Comparison of the Gut Microbiota of Preterm Infants Born before 32-Week Gestation with Feeding Intolerance

Article information

Neonatal Med. 2025;32(1):21-29

Publication date (electronic) : 2025 May 31

doi : https://doi.org/10.5385/nm.2025.32.1.21

Bo Kyeong Jin, MD

, Hyunsu Kim, MD

, Cho Ae Lee, MD

, Hye-Rim Kim, MD, PhD

Department of Pediatrics, CHA Bundang Medical Center, CHA University, Seongnam, Korea

Correspondence to: Hye-Rim Kim, MD, PhD Department of Pediatrics, CHA Bundang Medical Center, CHA University, 59 Yatap-ro, Bundang-gu, Seongnam 13496, Korea Tel: +82-31-780-3964 Fax: +82-31-780-1680 E-mail: hei23@cha.ac.kr

Received 2025 April 8; Revised 2025 May 7; Accepted 2025 May 12.

Abstract

Purpose

Feeding intolerance (FI) is a prevalent clinically sequential condition in preterm infants. To clarify its relationship with the gut microbiota, we compared microbial diversity and taxonomic composition at 2 and 4 weeks of age in infants born before 32 weeks of gestation.

Methods

Between August 2021 and December 2022, we prospectively enrolled infants who delivered before 32 weeks of gestation and were admitted to the neonatal intensive care unit at CHA Bundang Medical Center. Forty-four preterm infants were grouped based on the presence (n=16) or absence (n=28) of FI. Fecal samples were obtained at 2 and 4 weeks after birth and analyzed using 16S rRNA gene sequencing to determine microbial profiles.

Results

Microbial α-diversity and β-diversity did not differ significantly between groups at either time point. At the genus level, Staphylococcus was significantly more abundant in the FI group than in the feeding tolerance group at 2 weeks postnatal age (P=0.016). Linear discriminant analysis effect size revealed that Staphylococcus, Pseudomonas, and Escherichia were markedly enriched in the FI group at all time points.

Conclusion

Early colonization by potentially pathogenic genera, particularly Staphylococcus, may precede the development of FI in preterm infants. These findings highlight the potential microbial composition associated with FI and may provide preliminary insights for future microbiome-targeted research in neonatal care.

Keywords: Preterm infant; Microbiome; Feeding intolerance

INTRODUCTION

Feeding intolerance (FI) in preterm infants is a frequently encountered and clinically important challenge, characterized by abdominal distension, vomiting, and increased residual gastric volume after enteral feeding [1]. Among very low birth weight infants, FI affects approximately 60% to 70% of them, resulting in inadequate nutrient intake, postnatal growth failure, prolonged dependence on parenteral nutrition, increased susceptibility to nosocomial infections, and extended hospitalization periods in the neonatal intensive care unit (NICU) [2-6]. Despite its clinical importance, the mechanisms underlying FI are not fully understood, with emerging evidence suggesting disruptions in gut microbiota dysbiosis [7-9].

Neonatal intestinal microbiota plays several essential roles, including maturation and modulation of host immunity, defense against pathogens, and regulation of hormonal and metabolic pathways [10]. Preterm infants are at high risk of gut microbiota disruption due to their physiological immaturity and various environmental factors, including antibiotic exposure, mode of delivery, and nutritional practices [11-13]. Several reports have indicated that altered interactions between the gut microbiota and intestinal mucosa may predispose infants to FI [14-17]. To date, few clinical trials have characterized the gut microbial patterns specifically associated with FI in preterm infants [7,18,19], and the potential role of the gut microbiota in FI remains to be fully elucidated.

We aimed to investigate whether early microbial composition is associated with the development of FI in preterm infants born before 32 weeks of gestation by analyzing stool samples collected at 2 and 4 weeks after birth.

MATERIALS AND METHODS

1. Study population

From August 2021 to December 2022, we prospectively enrolled infants with a gestational age (GA) <32 weeks who were admitted to the NICU at CHA Bundang Medical Center. Exclusion criteria were as follows: major congenital anomalies, necrotizing enterocolitis (NEC), death, or transfer to other hospitals during the study period. All enrolled infants initiated enteral nutrition within 24 hours of life. Breast milk was prioritized as the primary source of nutrition, and preterm formula was administered when maternal milk was unavailable. None of the infants received probiotic supplementation during the study period. Those at high risk of early onset sepsis were treated with ampicillin and gentamicin after birth, and the course was based on blood culture. Piperacillintazobactam has been used to treat preterm infants with late sepsis.

2. Data collection

Information regarding maternal hypertension, diabetes, premature rupture of membranes (PROMs), and antenatal corticosteroid administration was collected from the medical records. Neonatal characteristics, such as GA, birth weight, sex, and mode of delivery, were recorded. Additional clinical outcomes included intraventricular hemorrhage (IVH), respiratory distress syndrome (RDS), sepsis, duration of antibiotic exposure, parenteral nutritional support, and length of NICU hospitalization.

3. Definitions

FI is defined as the inability to digest enteral feed, characterized by (1) a residual gastric volume exceeding 50% of the previous feeding (≥twice within 24 hours); (2) gastrointestinal symptoms, such as abdominal distension, emesis, or both; and (3) interruption or reduction of enteral feeding volume for >3 times a day, in accordance with prior literature [1]. Abdominal distension was defined as either an increase in abdominal girth or dilated bowel loops.

RDS was diagnosed on the basis of clinical signs of respiratory compromise (grunting, retraction, tachypnea, and increased oxygen demand) in conjunction with typical radiologic features and cases requiring surfactant replacement treatment. Severe IVH was defined as grade III or IV based on the Papile classification system criteria, with the worst grading results during hospitalization recorded [20].

4. Fecal sample collection

Fecal samples were collected directly from diapers using sterile disposable swabs at 2 and 4 weeks of age. Samples were immediately transferred into sterile tubes and stored at –80 °C until microbial deoxyribonucleic acid (DNA) was extracted for downstream microbiome analysis.

5. DNA extraction, polymerase chain reaction amplification, and 16S rRNA gene sequencing

Genomic DNA was isolated from fecal samples using a commercial DNA extraction kit according to the manufacturer's protocol (Supplementary Materials and Methods, Supplementary Table 1). The extracted DNA was prepared for sequencing according to the protocol of the Human Microbiome Project (HMP) consortium [16]. Polymerase chain reaction amplification was performed using 10 ng of extracted DNA and universal primers targeting the V3–V4 regions of the 16S rRNA gene (519F: 5′-CCTACGGGNGGCWGCAG-3′ and 806R: 5′-GACTACHVGGGTATCTAATCC-3′). The resulting amplicons were quantified using the KAPA Library Quantification Kit (Kapa Biosystems), and their quality was evaluated using the LabChip GX HT DNA High Sensitivity Kit (PerkinElmer). Libraries were normalized, pooled, and sequenced on an Illumina MiSeq platform.

The V3–V4 regions (519F–806R) were selected for sequencing based on their taxonomic resolution among the nine hypervariable regions of the 16S rRNA gene. DNA quality was validated prior to sequencing using PicoGreen reagent and a Nanodrop spectrophotometer (Agilent Technologies), and sequencing was conducted according to the Illumina 16S Metagenomic Sequencing Library protocol.

6. Data analysis and statistical analysis

Continuous variables were presented as mean±standard deviation, whereas categorical variables were expressed as absolute numbers and percentages. For comparisons between groups, normally distributed continuous variables were evaluated using Student’s t-test, while non-normally distributed variables were assessed using the Mann–Whitney U-test. Categorical variables were compared using Pearson’s chi-square test.

To assess microbial diversity, α-diversity was calculated using the Shannon index, which measures species richness and distribution uniformity. β-Diversity was analyzed through principal coordinate analysis based on Bray–Curtis dissimilarity to visualize differences in community composition. Microbial taxonomic comparisons were performed at the genus level using the Quantitative Insights Into Microbial Ecolog (QIIME) pipeline. For paired specimen comparisons, the Wilcoxon signed-rank test or Mann–Whitney U-test was used as appropriate.

To identify taxa that were differentially represented between groups, we utilized the linear discriminant analysis effect size (LEfSe) algorithm, which incorporates the nonparametric Kruskal–Wallis test followed by linear discriminant analysis (LDA) to estimate the effect size of each discriminative feature. All statistical computations were conducted using the SPSS software version 27.0 (IBM Corp.). Statistical significance was defined as a two-tailed P<0.05.

RESULTS

1. Study population

Between August 2021 and December 2022, 49 infants born before 32 weeks’ gestation were assessed for eligibility. After excluding five infants (one with a major congenital anomaly, two diagnosed with NEC, and two who either died or were transferred), 44 infants were included in the final analysis. Among the remaining 44 infants, the mean GA was 29⁺⁵±2⁺⁰ weeks, and the mean birth weight was 1,333±350 g. Sixteen infants (36%) met the criteria for FI and were allocated to the FI group, whereas the remaining 28 (64%) comprised the feeding tolerance (FT) group.

2. Comparison of clinical characteristics between the FT and FI groups

The maternal and neonatal characteristics were compared between the FT and FI groups (Table 1). The infants in the FI group had significantly lower birth weights than those in the FT group. Maternal hypertension was observed more frequently in the FI group than in the control group. The durations of antibiotic treatment, parenteral nutrition, and hospitalization were significantly longer in the FI group. However, when adjusted for confounding variables, such as birth weight, maternal hypertension, and PROM, these differences were not statistically significant.

Table 1.

Comparison of the Clinical Characteristics between the FT and FI Groups

3. Comparison of the microbiota between the FT and FI groups

1) Analysis of gut microbiota diversity

Comparison of the α-diversity revealed no significant difference in Shannon index between the FT and FI groups at postnatal weeks 2 and 4 (Figure 1). Furthermore, no difference in β-diversity based on Bray–Curtis dissimilarity was identified between the groups at postnatal weeks 2 and 4 (Figure 2).

Figure 1.

Alpha diversity of gut microbiota in the feeding tolerance (FT) and feeding intolerance (FI) groups. No difference in the Shannon index between the FT and FI groups at (A) 2 or (B) 4 weeks of life.

Figure 2.

β-Diversity of gut microbiota in the feeding tolerance (FT) and feeding intolerance (FI) groups. No difference in the β-diversity between the FT and FI groups at (A) 2 and (B) 4 weeks of life. Abbreviations: ANOSIM, analysis of similarities; PC, principal coordinates.

2) Analysis of microbial composition and abundance

We compared the main microbial abundance between the FT and FI groups and found no significant differences at the phylum level between the two groups at postnatal weeks 2 and 4. At the genus level, Staphylococcus was the most abundant genus in the FI group, and Enterococcus was the most abundant genus in the FT group 2 weeks after birth (Figure 3A). The relative abundance of Staphylococcus at this time point was significantly higher in the FI group than in the FT group (43.7% vs. 8.25%, P=0.016) (Figure 3A). After 4 weeks, Enterococcus remained the most abundant genus in both groups, and no significant differences in the top four genera were noted (Figure 3B).

Figure 3.

Relative abundance of microbiota between the feeding tolerance (FT) and feeding intolerance (FI) groups. (A) At the genus level, Staphylococcus was significantly more abundant in the FI group than the FT group at 2 weeks after birth (P=0.016). (B) No significant difference in the absolute abundances of the four most dominant microbiota at the genus level between infants with FT and FI at 4 weeks after birth.

At the genus level, the abundances of Staphylococcus, Pseudomonas, and Escherichia (LDA score >4) in the FI group were significantly greater than those in the FT group at 2 weeks postnatal age, according to LEfSe analysis. Klebsiella was less abundant in the FI group than in the FT group at 4 weeks postnatal age and received a score above 4 in the LEfSe analysis (Figure 4).

Figure 4.

DISCUSSION

This study examined the gut microbial diversity and composition in preterm infants born before 32 weeks of gestation, comparing those who experienced FI with those who did not at both 2 and 4 weeks postnatal age. While diversity indices (α- and β-diversity) did not significantly differ between groups, notable differences in taxonomic composition were identified. In particular, Staphylococcus abundance was significantly higher in the FI group at 2 weeks of age (P=0.016), and LEfSe analysis further indicated that Pseudomonas and Escherichia were more prevalent in this group at the same time point. These results provide insights into early microbial deviations that potentially precede the development of FI in preterm infants.

Microbial colonization during the neonatal period plays a pivotal role in intestinal development and physiological homeostasis [21,22]. Changes in the gut microbiota during the early postnatal period are essential for the maturation and regulation of intestinal motility, and influence energy delivery to the intestinal epithelium, particularly in preterm infants [14-17]. Several perinatal factors, including GA, maternal PROM, delivery mode, feeding, and immune maturity, affect microbial colonization [23,24]. In particular, preterm infants are vulnerable to microbial imbalances due to their immature immune systems and gut barriers, which may predispose them to FI [11].

In a study of preterm infants born before 30 weeks of gestation, no differences in the α-diversity or β-diversity between infants with FI or FT at 2 weeks after birth were determined [18]. In contrast, Hong et al. [25] reported a lower α-diversity and reduced bacterial richness in the microbiota of infants with FI at 3 to 4 weeks of life. These contrasting findings may be attributed to the variability in the timing of sample collection, cohort characteristics, or environmental conditions specific to each NICU. In our cohort, although the observed differences did not reach statistical significance, a consistent trend toward reduced microbial diversity was noted in the FI group at both 2 and 4 weeks.

This trend toward reduced diversity in the FI group may reflect early instability or delayed maturation of the gut microbiota. Previous studies have demonstrated that the gut microbiome diversity in preterm infants is initially low increases daily after birth during the first month of life [26,27]. Subtle shifts in microbial composition, such as the expansion of facultative anaerobes or delayed colonization by beneficial taxa, may precede detectable changes in overall diversity and play critical roles in the pathogenesis of FI. Additionally, the use of antibiotics and parenteral nutrition, both of which were more frequent in the FI group, are known to alter microbial succession and suppress diversity [28,29]. However, the interindividual variability in our small cohort may have limited the statistical power to detect subtle but biologically meaningful differences in diversity.

Existing literature on the relationship between gut microbiota and FI remains limited; however, a few studies have highlighted alterations in microbial profiles among preterm infants with FI [7,18,19]. Compared to term infants, preterm infants typically exhibit a microbiome with decreased species richness [8,30], a dominance of facultative anaerobes (e.g., Staphylococcus, Enterococcus, Klebsiella, Enterobacter, and Escherichia), and reduced levels of beneficial genera such as Bifidobacterium and Lactobacillus [31,32]. Such microbial imbalances, characterized by reduced protective taxa and increased opportunistic pathogens, are thought to contribute to FI development [33,34]. In the present study, infants with FI presented a significantly greater abundance of Staphylococcus, Pseudomonas, and Escherichia than infants in the FT group at 2 weeks after birth, according to the LEfSe analysis. Furthermore, Staphylococcus was significantly more abundant in infants with FI than in those with FT 2 weeks after birth. While this observation does not establish causality, it aligns with the existing literature, suggesting a potential pathogenic role of Staphylococcus in preterm infants. Brehin et al. [35] observed elevated levels of Staphylococcus in FI cases during the first postnatal month. Another study demonstrated that Staphylococcus species were associated with increased intestinal permeability and mucosal inflammation [36]. These findings suggest that Staphylococcus overgrowth contributes to impaired gut barrier function and mucosal inflammation in preterm infants. Additionally, Yuan et al. [7] reported that FI alters the gut microbiota of preterm infants, with significant differences in the microbial composition between the FI and FT groups. These studies suggest that Staphylococcus enrichment may play a mechanistic role in FI development by altering intestinal integrity and delaying microbial maturation.

Escherichia species were more abundant in infants with FI than in those with FT at 2 weeks after birth, according to the LEfSe analysis. This genus has been widely implicated in gastrointestinal diseases including gastroenteritis and NEC [37,38]. In a recent pilot study, Hsu et al. [34] reported that the gut microbiota of preterm infants with FI was notably enriched in potentially pathogenic Escherichia–Shigella species, along with a marked reduction in beneficial genera, such as Lactobacillus and Streptococcus. These findings are consistent with our results, which demonstrated a trend toward greater Escherichia abundance in the FI group. Recent metagenomic analyses with strain-level resolution have further implicated uropathogenic Escherichia coli strains in the development of NEC and in increased mortality in preterm infants, highlighting the potential role of specific Escherichia coli lineages in the development of NEC [39].

In our study, we also observed a significantly higher abundance of Pseudomonas in the FI group 2 weeks after birth, a genus that has received comparatively less attention in association with FI in preterm infants. Pseudomonas aeruginosa is an opportunistic pathogen in NICUs and is known for its ability to produce proteases and exotoxins that can compromise epithelial barrier integrity [40,41]. In experimental models, colonization with Pseudomonas in the gut exacerbated dextran sulfate sodium-induced intestinal injury in mice, leading to increased gut permeability, bacterial translocation, and systemic inflammation [27]. These findings suggest that the observed association between increased Pseudomonas abundance and FI in our study may be due to the ability of the bacterium to impair gut barrier function, thereby contributing to the pathogenesis of FI in preterm infants. Interestingly, both Escherichia and Pseudomonas, which were significantly enriched in the FI group at 2 weeks of age, did not retain their discriminative power at 4 weeks, as indicated by the reduced LDA score in the LEfSe analysis. This temporal pattern suggests that the initial over-representation of potentially pathogenic taxa in the FI group diminished over time, potentially reflecting partial recovery or delayed microbial maturation following early dysbiosis.

One strength of our study is its prospective design, in which fecal samples and clinical data were collected in a standardized manner. Nonetheless, this study has several limitations. First, we were unable to assess the dynamic microbiota changes during actual FI episodes. Second, although we identified associations between gut microbiota and FI, causality could not be established. Third, our analysis was restricted to the genus level, which may have obscured strain-specific pathogenic roles. Further studies employing strain-level metagenomics are required. Finally, the relatively small sample size, particularly the limited number of infants in the FI group (n=16), constrained statistical power and may limit the generalizability of our findings. Future multicenter studies with larger cohorts are needed to validate these observations and improve the external validity.

In conclusion, our results align with the accumulating evidence suggesting an association between early gut microbial dysbiosis and FI development in preterm infants. These findings underscore the potential value of microbiome-targeted strategies in supporting intestinal health and improving outcomes in vulnerable populations. Further investigation is necessary to better understand the complex interactions among gut microbiota, intestinal function, and gastrointestinal physiology during early development.

SUPPLEMENTARY MATERIALS

Supplementary materials related to this article can be found online at https://doi.org/10.5385/nm.2025.32.1.21.

SUPPLEMENTARY MATERIALS AND METHODSnm-2025-32-1-21-Supplementary-Materials and Methods.pdf

Supplementary Table 1.

Adjusted Odds Ratios and P-values for Perinatal Factors of FI Group Compared to FT Group

nm-2025-32-1-21-Supplementary-Table-1.pdf

Notes

Ethical statement

This study was performed with informed consent of the participants in compliance with the Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of the CHA Bundang Medical Center (IRB No. CHAMC 2021-03-062).

Conflicts of interest

No potential conflict of interest relevant to this article was reported.

Author contributions

Conception or design: B.K.J., H.R.K.

Acquisition, analysis, or interpretation of data: B.K.J., H.R.K.

Drafting the work or revising: B.K.J., H.K., C.A.L., H.R.K.

Final approval of the manuscript: All authors read and approved the final manuscript.

Funding

This study was supported by the Basic Research Program of the National Research Foundation of Korea, funded by the Korean Government (MSIT) (NRF-202001110003). The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.

Acknowledgments

None

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Characteristic	FT (n=28)	FI (n=16)	P-value
Gestational age (wk)	29.9±1.7	28.9±2.4	0.130
Birth weight (g)	1,458.6±281.8	1,112.2±357.4	<0.001
Male sex	14 (50.0)	11 (68.8)	0.227
Cesarean section	23 (82.1)	15 (93.8)	0.280
Maternal hypertension	3 (10.7)	6 (37.5)	0.034
Maternal diabetes	6 (21.4)	3 (18.8)	0.832
Maternal PROM	17 (60.7)	2 (12.5)	0.002
Antenatal corticosteroids	12 (42.9)	7 (43.8)	0.954
RDS	24 (85.7)	14 (87.5)	0.868
IVH (≥grade 3)	1 (3.5)	1 (6.3)	0.682
Sepsis	1 (3.5)	3 (18.8)	0.092
Duration of antibiotic use (d)	10.5±6.0	15.4±6.4	0.016
Duration of Parenteral nutrition (d)	9.90±2.9	16.2±6.8	0.002
Length of NICU admission (d)	66.0±23.6	86.6±37.8	0.031