Infants with a very low birth weight (VLBW) of 1,000-1,500 g and an extremely low birth weight (ELBW) of 500-999 g are characterized by the morphological and functional immaturity of organs and systems. Premature babies do not always manage to maintain their intrauterine growth rate after birth. Malnutrition in the early stages of development leads to long-term short stature, delayed organ growth, and a deficit in the number of neurons and dendritic connections, as well as later behavioral disorders (1). In addition to the immaturity of the gastrointestinal (GI) tract, reduced motility and the decreased activity of intestinal enzymes, preterm newborns often present with severe comorbid conditions. These conditions are accompanied by inflammatory processes associated with bronchopulmonary dysplasia, necrotizing enterocolitis (NEC) and intraventricular hemorrhages (2,3). In their study, Chen et al (4) identified risks and predicted a pattern of feeding intolerance (FI) in children. These children have a low gestational age, a low birth weight, a history of fetal distress, a history of the use of aminophylline, and an interval of >3 days between bowel movements (4).

In recent years, the intestinal microbiome in newborns has been a focus of neonatology due to its critical role in the proper functioning of the body and neonatal health. Newborns have less diversity in their intestinal flora than adults. Intestinal colonization factors in newborns are closely related to gestational age at birth, the mode of delivery and antibacterial therapy (5-7).

According to Neu and Rushing (8), newborns born vaginally acquire bacteria resembling the vaginal microbiome of their mother, mainly Lactobacillus and Prevotella. On the contrary, newborns born by cesarean section acquire bacteria similar to the skin microbiome of their mother, primarily Staphylococcus aureus (8).

Pace et al (9) demonstrated the changes in the vaginal microbiome with gestational age. The relative content of Lactobacillus spp. in the mother's vaginal microflora increases towards full-term pregnancy. Thus, Lactobacillus spp. predominates and determines the spectrum of microorganisms colonizing the newborn during its passage through the birth canal (9).

Factors, such as antibacterial therapy, mechanical ventilation, delayed breastfeeding and low enteral feeding volumes delay the establishment of a normal gut microbiota in infants born prior to 28 weeks of gestation. The resulting prevalence of Escherichia coli, Klebsiella, Staphylococcus, Propionibacterium and Corynebacterium in premature babies has long-term consequences for their health, including an increased susceptibility to developing NEC (10,11). Normal intestinal biocenosis prevents the development of opportunistic microflora, producing vitamins B, K, and D, and promoting the absorption of iron and calcium (12).

According to the study by Yao et al (13), the intestines of preterm infants are colonized with hospital-acquired flora due to their prolonged periods of hospitalization in intensive care units (ICUs) and the long-term use of antibiotics. They emphasized that gut colonization in preterm infants differs significantly from that in full-term infants (13). The inverse association between age at NEC diagnosis and gestational age at birth reflects the accumulation of risk factors, such as dysbiosis and developmental features of the intestinal immune system (14).

FI in preterm infants is directly associated with changes in the gut microbiota. Children with FI have a lower microbial diversity, fewer beneficial bacteria and increased numbers of pathogenic bacteria (15).

A recent literature review demonstrated that intestinal dysbiosis can cause acute illnesses in preterm infants, including NEC, sepsis, bronchopulmonary dysplasia and retinopathy of prematurity, and can affect their neuropsychological development and somatic growth (16).

Altered gut microbiota and its metabolites directly affect nutrient absorption, gut development, inflammation and hormonal signaling, thereby affecting growth, and promoting the development of metabolic disorders in preterm infants. This supports the connection between pathologic gut colonization and the development of bacteremia in preterm infants in ICUs (17). These data highlight the key role of the gut microbiota in the development of inflammatory complications, including NEC, in preterm infants.

The pathogenesis of NEC begins with an inflammatory process that damages the intestinal mucosa colonized by microflora, the immature intestinal epithelium and the intestinal circulation, leading to an excessive inflammatory response (18).

The high incidence and mortality rates associated with FI and NEC among preterm infants (19) emphasize the need for the early identification of risk groups and the development of effective methods for their prevention and early diagnosis. Previous studies have highlighted the role of the gut microbiota in the pathogenesis of these conditions, with a particular focus on Enterobacter, Klebsiella, Citrobacter and Acinetobacter Gram-negative bacteria (10,19). These microorganisms, often nosocomial pathogens, can colonize the intestine in humans with immature immunity and impaired barrier function, contributing to the development of systemic infections and the exacerbation of inflammatory processes (20). The clinical consequences of a disrupted intestinal microbiome in preterm infants are of particular interest. Prospects for microbiome diagnostics and interventions to improve the health of preterm infants have been discussed (21,22).

Mai et al (23) reported increases in Proteobacteria (by 34%) and decreases in Firmicutes (by 32%) within 72 h prior to the onset of the clinical symptoms of NEC. They suggested that such a change in microbiota may represent a pattern of the manifestation of NEC (23).

Almost all preterm infants are prescribed initial antibiotic therapy. It reduces overall microbial diversity and depletes beneficial microorganisms, such as Bifidobacteriaceae, which are early colonizers of the intestine. Bifidobacteriaceae efficiently ferment breast milk oligosaccharides, which serve as substrates for the synthesis of short-chain fatty acids, an important nutrient source in the intestine. The depletion of protective microorganisms disrupts microbial homeostasis and increases the proportion of pathogenic enterobacteria, such as Enterobacteriaceae, which are often associated with antimicrobial resistance and the risk of developing sepsis and NEC (24,25). The high levels of antibiotic resistance and clinical significance of pathogenic microorganisms urge us to understand the factors that contribute to their growth and pathogenicity, to improve the prevention and treatment of NEC and FI.

Both qualitative and quantitative assessments of gut microbiota are critical for understanding its role in FI and NEC. The quantification of bacterial colonization is usually performed in ‘colony-forming units per gram’ (CFU/g). This approach, based on standard seeding methods followed by colony counting, is considered reliable for identifying and quantifying bacterial cultures in the feces of newborns (26,27).

A search is currently underway for a simple, rapid, reliable, sensitive and safe diagnostic tools which can be used to detect and monitor intestinal inflammation in neonates. Fecal calprotectin (FC) analysis is considered a promising non-invasive marker of inflammation (28). When measured in feces, calprotectin is markedly associated with the neutrophil infiltration of the intestinal mucosal surface and within the gut lumen, and is a hallmark of digestive inflammatory pathology (29,30). The interpretation of FC levels in neonates, particularly preterm infants, should consider age-dependent factors and the degree of intestinal maturity. Some clinical studies have demonstrated that FC levels can vary significantly during the first weeks of life, including physiologically high values, particularly at 3-78 days (31,32). Such values may be associated not only with an inflammatory reaction, but also with the processes of primary colonization of the intestine by opportunistic microflora (31,32).

There is ample evidence of higher neonatal FC levels in full-term and premature newborns compared to reference values in adults (<50 µg/g) and older children. FC levels can reach 600 mcg/g in clinically healthy newborns and even more in preterm infants, with high interindividual variability. However, FC levels tend to decrease as the intestines adapt postnatally. Notably, the method of feeding (breast or formula) does not significantly affect FC levels (33-36).

Cekovic et al (37) reported a positive correlation between high FC levels and intrauterine growth retardation, a prominent arterial duct, intolerance to enteral feeding and the prolonged use of broad-spectrum antibiotics in the postnatal period (37).

Preterm infants with a VLBW of ELBW, particularly those in neonatal ICUs, are exposed to invasive procedures, antibacterial therapy and hospital microflora, which can prevent the development of normal gut microbiota and increase the vulnerability of the intestine to inflammatory and infectious complications. In this population, the characteristics of the gut microbiota and intestinal inflammation require targeted analyses as potential key factors in the pathogenesis of FI and NEC.

The present study used FC as a marker of intestinal wall inflammation and a key additional indicator of the inflammatory process. Preterm infants with a VLBW or ELBW are at a risk of developing more severe FI and NEC. Therefore, the present study aimed to assess the possible role of the gut microbiota and FC levels in the development of inflammatory bowel changes and NEC analysis in underweight preterms with FI.

In the present study, 78 preterm infants with a birth weight <1,500 g were included, comprising 21 healthy preterm infants and 57 preterm infants with feeding intolerance. The overall clinical and demographic characteristics are summarized in Table I. There were no statistically significant differences between the groups with respect to sex, mode of delivery (cesarean section or vaginal delivery), or use of antenatal steroids (P>0.05 for all comparisons). However, infants with feeding intolerance had a significantly lower gestational age and birth weight compared with healthy preterm infants (26.0±1.9 vs. 28.0±1.03 weeks and 798±137 g vs. 1,190±102 g, respectively; both P<0.001).

Microbiological tests revealed fairly diverse microbial associations in the intestines of the examined premature newborns (Fig. 2). The initial microbiological landscape represents pooled data rather than comparative analysis across subgroups.

The microbial landscape was dominated by normal flora at a concentration of at least 10⁷-10⁸ CFU/g, including Bifidobacterium spp. in 78 (100%) of the infants and Lactobacillus spp. in 68 (87.2%) of the infants; Escherichia coli with normal enzymatic activity was observed in 44 (56.4%) of the infants and with reduced enzymatic activity in 27 (34.6%) of the infants. The Gram-positive lactic acid microorganism, Enterococcus faecalis, a component of the normal microflora of the gastrointestinal tract in breastfed newborns, was found in 36 (46.2%) of the infants, and Staphylococcus epidermidis in 17 (21,8%) of the examined infants. The remaining opportunistic flora was observed in isolated cases (Fig. 2).

Notably, 57/78 (73%) of the preterm infants presented pathogenic intestinal flora that can impair the GI tract function and promote the development of FI symptoms. The normal flora composition was almost similar in the study group and the control group; however, only children with FI had pathogenic flora within 10⁷-10⁸ CFU/g (Fig. 3).

Statistical analysis revealed a statistically significant difference in the presence of Escherichia coli with normal enzymatic activity, lactobacilli and pathogenic flora between the healthy preterm infants and preterm infants with FI. The comparative analysis using Fisher's exact test revealed statistically significant differences in the intestinal microbiota composition between the study groups (Table II). In the preterm infants with FI, lactobacilli were less frequent compared with the healthy preterm infants (82.4 vs. 100%; P=0.039 analyzed using Fisher's exact test), as were Escherichia coli with normal enzymatic activity (23.8 vs. 68.4%; P=0.001). At the same time, preterm infants with FI had a higher proportion of opportunistic microorganisms. Pathogenic flora were present in all infants with FI (100%) and absent in the healthy preterm infants (P<0.001 analyzed using Fisher's exact test), indicating a markedly more pronounced dysbiosis in the study group.

The analysis confirmed a markedly higher colonization of the GI tract by pathogenic microflora in the newborns with FI. In the present study, the pathogenic microflora in the newborns with FI (n=57) was presented by Klebsiella pneumoniae in 17 (29.8%) of the infants at 10⁷ CFU/g, Klebsiella oxytoca in 13 (22.8%, 10⁷ CFU/g), Enterobacter cloacae in 10 (17.6%, 10⁷ CFU/g), Enterobacter aerogenes in 9 (15.8%, 10⁸ CFU/g), Citrobacter koseri in 6 (10.5%, 10⁷ CFU/g), Candida albicans in 4 (7.0%, 10⁵ CFU/g) and Acinetobacter baumannii complex in 1 (1.7%, 10⁸ CFU/g) of the infants (Fig. 4). Such a pronounced colonization of the intestine with pathogenic and opportunistic flora may have clinical significance for the development of food intolerance in this patient category and may indicate a disturbance in the microbiocenosis.

The measurement of FC levels using ELISA in stool samples was used as a non-invasive marker to quantify the intestinal condition in infants with a VLBW or ELBW. This method was safe and simple, and allowed the quantitative determination of FC levels expressed in µg/g. As illustrated in Fig. 5, FC levels in preterm infants were consistent with their condition. The majority of healthy preterm infants (n=21; 47.6 and 52.4%, respectively) had low to moderate FC levels (50-350 µg/g), with none >350 µg/g. In preterm infants with FI (n=47), low and moderate levels prevailed (59.6 and 21.3%, respectively); however, some had elevated calprotectin concentrations (≥350 µg/g: 8.5% with 350-500 µg/g and 10.6% with >500 µg/g). The most pronounced increase was observed in the preterm infants with NEC (n=10): All had high FC levels (≥350 µg/g), with 40.0% in the 350-500 µg/g range and 60.0% exceeding 500 µg/g, reflecting a significant increase in intestinal inflammation in NEC. Thus, in the study groups, FC levels, evaluated against the background of microbiological changes, were associated with identified disturbances of the gut microbiota (dysbiosis).

The association between the clinical group and FC levels was evaluated using Fisher's exact test for an R x C contingency table with the Monte-Carlo estimation (10,000 simulations), which demonstrated a statistically significant association (P<0.001). The NEC group had the highest proportion (60.0%) of infants with markedly elevated FC levels (>500 µg/g), compared with 10.6% among preterm infants with feeding intolerance. No FC elevation was observed among the healthy preterm infants, in whom FC levels were not >350 µg/g (Table III).

The association between clinical groups and FC categories is presented in Fig. 6, as determined by correspondence analysis based on χ²-derived distances. For this analysis, the same contingency table as in Table III was used: χ² distances between row and column profiles of this table were calculated and then plotted in a reduced two-dimensional space. A sharp angle indicates a positive association with FC levels >500 µg/g. The plot illustrates a statistically significant association [χ²(6)=43.536; P<0.001]. The obtained results demonstrate that unfavorable intestinal colonization patterns in preterm infants (a dysbiotic shift toward pathogenic flora) are associated with elevated FC levels. This confirms the potential diagnostic and differential diagnostic value of FC levels for identifying and stratifying the severity of inflammatory bowel disease in preterm infants, particularly for differentiating between functional FI and NEC.

FI and NEC are fundamentally different, yet clinically similar conditions in preterm infants. FI often reflects the functional immaturity of the GI tract and dysbiotic changes, whereas NEC is a severe, inflammatory, and necrotic intestinal condition with a high risk of adverse outcomes. Since both FI and NEC are closely dependent on the specifics of intestinal microbial colonization, qualitative and quantitative studies of the microbiota composition in preterm infants are particularly important. The identification of bacterial (and fungal) infections allows for the characterization of the intestinal biocenosis and the identification of potential pathogens associated with NEC development.

In the present study, a comparative analysis of the intestinal microbiological profile in the examined newborns revealed that representatives of the Enterobacteriaceae family, including Klebsiella pneumoniae and Enterobacter cloacae, as well as fungal flora (Candida albicans), predominated in the fecal cultures of children with NEC. These results align with those of prior publications on the microbiota in preterm infants. For example, Coleman et al (38) reported a predominance of Klebsiella spp. in the intestines of infants who developed NEC compared with a predominance of commensal Escherichia spp. in healthy neonates. In addition, a metagenomic study in India also revealed a significant increase in the proportion of Enterobacteriaceae and the presence of virulence genes associated with impaired intestinal barrier function (39).

Harmful bacteria, including Enterococcus, Klebsiella and Acinetobacter, dominate the intestinal microflora of premature infants. Premature infants with an ELBW have less beneficial bacteria and more potentially harmful bacteria in the gut. These potentially harmful bacteria may serve as potential biomarkers of intestinal dysbiosis and increased risk of NEC in preterm infants with an ELBW.

The role of Candida in the development of NEC in preterm infants remains poorly understood, despite several studies confirming its involvement in the condition. Thus, according to the study by Pappas et al (40), 25% of premature babies with prolonged antibiotic use presented with Candida, as well as Klebsiella and Enterococcus. Dermitzaki et al (41) revealed that newborns, particularly preterm infants, admitted to ICUs, had pronounced risk factors for invasive Candida infections, and the use of antifungal prophylaxis is particularly justified for this category of patients. It has been reported that Acinetobacter baumannii has become a common pathogen causing hospital-acquired infections worldwide (24,42). As a typical nosocomial pathogen, it can colonize the skin and GI tract of humans (42). Currently, Acinetobacter spp. increasingly causes severe illness in newborns worldwide. The reason for such an escalation is unclear; however, it may be related to a combination of host, pathogen and environmental factors (43).

In previous a review of factors influencing the gut microbiota in newborns, Srinivasjois et al (44) identified several key determinants of microbiota formation: The effects of antibiotics, feeding type, probiotic use and prolonged infant stays in neonatal ICUs. Preterm infants are particularly susceptible to intestinal dysbiosis, which is associated with conditions such as NEC, growth retardation, cognitive impairments and brain damage (45). Accumulated data indicate that functional FI and NEC in preterm infants are associated with severe disturbances in intestinal microbial colonization and activation of the local inflammatory response.

This places special emphasis on non-invasive diagnostic methods, allowing for the timely detection of early signs of inflammation without traumatic interventions. FC reflects the local inflammatory response of the intestinal mucosa. Determining the FC level is a safe and convenient method for early diagnosis of inflammatory and necrotic intestinal lesions, especially in preterm infants, in whom invasive procedures are limited by clinical condition. The present study used FC as a non-invasive marker confirming inflammatory changes in the intestines of the examined newborns. FC levels were reliably higher in infants with suspected NEC than in infants with signs of functional GI disorders and in the control group.

Recent foreign studies have reported similar data. A previous study demonstrated that FC levels were statistically significantly higher in preterm infants with NEC than in the control group; they were directly associated with the severity of inflammatory changes in the intestinal mucosa (46).

In another study, an elevated FC level was used as an early diagnostic criterion for NEC. In the NEC group, FC levels were higher than in the FI group and healthy children (P<0.05); FC ranged from 250 to 400 µg/g in children with mild inflammation and exceeded 800 µg/g in children with severe NEC (47), which is comparable with the results of the present study.

FC levels may also depend on microbiota composition. In a previous study, children with a lower gestational age (<32 weeks) exhibited a greater variability in FC and inflammatory metabolite levels (48). This could be due to the immaturity of the gut microbiota (48). These results are consistent with the observations of the present study: Extremely preterm babies (>32 weeks) had higher FC levels than healthy preterm infants, suggesting varying degrees of intestinal colonization and immune response development.

The strengths of the present study included a well-defined cohort of extremely preterm infants with a VLBW of ELBW, a unified feeding strategy based exclusively on expressed breast milk, and a combined assessment of gut microbiota using culture methods and FC levels as an objective, non-invasive marker of intestinal inflammation. However, the present study had some limitations which should be mentioned. First, the single-center, observational nature of the study and a relatively small sample size (78 premature infants, including 10 who developed NEC) limit the statistical power of the subgroup analysis and the generalizability of the results. Second, although the groups were comparable by sex and method of delivery, the authors did not systematically collect or adjust for important maternal and perinatal factors (e.g., maternal infections, prenatal antibiotic exposure, chorioamnionitis, overall disease severity). Third, all infants received empirical antibiotic therapy, caffeine citrate and glucocorticoids, and the isolates were not tested for antibiotic susceptibility. Finally, the present study did not provide a detailed description or systematic analysis of the complete clinical picture (including the evolution of gastrointestinal and systemic symptoms and the severity of the disease); the main focus was on microbiological data and FC levels.

In conclusion, the present prospective observational study of the gut microbiota revealed the presence of pathogenic flora represented by Klebsiella pneumoniae (29.8%), Klebsiella oxytoca (22.8%), Enterobacter cloacae (17.6%), Enterobacter aerogenes (15.8%), Citrobacter koseri (10.5%), Acinetobacter baumannii complex (1.7%) and Candida albicans fungi (7.0%) in preterm infants with a VLBW or ELBW and with FI (n=57) compared to healthy preterm infants. A total of 10 of the 57 infants (17.5%) subsequently developed NEC, and a pronounced inflammatory process in the intestine was accompanied by an increase in FC level. Disturbances in the intestinal microbiocenosis should be considered a key pathogenetic risk factor for the development of NEC in preterm infants with a VLBW or ELBW and with FI in ICUs. For preterm infants with a VLBW or ELBW in ICUs, the early identification of pathogenic gut colonization and elevated fecal calprotectin may help stratify the risk of FI and NEC and guide targeted preventive strategies.