gastrointestinal complications in this population. The pathogenesis of NEC is multifactorial and involves an intricate interplay of environmental, host, and microbial factors. This study aimed to elucidate the role of microbial dysbiosis and clinical risk factors in the development of NEC in preterm infants. Our findings provide valuable insights into the microbial shifts associated with NEC and identify key clinical risk factors that exacerbate disease progression.

Microbial Dysbiosis and NEC Pathogenesis

Our study identified significant differences in the gut microbiome composition between preterm infants who developed NEC and those who did not. Specifically, we observed a reduction in microbial diversity (both alpha and beta diversity) in the stools of infants who developed NEC. Lower microbial diversity is a hallmark of dysbiosis, which has been previously implicated in a variety of neonatal diseases, including NEC. A healthy, diverse microbiome is critical in promoting intestinal immune homeostasis and protecting the gut from pathogen colonization. In contrast, microbial imbalances in preterm infants, characterized by a predominance of harmful bacteria and a reduction in beneficial microbes, may predispose the intestine to inflammation and necrosis.

In our cohort, the microbial composition of infants who developed NEC was dominated by potentially pathogenic species such as Enterobacteriaceae and Clostridia, while beneficial genera such as Bifidobacterium and Lactobacillus were significantly reduced. These findings are consistent with previous studies that have demonstrated an overrepresentation of Enterobacteriaceae (e.g., Escherichia coli and Klebsiella) and Clostridia species in the gut microbiomes of preterm infants with NEC. These pathogens have been shown to produce pro-inflammatory signals and disrupt the intestinal mucosal barrier, thereby contributing to the onset of NEC. On the other hand, Bifidobacterium and Lactobacillus are known for their protective roles in the gut, including the modulation of immune responses and maintenance of gut barrier integrity. The reduction of these beneficial microbes in NEC infants highlights the potential protective role of these taxa in preventing NEC development.

Our findings also align with emerging research that highlights the critical role of microbial diversity in regulating the gut-immune system interaction. A more diverse microbiome fosters a balanced immune response, limiting excessive inflammation. In contrast, microbial dysbiosis may trigger a dysregulated immune response, which could predispose to the development of NEC. Several studies have shown that a diverse microbiome enhances the maturation of neonatal gut-associated lymphoid tissue (GALT) and improves mucosal immunity, both of which are crucial for preventing NEC.

Clinical Risk Factors and Their Role in Microbial Dysbiosis

In addition to microbial dysbiosis, our study identified several clinical risk factors that were strongly associated with the development of NEC. These include low birth weight, chorioamnionitis, mechanical ventilation, antibiotic use, and feeding practices. The role of these factors in the pathogenesis of NEC is well-documented, and our findings further underscore their significance.

Low Birth Weight and Gestational Age:

Infants with lower birth weight and earlier gestational age have been shown to have an immature immune system and underdeveloped gastrointestinal tract, both of which increase their susceptibility to infections and gut dysbiosis. In our cohort, infants who developed NEC had significantly lower birth weights and were more likely to be of younger gestational age. These findings are consistent with the literature, which reports that preterm infants with very low birth weight (VLBW) are at the highest risk for NEC. The immaturity of the intestinal barrier and the increased intestinal permeability in these infants may facilitate the translocation of pathogens from the gut to the bloodstream, exacerbating the inflammatory response that characterizes NEC.

Chorioamnionitis and Inflammation:

Chorioamnionitis, a bacterial infection of the placental tissues, has long been associated with an increased risk of NEC. In our study, we observed a higher incidence of chorioamnionitis among infants who developed NEC compared to those who did not. This finding suggests that the inflammatory milieu induced by chorioamnionitis may disrupt the neonatal gut microbiome and predispose infants to gut inflammation. Chorioamnionitis can trigger systemic inflammation, including the release of cytokines and other pro-inflammatory molecules, which may enhance the susceptibility of the gut to bacterial invasion and necrosis. The inflammatory environment may also favor the overgrowth of pathogenic bacteria while suppressing the growth of beneficial microbes.

Mechanical Ventilation and Antibiotic Use:

Mechanical ventilation and prolonged antibiotic use are standard interventions in the care of preterm infants but have been shown to negatively impact the neonatal microbiome. In our study, infants who required mechanical ventilation for prolonged periods had a higher incidence of NEC. The disruption of normal gut function caused by mechanical ventilation, including altered gastric pH and delayed gastric emptying, may facilitate the colonization of pathogenic bacteria and the onset of NEC. Similarly, antibiotic use, especially broad-spectrum antibiotics, has been shown to significantly alter the microbiome by reducing the abundance of commensal bacteria and promoting the growth of opportunistic pathogens. In our cohort, prolonged antibiotic therapy was associated with a higher incidence of NEC, and infants who received antibiotics had a significantly altered microbiome characterized by an overrepresentation of Enterobacteriaceae and Clostridia species.

Feeding Practices:

Feeding practices, particularly the choice between exclusive breastfeeding and formula feeding, have long been recognized as critical factors influencing the gut microbiome. Breast milk contains a diverse array of prebiotics, immune factors, and antimicrobial proteins that promote the growth of beneficial gut bacteria and protect against intestinal inflammation. In contrast, formula feeding has been associated with a less diverse microbiome and an increased risk of NEC. In our study, infants who were exclusively breastfed had significantly lower rates of NEC and higher microbial diversity compared to those who received formula. This finding supports the growing body of evidence that emphasizes the protective role of breast milk in preventing NEC.

Interplay Between Microbial Dysbiosis and Clinical Risk Factors

The results of this study suggest that microbial dysbiosis and clinical risk factors act in a synergistic manner to influence the development of NEC. For example, the combination of antibiotic therapy and low microbial diversity may create an environment conducive to the growth of pathogenic bacteria, which in turn exacerbates intestinal inflammation and damage. Similarly, clinical factors such as low birth weight, mechanical ventilation, and chorioamnionitis may further promote microbial imbalances by disrupting the gut environment and immune function. The interaction between these factors is complex and likely involves multiple pathways, including immune modulation, gut barrier dysfunction, and pathogen-host interactions.

This study also highlights the potential for microbiome-based interventions to mitigate the risk of NEC in preterm infants. Probiotics, prebiotics, and fecal microbiota transplantation (FMT) have all been explored as potential therapeutic strategies to restore microbial balance and prevent NEC. While the evidence for the efficacy of these interventions remains mixed, the identification of specific microbial taxa associated with NEC development provides a valuable opportunity for targeted microbiome-based therapies in the future.

This study provides important insights into the role of microbial dysbiosis in the pathogenesis and outcomes of NEC in preterm infants. The identification of specific microbial taxa associated with the disease, along with the recognition of clinical risk factors, offers potential targets for preventive and therapeutic interventions aimed at reducing the incidence and severity of NEC. Further research is needed to explore the potential for microbiome-based therapies in preventing NEC and improving outcomes in this vulnerable population.

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