Seminars in Fetal and Neonatal Medicine xxx (xxxx) xxx–xxx

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Seminars in Fetal and Neonatal Medicine

journal homepage: www.elsevier.com/locate/siny

Necrotizing enterocolitis: The intestinal microbiome, metabolome and

inflammatory mediators
Josef Neua,∗, Mohan Pammib
a
Section of Neonatology, Department of Pediatrics, University of Florida, Gainesville, FL, USA
b
Section of Neonatology, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Necrotizing enterocolitis (NEC) is a disease of preterm infants and associated with significant mortality and
Microbiome morbidity. Although the pathogenesis of NEC is not clear, microbial dysbiosis, with a bloom of the phylum
Intestinal Proteobacteria, has been reported. Antibiotics and the use of H2 blockers, which affect the gut microbiome, are
Preterm associated with increased incidence of NEC. In association with dysbiosis, inflammatory processes are upregu-
Necrotizing enterocolitis
lated with increased Toll-like receptor signaling, leading to translocation of nuclear factor kappa-β, a tran-
scription factor that induces transcription of various pro-inflammatory cytokines and chemokines. Microbial
metabolites, short chain fatty acids including acetate and butyrate, may modulate immunity, inflammation,
intestinal integrity and regulate transcription by epigenetic mechanisms. Evaluation of microbiome and meta-
bolome may provide biomarkers for early diagnosis of NEC and microbial therapeutic approaches to correct
microbial dysbiosis.

1. Necrotizing enterocolitis
Necrotizing enterocolitis (NEC) is a major cause of mortality in
preterm infants who survive the first few days after birth [1,2]. NEC is
seen in 7% of very low birth weight infants (birth weight < 1500 g) and
up to 5% of admissions to the neonatal intensive care unit (NICU) [2,3].
NEC is associated with a mortality of 15–30% and a significant long-
term neurodevelopmental morbidity but we have made little progress
for 60 years [3–5]. The pathogenesis of NEC is not clear but microbial
dysbiosis, formula feeding and excessive inflammation have been im-
plicated [3]. Furthermore, a clear definition of this disease remains
elusive and it likely represents several different diseases with the final
outcome of intestinal injury or necrosis [6].
Postmenstrual age is correlated with the development of NEC, the
peak age being 29–31 weeks (peak 31 weeks) (based on last menstrual
period or conception) [7–9]. Fig. 1 summarizes several data sets on the
post-conceptual timing of NEC (Fig. 1) [10]. Prematurity is the major
risk factor, but, since not all preterm infants develop this disease, sev-
eral other environmental factors including intestinal dysbiosis may be
involved.
2. Microbiome and human health
The microbial communities on the surface of the human body
outnumber human cells and genes. Host–microbe interactions on in-
testinal and respiratory mucosal surfaces play a major role in the pre-
vention of infections and development of immunity. Intestinal micro-
biota, the best studied, has a bacterial load in the magnitude of 1014
bacteria/mm3, which makes it the most densely colonized surface of the
human body. Bacteroidetes represent the most abundant phylum, fol-
lowed by Firmicutes [11,12]. In healthy adults, Bacteroides, Faecali-
bacterium, and Bifidobacterium are the most prevalent genera [12].
The lower respiratory tract is one of the least-populated surfaces of the
human body with an estimated number of 10–100 bacteria per 1000
human cells [13]. The “core microbiota” of healthy individuals consists
mainly of genera Pseudomonas, Streptococcus, Prevotella, Fuso-
bacteria, Veillonella, Haemophilus, Neisseria, and Porphyromonas
[14–16].
2.1. Normal development of a preterm intestinal microbiome
Before birth, the fetus is bathed by the amniotic fluid that may not
be sterile [17]. Rather, the fetal–maternal unit is constantly exposed to
microbes and microbial metabolites that may originate from the vagina,
and mother's gastrointestinal tract including the mouth [18]. Recent
studies suggest that amniotic fluid contains microbes that can most
readily be detected with non-culture based techniques and that the
placenta harbors microbial DNA [17,18]. The fetus ingests large

∗
Corresponding author. Section of Neonatology, Department of Pediatrics, University of Florida, 1600 SW Archer Road, Gainesville, FL, 32607, USA.
E-mail address: neuj@peds.ufl.edu (J. Neu).

https://doi.org/10.1016/j.siny.2018.08.001

1744-165X/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: Neu, J., Seminars in Fetal and Neonatal Medicine (2018), https://doi.org/10.1016/j.siny.2018.08.001
J. Neu, M. Pammi
Fig. 1. Post-conceptual timing of necrotizing enterocolitis (NEC) diagnosis.
CGA, corrected gestational age.
[y-Axis label: ‘No. of NEC cases’].
quantities, up to 150 mL/kg/d, of amniotic fluid and the fetal gastro-
intestinal tract is exposed to large number of microbes and microbial
components during gestation. Several studies demonstrate that the
meconium contains microbial DNA as well as live microbes [19,20].
Studies suggest that the composition of these microbes in amniotic
fluid, meconium and placenta differ depending on gestational age of
delivery. Meconium microbes most likely reflect the in-utero rather
than the extrauterine environment. The microbial milieu in the meco-
nium of babies may determine the development of sepsis and NEC [19].
After birth, the preterm intestinal microbiome varies with post-
menstrual age and undergoes a patterned progression and increase in
anaerobes (around 36 weeks corrected gestational age (CGA)), in-
dicating maturation of the microbiome [21]. An enumeration of mi-
crobes of stools from preterm infants using 16SrRNA sequencing, has
determined that the microbiota progresses through a succession of the
bacterial classes from Bacilli, to Gammaproteobacteria, to Clostridia
[21]. By the time the infants approach 33–36 weeks post conceptual
age, anaerobes constitute a large component of the gut colonization.
2.2. Microbial dysbiosis and NEC
The concept of NEC associated with inappropriate colonization of
the premature intestine was introduced by Claude and Walker [22].
This was because “epidemics” of NEC had occurred, but no pathogen
had been associated with NEC. The authors posited that, rather than a
direct infection, NEC may be the result of a secondary inflammation in
response to microbial colonization or infection. Histologic sections of
intestine from infants with NEC often showed evidence of necrosis and
inflammation in addition to bacterial overgrowth. They suggested a
cycle of microbe-induced neutrophil activation leading to inflammatory
mediator release, vasoconstriction, and disruption of the intestinal
barrier that was initially caused by interaction of a “dysbiotic” in-
testinal microbiota and an immature intestine. They also suggested that
various factors such as breastfeeding could reduce colonization by pa-
thogenic organisms but induce colonization by commensal organisms,
which in turn modulate inflammatory reactions and decreased in-
testinal injury. Other studies have implicated microbial dysbiosis in the
gastrointestinal tract and an exaggerated inflammatory response in the
development of NEC [23–25]. Immune dysregulation in association
with microbial dysbiosis has also been implicated in the pathogenesis of
NEC. Excessive Toll-like receptor-4 (TLR4) signaling in response to li-
popolysaccharide (LPS) [26,27] and an exaggerated inflammatory re-
sponse [24,25] in preterm infants have been reported.
Prior to the last decade, several studies have searched for a specific
microbial origin of NEC using culture-based techniques [28–30]. Re-
cently, culture-independent techniques have been employed to evaluate
the microbial environment. Studies have reported dysbiotic microbial
patterns rather than a single pathogen causing NEC [10]. Compared to
term infants, the intestinal microbiota in preterm infants has fewer
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Seminars in Fetal and Neonatal Medicine xxx (xxxx) xxx–xxx
bacterial species, less diversity and increased proportion of potential
pathogens [31,32]. The microbial dysbiosis theory of NEC is supported
by the fact that NEC cannot be produced in germ-free animals [26,33].
It is also suggested by an association between early antibiotic use and
NEC [34,35].
2.3. The intestinal microbial milieu prior to the diagnosis of NEC
Early studies utilizing 16S rDNA for microbiome evaluation sug-
gested that relative increases in the phylum Proteobacteria when
compared to other major microbial phyla such as Firmicutes,
Bacteroides and Negativicutes are associated with the development of
NEC [36,37]. This intriguing relationship suggests that these may be-
come targets of microbial intervention against NEC, but true causality
has not yet been demonstrated and will be a requisite in the determi-
nation of whether a certain individual microbe or microbial pattern is
responsible for the disease. The microbial version of Koch's postulates
needs to be fulfilled for causality to be established.
There are issues with 16S rRNA gene sequencing in the evaluation of
the human microbiome. Taxonomic resolution is limited, and strain-
level resolution and tracking are not possible. The metabolic functions
of the microbiome cannot be assessed. In contrast, metagenomics or
whole genome shot-gun sequencing can provide strain-level resolution.
In addition to understanding the microbial community structure, the
metabolic potential of the microbial communities can also be assessed
[38,39]. 16S rRNA gene evaluation is based on existing information of
conserved sequences, and unusual sequences are missed. The metage-
nomic approach enables an unbiased evaluation of the microbial
communities even those with unusual RNA sequences or plasmids.
Meta-genomic approach may also be useful in the diagnosis of a disease
due to an unusual or unsuspected organism [40]. Metagenomic ap-
proaches are labor intensive and much more expensive than the 16S
rRNA gene sequencing methods. Metagenomic studies on NEC are
scarce although some small-sample studies have been published
[41,42]. Raveh-Sidka et al. studied the stool microbiome in preterm
infants (five with NEC and five controls) by the meta-genomic approach
and reported that although many microbial species were found in many
infants, the strains colonizing each infant were distinct, indicating a
paucity of shared gut colonization [41]. In another meta-genomic study
of the early intestinal microbiome of 144 preterm and 22 term infants,
NEC and mortality were associated with uropathogenic E. coli coloni-
zation [43].
La Rosa et al. observed an orchestrated patterned progression of gut
microbiota towards an abundance of Clostridia with increasing gesta-
tional age [21]. Antibiotics, feeding or mode of delivery caused abrupt
shifts in microbiota but did not change this predestined progression.
Other studies comparing gut microbiota in infants with NEC and con-
trols have discussed a Proteobacteria bloom positively associated [44]
and anaerobes (especially Negativicutes) negatively associated with
NEC [37]. In our meta-analyses of microbiome studies in preterm in-
fants – NEC versus controls who did not develop NEC – we found
gradual shifts in the relative abundances of multiple phyla at about 27
weeks CGA, where decreased abundances of Firmicutes and Bacter-
oidetes and increased abundances of Proteobacteria preceded the di-
agnosis of NEC [10] (Fig. 2).
2.4. Factors that alter the microbiome and may predispose to NEC
Several studies evaluating antibiotic use in preterm infants suggest
an increased odds ratio in the development of NEC, which is directly
related to the days of antibiotic usage [35,45,46]. Studies by La Rosa
et al. and Murgas et al. suggest a relationship between antibiotic use in
preterm infants, the microbial composition of the gastrointestinal tract,
and the subsequent development of NEC [21,47]. Additional studies
suggest that the acid–base environment of the gastrointestinal tract may
also play a significant role in the development of NEC [48]. This may
J. Neu, M. Pammi Seminars in Fetal and Neonatal Medicine xxx (xxxx) xxx–xxx

Fig. 2. Microbial profiles in necrotizing enterocolitis (NEC).

3
J. Neu, M. Pammi
also relate to the types of microbe present in the gastrointestinal tract
because they respond differently to the acid–base environment in terms
of their growth. H2 blockers favor the proliferation of the Proteo-
bacteria over Firmicutes in the gastrointestinal tract [49]. This re-
lationship is highly intriguing since the Proteobacteria to Firmicute
ratios also are altered in those infants who subsequently develop NEC
[10]. Animal studies in rabbits who were fed acidified milk had de-
creased gut colonization and translocation, emphasizing the protective
effects of gastric acidity on microbial colonization of the gut [50,51].
Human studies in which a formula was acidified resulted in a lower
incidence of NEC [52]. Retrospective database reviews and secondary
analysis of large multi-center studies show that the use of antacids, both
H2 blockers and proton pump inhibitors increase the risk for the de-
velopment of NEC [53,54]. Bovine lactoferrin supplementation miti-
gates this effect [53].
The well-known phenomena that babies receiving their own mo-
ther's milk have a decreased incidence of NEC is likely to relate to many
factors in the milk, including its macronutrient composition, the com-
position of polyunsaturated fatty acids, lactoferrin, various immune
cells, immunoglobulins, and microbes. The fact that human milk con-
tains microbes that appear to be personalized for each mother's own
infant [55] is a fascinating phenomenon that may also relate to the
decreased risk of NEC in babies fed their own mothers' milk. The fact
that donor milk is usually pasteurized and thus devoid of cellular ma-
terial as well as live microbes could be highly germane in this regard.
Studies utilizing only donor milk in comparison to commercial formula
have not overwhelmingly favored the donor human milk and the pre-
vention of NEC. Some of the studies included in meta-analyses of donor
milk versus formula are nearly 30 years old and it is difficult to discern
the criteria utilized in these studies to diagnose NEC in these older
studies.
3. Microbiome immunity and inflammation
3.1. Microbial–host interactions and innate immunity, inflammation
The relationship between microbes and the innate immune system
in the gastrointestinal tract has been reviewed [56,57]. Intestinal cells
including the intestinal epithelium harbor receptors to microbial com-
ponents, TLRs, which play a major role in innate immunity [26,58].
Activation of these Toll-like receptors results in signaling cascades that
induce nuclear translocation of nuclear factor kappa-β (NFKβ), a tran-
scription factor that induced transcription of various pro- and anti-in-
flammatory cytokines and chemokines [25]. For example, activation of
the chemokine interleukin (IL)-8 attracts neutrophils to trigger areas of
the intestine, where they undergo phagocytosis, and other in-
flammatory responses which subsequently cause microvascular con-
striction, ischemia and injury to the intestine [59]. Certain microbes
may also affect dendritic cells, which are of the monocyte/macrophage
lineage. These cells can traverse the intestinal epithelium, grasp anti-
gens from the lumen, carry them to the sub-epithelium and act as an-
tigen presenting cells to undifferentiated lymphocytes in the intestinal
lamina propria. Depending on the surface receptors that are stimulated
by the distinct microbial patterns to which these cells are exposed, in-
teraction between dendritic cells may result in the differentiation of T-
cells into effector versus tolerogenic cell types. The precise mechanisms
of these interactions as they relate to NEC are currently poorly under-
stood.
In our meta-analyses of microbiome studies in NEC, Proteobacteria
abundances increase in babies who develop NEC compared to controls
[10]. LPS produced by Proteobacteria is recognized by TLR4 in the
intestine, which triggers intestinal inflammation and enterocyte injury
that may lead to the development of NEC in preterm infants [60]. TLR4
expression is higher in the intestinal tract of preterm neonates when
compared to terms, and thus may regulate the balance between in-
testinal repair and injury [26]. It is interesting to note that TLR4 mutant
4
Seminars in Fetal and Neonatal Medicine xxx (xxxx) xxx–xxx
mice and TLR4 knockout mice are protected from NEC [61–64]. It is
possible that inhibition of the TLR4 pathway may be a novel strategy in
the treatment or prevention of NEC.
4. Metabolome in necrotizing enterocolitis
Microbiota-derived metabolites, short chain fatty acids (SCFAs),
butyrate, propionate and acetate induce IL-18 production from the in-
testinal epithelial cells (IECs) through activation of NOD-like family,
receptors (NLRs) [65]. Acetate produced by Bifidobacteria promotes
epithelial cell barrier function by inducing an anti-apoptotic response in
the IECs. Microbiota-derived sphingolipids presented on CD1d by
dendritic cells inhibit colonic invariant natural killer T-cell develop-
ment. Thus microbiota and their metabolites mediate immune response
via the IECs and immune cells [66,67]. Germ-free mice have lower le-
vels of SCFAs compared to conventionally housed animals [68,69].
Butyrate also is a major fuel for colonocytes and is a major stimulus for
tight junction formation and integrity [70]. SCFAs have also been
shown to stimulate histone acetylation of FoxP3 (forehead box P3)
locus on naïve CD4+ T-cells, increase FoxP3 expression and promote
differentiation of Tregs (anti-inflammatory effects) [65,68,71]. Faecali-
bacterium prausnitzii and Eubacterium rectale/Roseburia species (Firmi-
cutes) are major contributors of butyrate which regulates gene ex-
pression by histone modifications [72]. LPS – an inflammatory marker
for cardiovascular disease – may also have a role in epigenetic regula-
tion of intestinal and immune cells [73].
4.1. Clinical studies of the metabolome in neonates
‘Metabolomics’ was coined in 1998 by Oliver et al. [74] and is the
science of detecting small molecules, the result of metabolic pathways
from biological specimens such as plasma, serum, urine and tissues, and
is the latest of the ‘omics’ technologies. Metabolomics detects the pro-
ducts of the metabolic pathways in an organism which may be useful in
diagnosis, prediction, prognosis or assigning disease status (biomarker
detection). Metabolomics allows identification of distinct patterns of
small molecules generated during both host and microbial cellular
metabolism and may be useful in searching for biomarkers of micro-
biome patterns and dysbiosis [75–79]. Metabolite patterns are dy-
namic, changing with gestational age, time or disease process and at
any time give us a snapshot of the metabolic milieu of the organism.
The complexity and the numerous metabolites to be measured need
sophisticated analytical techniques. Nuclear magnetic resonance (NMR)
spectroscopy and mass spectrometry (MS) are the techniques most
widely employed.
Very few studies have performed on metabolomic patterns in NEC
patients, compared to controls or healthy preterm infants [80–84].
Although there is no unifying metabolomic signature in NEC, these
studies have reported interesting results. A multi-center evaluation of
the gut microbiome and metabolome named ‘Mechanisms affecting the
gut of preterm infants in enteral feeding trials (MAGPIE)’ study, funded
by the UK NIHR Efficacy and Mechanistic Evaluation program, is in
progress [85].
Morrow et al. evaluated urinary metabolome patterns in association
with gut dysbiosis preceding NEC and found that three urinary meta-
bolites differed in those with NEC compared to those without the dis-
ease [81]. Alanine in the urine was directly correlated with the relative
abundance of Firmicutes (P = 0.009), and was inversely correlated
with the relative abundance of both Proteobacteria (P = 0.027) and
Propionibacterium (P = 0.015) [81]. Histidine was not independently
associated with microbial community characteristics, but ratio of ala-
nine to histidine was positively associated with overall NEC (Krus-
kal–Wallis, P = 0.001) and inversely associated with the relative
abundance of Propionibacterium (Spearman's rho = −0.57,
P = 0.002).
Wilcock et al. described metabolomic differences in serum between
J. Neu, M. Pammi
preterm infants who developed NEC (five preterm
infants < 28 weeks GA) and those who did not (seven
preterm < 29 weeks GA and eight term infants) [82]. Serum samples in
the first week of life in these preterm infants did not predict who would
later develop NEC. However, when the infants were on complete
feeding, analyses revealed differences in 12 metabolites related to
carbohydrate, lipid metabolism and intracellular signaling, between
NEC and control infants. Based on the metabolites identified, an in-
hibition of the pro-insulin and upregulation of IL-1β were correlated
with the development of NEC.
Stewart et al. studied a large cohort of preterm infants (641 samples
from 35 infants from a cohort of > 300 infants) and compared preterm
infants with NEC (n = 7) to those without (n = 28) by 16S rRNA gene
profiling and metabolomic analysis of stools [84]. A subset of 75
samples (n = 16 patients) were also subjected to metabolomic analysis
by ultra-performance liquid chromatography mass spectrometry. At the
time of NEC diagnosis, five metabolites were significantly different in
preterm infants with NEC, two metabolites from C21-steroid hormone
biosynthesis and linoleate metabolism pathways, two metabolites from
the linoleate metabolism pathway, and one metabolite from the leu-
kotriene metabolism and prostaglandin formation from arachidonate
pathway. Stewart et al. also reported a proteomic and metabolomic
analysis of the serum in preterm infants who developed NEC with
controls but did not identify any protein or metabolite associated with
the development of NEC [80].
De Magistris et al. compared six infants who developed NEC with 12
controls by NMR-based urinary metabolic profiles. The investigators
found increased urinary gluconic acid in NEC patients. Gluconic acid
can be a byproduct of calcium gluconate administration or a byproduct
of bacterial metabolism by alternate metabolic pathways
(Entner–Doudoroff pathway). It is possible that production of gluconic
acid can favor bacteria that utilize gluconic acid but are more virulent
[83].
There is no unifying hypothesis related to altered metabolism in
NEC from published studies, and the use of metabolomic techniques in
understanding NEC pathogenesis is at its infancy. Integration of all
omics platforms is needed to understand the pathophysiology in NEC.
5. Future directions
For the prediction and diagnosis of NEC, diagnostic biomarkers in-
clude those that may not be specific for NEC such as the C-reactive
protein, white blood cell count, and platelet count. However, more
specific biomarkers are being developed which include tight junction
proteins such as claudin-3, intestinal epithelial cell proteins such as
intestinal fatty acid binding protein (IFAB), both of which can be
analyzed non-invasively in the urine. More predictive biomarkers using
the proteomic, metabolomic and genomic platforms are being eval-
uated. A novel “electronic nose” technology is highly intriguing, but
studies that critically evaluate its predictive and diagnostic potential
remain to be done.
The fact that certain microbial patterns as well as single microbes
are being found to be highly associated with NEC is intriguing.
However, the finding of an association is not adequate to directly base
therapeutic interventions. More rigorous studies of the causality will
need to be done. These will need to be based on in-vitro as well as in-
vivo techniques to recapitulate the disease in cell culture, and/or an-
imal models. Once these are established, directed therapies using highly
specific antibiotics, bacteriophages, probiotics or other microbial ther-
apeutic techniques may be further evaluated for safety and efficacy.
Several studies suggest that a microbial therapeutic approach to
NEC may be useful in the prevention of NEC. However, the most rig-
orous studies using probiotics to date are not supportive even though
numerous meta-analyses suggest efficacy. The fact that numerous dif-
ferent probiotics, which are very different in their character, have been
utilized in these meta-analyses has been subject of the criticism.
5
Seminars in Fetal and Neonatal Medicine xxx (xxxx) xxx–xxx
Furthermore, US Food and Drug Administration-based drug standards
should be utilized if these agents are to be used for prevention of NEC.
Whether fecal microbial transplant-like approaches such as those being
used in the treatment of Clostridium difficile infections may be of benefit
in the prevention of NEC could be a basis of future investigation.
Transfaunation of donor milk using small amounts of the baby's own
mother's milk may also be a means to “personalize” the donor milk to
provide a means to protect the infant's intestinal tract from the mi-
crobial environment that predisposes to NEC [86].
Conflicts of interest
None declared.
Funding sources
None.
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