Introduction
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.
Subjects and methods
Study design, area and period
The present prospective cohort study was approved by
the Local Ethics Commission of Asfendiyarov Kazakh National Medical
University (Minutes no. 14 of October 28, 2021). The study included
preterm infants with a VLBW and ELBW observed in the Neonatal ICU
of the Almaty City Perinatal Center (Almaty, Kazakhstan) from
October, 2021 to October, 2022.
Inclusion and exclusion criteria
The inclusion criteria were preterm infants with a
birth weight <1,500 g (VLBW or ELBW); an intolerance to enteral
feeding (for the study group), and those >14 days after birth.
The exclusion criteria were those with congenital intestinal
malformations and other severe congenital malformations, and those
with incomplete laboratory data, failure to comply with sample
collection, storage, and delivery procedures.
Of the 97 preterm infants, 19 were excluded due to
poor specimen collection (insufficient stool volume, late delivery
and/or violation of storage conditions), which could affect the
bacteriological examination results. A total of 78 patients
participated in the study: A total of 57 in the study group and 21
in the control group. The average age of the infants was 35 days.
Children in the study group presented with signs of GI disorders,
including bloating (flatulence), regurgitation, loose stools with
mucus and stool discoloration, as well as insufficient weight gain
or weight loss. The control group consisted of preterm infants with
a birth weight of 1,000-1,500 g with no clinical signs of GI
dysfunction (no regurgitation, no symptoms of flatulence, adequate
daily weight gain).
All participants in the study received breast milk
expressed by the mother, administered via an orogastric tube. All
babies received initial antibiotic therapy, as well as caffeine
citrate and glucocorticosteroids to prevent and treat
bronchopulmonary dysplasia. Ampicillin was administered at a
starting dose of 50 mg/kg every 12 h and gentamicin at 5 mg/kg
every 48 h for 8-12 days of life. Caffeine citrate was administered
as a loading dose of 20 mg/kg followed by a maintenance dose of
5-10 mg/kg/day once daily until clinical resolution of apnea of
prematurity. Dexamethasone was administered according to the
following tapering regimen: 0.15 mg/kg/day on days 1-3, 0.10
mg/kg/day on days 4-6, 0.05 mg/kg/day on days 7-8, and 0.02
mg/kg/day on days 9-10. None of the patients received probiotics,
antacids, or other medications that may affect the GI tract.
During the study, 10 participants (17.5%) in the
study group experienced deterioration in clinical condition and
developed signs of NEC, including progressive abdominal distension,
symptoms of intestinal paresis, and characteristic X-ray changes.
These patients were allocated to a separate subgroup with
established NEC. The study group design is illustrated in Fig. 1.
Samples and data collection
The study objects were the feces of newborns with a
VLBW or ELBW or who were treated in the neonatal ICU and included
in the study due to clinical symptoms of FI. Control samples were
obtained from premature newborns with a VLBW or ELBW without the
clinical symptoms of FI.
The material was collected immediately following the
physiological act of defecation, the inner layer of a baby diaper
using a sterile spatula, and placed into two sterile containers.
Container no. 1 contained Cary-Blair transport medium (Cary-Blair
Medium: HiMedia Laboratories) for microbiological investigations
(culture, quantitative analysis of microbiota). Сontainer no. 2 did
not include a nutrient medium for determining the FC level. The
containers had a tightly closing lid and a built-in spatula. Each
sample was labeled with an individual patient code, date and the
time of collection.
Samples were collected no later than 5 min following
defecation to minimize the possible absorption of the liquid stool
into the diaper and to avoid the degradation of biomarkers,
including calprotectin. To ensure proper processing, the samples
were stored in a refrigerator at 2 to 4˚C for no more than 2-3 h
prior to transport to the laboratory. The samples were delivered to
the laboratory in a special cooling bag, ensuring a stable
temperature.
In the laboratory, samples from container no. 1 were
incubated in a thermostat at +37˚C for 4-7 days. Colony growth was
then assessed, and the microbial load was determined (in CFU/g).
Samples from container no. 2 were analyzed using ELISA for FC
levels in the stool extract (in µg per 1 g of feces).
Such an approach to collecting and transporting
samples allowed for the maintenance of the integrity of the
microbial composition and ensuring the reliability of determining
FC levels, which is critical when working with newborns and small
volumes of biomaterial.
The bacteriological culture of feces and
determination of fecal calprotectin (FC) levels were performed on a
contractual basis in the accredited commercial laboratory
‘Invivo.kz’ (Almaty, Kazakhstan; Contract no. 230879 dated October
5, 2022). All analyses were carried out using standard validated
methods in accordance with the manufacturers' instructions. Stool
samples were cultured on Salmonella-Shigella agar (SS agar; HiMedia
Laboratories Pvt. Ltd.) and analyzed using the MicroScan WalkAway
96 Plus automated microbiology system (Beckman Coulter, Inc.) for
bacterial identification.
Fecal calprotectin concentrations were determined
using a Calprotectin ELISA kit (cat. no. EQ 6831-9601 W, EUROIMMUN
Medizinische Labordiagnostika AG) on an ImmunoChem-2100 microplate
photometer (High Technology, Inc.), with all analyses performed
according to the manufacturers' validated protocols.
Statistical analysis
Statistical analysis was performed using STATISTICA,
version 12 (StatSoft Inc., USA). Baseline clinical and demographic
characteristics were compared between two independent groups
(healthy preterm infants and preterm infants with feeding
intolerance). Continuous variables (gestational age and birth
weight) were expressed as mean ± standard deviation (SD) and
compared using the independent samples Student's t-test.
Categorical variables (sex, mode of delivery, use of antenatal
steroids, etc.) were compared using Pearson's χ² test. The
composition of the intestinal microbiota was compared in the same
two independent groups. Fisher's exact test was used to compare the
proportions due to the small number of some cells included in the
contingency analysis (<5). Fisher's exact test was also used to
assess the association between the clinical group and FC levels.
Statistical significance was assessed using the Monte Carlo method
(10,000 simulations), as the expected number of observations was
<5 in >20% of cells. Pearson's χ2 test was also
used and data are presented as a descriptive measure of the
association. All tests were two-tailed and a P-value <0.05 was
considered to indicate a statistically significant difference.
Correspondence analysis was conducted to visualize the association
between clinical groups and FC categories. This method is based on
the χ2 statistic and represents the rows and columns of
the contingency table as points in a low-dimensional space.
Results
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).
 | Table IBaseline characteristics of the
infants included in the study. |
Table I
Baseline characteristics of the
infants included in the study.
| Characteristic | Healthy preterm
infants, n=21 | Preterm infants
with feeding intolerance, n=57 | P-value |
|---|
| Male sex, n
(%) | 10 (47.6%) | 29 (50.9%) | 0.81 |
| Female, n (%) | 11 (52.4) | 28 (49,.1%) | 0.81 |
| Cesarean delivery,
n (%) | 9 (42.9%) | 27 (47,.4%) | 0.71 |
| Antenatal steroids,
n (%) | 13 (61.9%) | 30 (56.1%) | 0.64 |
| Birth weight, g,
mean ± SD | 1,190±102 | 798±137 | <0.001 |
| Gestational age,
weeks, mean ± SD | 28.0±1.03 | 26.0±1.9 | <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.
| Table IIComparison of the intestinal
microbiota in healthy preterm infants and preterm infants with
feeding intolerance. |
Table II
Comparison of the intestinal
microbiota in healthy preterm infants and preterm infants with
feeding intolerance.
| Microorganisms | Healthy preterm
infants, % (n=21) | Preterm infants
with feeding intolerance, % (n=57) | P-value |
|---|
|
Bifidobacteria | 100.0 | 100.0 | 0,.999 |
| Lactobacilli | 100.0 | 82.4 | 0.039a |
| Escherichia
coli with normal enzymatic activity | 23.8 | 68.4 | 0.001b |
| Escherichia
coli with reduced enzymatic activity | 23.8 | 38.8 | 0.217 |
|
Enterococci | 57.1 | 42.1 | 0.238 |
| Staphylococcus
epidermidis (S. epidermidis) | 28.5 | 19.3 | 0.383 |
| Pathogenic
microorganisms | 0.0 | 100.0 |
<0.001b |
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).
| Table IIIDistribution of fecal calprotectin
levels in preterm infants by clinical group. |
Table III
Distribution of fecal calprotectin
levels in preterm infants by clinical group.
| Clinical group | 50-100 µg/kg | 100-350 µg/kg | 350-500 µg/kg | >500 µg/kg | Total |
|---|
| Healthy preterm
infants | 10 (47.6%) | 11 (52.4%) | 0 (0%) | 0 (0%) | 21 |
| Preterm infants
with feeding intolerance | 28 (59.6%) | 10 (21.3%) | 4 (8.5%) | 5 (10.6%) | 47 |
| Preterm infants
with necrotizing enterocolitis | 0 (0%) | 0 (0%) | 4 (40.0%) | 6 (60.0%) | 10 |
| P-value | 0.002 | 0.003 | 0.006 | 0.0001 |
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.
Discussion
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.
Acknowledgements
The authors express their sincere gratitude to the
City Perinatal Center of Almaty, Kazakhstan, for its support in
organizing the study, and to the physicians and nurses of the
intensive care and neonatal intensive care units who voluntarily
assisted in the collection of biological samples.
Funding
Funding: The present study was supported by the Within the
framework of the intra-university grant for scientific research in
the field of medicine and public health (project on ‘New ways of
prediction, prevention and methods of correction of necrotizing
enterocolitis in premature infants’), based on Order Nno. 312;
dated June 18, 2021.
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
GB conceived and designed the study, collected data,
drafted the initial version of the manuscript, and critically
reviewed and revised the manuscript. KZ collected data and
critically reviewed and revised the manuscript. GХ assisted with
the statistical analysis and critically reviewed the manuscript. AK
provided consultations on the interpretation of the laboratory
tests and contributed to the literature search related to the
study. MА and NV collected and transported biological samples,
entered patient data into Excel spreadsheets, and managed the
databases. KZ and GX confirm the authenticity of all the raw data.
All authors have read and approved the final version of the
manuscript and agree to be accountable for all aspects of the
work.
Ethics approval and consent to
participate
sThe present study was conducted from August, 2022
to December, 2023 and was approved by the Local Ethics Commission
of the Kazakh National Medical University. S.D. Asfendiyarov
(Ethical no. 1331 dated October 28, 2021).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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