REVIEW

URRENT
C
OPINION

The neonatal bowel microbiome in health

and infection
Janet E. Berrington a, Christopher J. Stewart b, Stephen P. Cummings b, and
Nicholas D. Embleton a

Purpose of review
In newborns, interactions between the host and the microbiome operate synergistically, modulating host
immune function and shaping the microbiome. Next generation molecular sequencing methodologies in
tandem with modeling complex communities allow insights into the role of the microbiome in health and
disease states. Infection-related disease states in which dysbiosis is integral include late-onset sepsis (LOS)
and necrotizing enterocolitis (NEC), which still cause deaths and morbidity. Understanding microbiomic
interactions may lead to alternative prevention, monitoring or treatment strategies, and modulation of longterm health outcomes especially in the preterm population. Recent studies have advanced understanding of
the microbiome in NEC and LOS.
Recent findings
Mechanisms of hostmicrobiome interaction have been demonstrated. Patterns of microbiomic change in
association with NEC and LOS have been observed, with community changes dominated by
Proteobacteria and Firmicutes appearing to precede NEC, and very early microbiomic signatures
influencing LOS. Data on viral and fungal elements are emerging.
Summary
Greater understanding of the neonatal bowel microbiome may allow tailored clinical practice and
therapeutic intervention. Data handling and interpretation is challenging. Mechanistic studies of clinical
interventions that affect the gut microbiome are important next steps.
Keywords
health, infection, microbiome, necrotizing enterocolitis, neonatal

INTRODUCTION
The complex microbial community (microbiome)
in the human gut is viewed as a superorgan involved
in many aspects of health and disease in which the
microbiome exerts metabolic, nutritional, and
immunological effects on the host [1]. In healthy,
vaginally delivered, term newborns (HVDTNB) the
gut and adaptive immune system tolerate and
regulate this community; the microbiome in turn
sculpts gut immune and metabolic function [2]. The
establishment of the microbiome proceeds predictably, reflecting the coevolution with their host over
millennia [3,4]. However, this dynamic process is
readily disrupted, especially by events related to
prematurity [5]. Such disruption, termed dysbiosis,
is implicated in development of late-onset sepsis
(LOS) and necrotizing enterocolitis (NEC), which
are both major causes of mortality and morbidity
[6]. Therapeutic manipulation and even microbiome
transplantation are possible but the mechanistic
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interactions involved are complex and poorly understood. This review summarizes how advances in molecular technologies can offer mechanistic insights
into hostmicrobiome interactions and reviews
recent work suggesting a microbiome pattern representative of health and changes that precede LOS and
NEC. We review progress toward our understanding
of the functional contribution of bacterial community members, and the significance of nonbacterial organisms (fungi, viruses, and archaea). Insights
a
Newcastle Neonatal Service, Newcastle upon Tyne Hospitals NHS
Foundation Trust and Newcastle University and bFaculty of Health and
Life Sciences, University of Northumbria, Newcastle, UK

Correspondence to Janet Elizabeth Berrington, Newcastle Neonatal

Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Queen
Victoria Road, Newcastle, NE1 4LP, UK. Tel: +44 191 2825197; fax:
+44 191 2825048; e-mail: j.e.berrington@ncl.ac.uk
Curr Opin Infect Dis 2014, 27:236243
DOI:10.1097/QCO.0000000000000061
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The neonatal bowel microbiome Berrington et al.

KEY POINTS

Very early microbe exposures and microbehost
interactions are key to the development of NEC and
LOS, and therapeutic manipulations require
underpinning with mechanistic evaluation.

DNA extraction before universal PCR primers allow

amplification of intervening hypervariable regions.
These amplicons then require differentiating into
sufficiently similar groups to be classified together
into an operational taxonomic unit (OTU). Different techniques have been used and continue to
develop (Fig. 1 [8 ]). Bacterial classification is determined by using open access sequence databases.
Methodologies for nonbacterial constituents are
less robust or well developed [9] but fungal, archaeal, protozoal, and viral microbiomic studies now
exist. The functional capacity of the microbiome is
also increasingly acknowledged: metabolomic and
proteomic techniques offer insight into functional
microbiomic changes associated with disease [10].
&

Patterns of bacterial dysbiosis are emerging:

Proteobacteria and Firmicutes appear pivotal to
changes that predict or precede NEC or LOS
development.

Nonbacterial elements may also be important and
require further study in carefully sampled populations
with careful exposure data.

from interventions intended to manipulate the

microbiome are considered and a brief examination
of residual research needs and priorities are outlined.

METHODOLOGICAL CONSIDERATIONS
Over time the gut microbiota changes in constituent
members, relative abundance, gene expression, and
metabolic activity. Measuring this dynamic complexity poses significant challenges: analysis typically involves passed stool (rarely ileal fluid or tissue)
and is thus limited by spontaneous stooling (which
occurs less frequently when infants are ill) and
reflects alterations that are historical and dependent
on transit times. Analyzing and presenting such
complex dynamic data sets also present many
challenges.

Data handling
Next-generation sequencing techniques (Fig. 1)
generate large and complex datasets. Indeed, the
subsequent bioinformatic and statistical analysis
of these datasets have become the bottleneck for
microbial community analyses [11]. Communities
are described in terms of richness (their constituents), evenness (dominance by particular OTUs),
and diversity (a combination of richness and evenness). Ordination analyses and constrained or
redundancy analyses allow exploration of potential
mediators of structure and changes [12], approaches
that are particularly valuable when attempting to
relate microbiomic data to the patients demographic and clinical details. New methods of handling and interpreting such complex datasets are in
evolution [13 ].
&&

Molecular techniques
Culture independent analysis exploits the presence
of the ubiquitous and evolutionarily conserved 16S
ribosomal RNA (16S rRNA) gene, by which prokaryotic taxa can be differentiated. Molecular analyses
are facilitated by recent work showing that once
sampled, the stool microbiome is stable at room
temperature for several weeks [7]. Samples undergo

HOSTMICROBIOME INTERACTIONS
The neonatal period uniquely challenges the host:
pioneering organisms are encountered by nave host
gut defences, educate and modulate immune
responses, and thus regulate the microbiome development. The bacterial community structure and
host defences modulate gut endothelial cells,

First generation techniques: separation by physical means using denaturing or

temperature gradient gel electrophoresis (D/TGGE) - fingerprinting techniques
Next generation sequencing (NGS): combinations of enzymology, chemistry, highresolution optics, hardware, and software engineering. Generate enormous amounts of
data and sequence to great depth - examples: 454 pyrosequencing (Roche), HiSeq and
bench top MiSeq (Illumnia) platforms. The MiSeq was recently found to have the
highest throughput and lowest error rate of its type [8].
Third generation platforms: capable of generating reads greater than 400 base pairs,
facilitating identification to species level.

FIGURE 1. Sequencing techniques.

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Paediatric and neonatal infections

modifying function, enhancing protection and

maintaining barrier function. Careful regulation
of pro- and anti-inflammatory effects is needed to
maintain health [14]. Recent mechanistic insights
are outlined below.

Competitive evolutionary strategies

Studies in a murine model suggest direct pathogen
control occurs through commensal bacteria controlling virulence factor expression in pathogenic bacteria, and by competition modulated through
shared carbohydrate requirements [15 ]. Environmental acidification by Bifidobacteria as a means
of controlling Escherichia coli has also been demonstrated, a mechanism of potential import for probiotic reduction of incidence of NEC [16].
&

Barrier integrity
The traditional top-down hypothesis of a breach of
barrier integrity, with breakdown in endothelial
tight junctions leading to loss of regulation of factors intrinsic to a balanced pro- and anti-inflammatory response has recently been challenged. An
alternative hypothesis, that of Paneth cell dysregulation, has been proposed that challenges many
traditional assumptions [17 ]. The antibacterial
lectin RegIII gamma has been shown to modulate
both total bacterial load and contact of luminal
bacteria with host epithelium [18].
&&

shown to induce extrathalamic Treg differentiation

[22 ,23 ].
&

&

DEFINING THE HEALTHY NEONATAL GUT

MICROBIOME
A healthy gut microbiome is the intestinal
microbial community that assists the host to maintain a healthy status under certain environmental
conditions [24]. If this could be defined for the
neonate, a comparison with disease states could
be made, allowing insights into pathogenesis or
guiding preventive or treatment options.

Term neonates
The full-term vaginally delivered breast-fed (FTVDBF)
infant is considered ideal from a microbiomic
perspective and was initially studied in 14 infants
[25]. The more comprehensive Norwegian Microflora
Study (NOMIC) enrolled 246 term infants at day 4,
10, 30, and 120 of life and showed links between early
colonization patterns and later growth patterns
[26 ]. Specifically, Bacteroides appeared protective
against obesity [27]. Three phyla Bacteroidetes,
Proteobacteria, and Firmicutes exert the greatest
effects on community dynamics [4,28]. Bifidobacterium appears specifically important to the neonate:
six species in breast milk showed properties expected
of commercial probiotics [29], and specific probiotic
formulations for preterm infants could be formulated
with this knowledge [30].
&&

Host immunomodulation
Several mechanisms have recently been demonstrated through which intraluminal bacteria modify
host responses. Initial neonatal bacterial exposure
within the gut leads to activation of Toll-like receptor (TLR)-4, which has been shown to activate an
inhibitory loop through the expression of miR-146a
(a small noncoding mRNA), affecting the response
to TLR agonist [19 ]. Some Bifidobacterium bifidum
can generate a proinflammatory Th17 immune
response (involved in protection against mucosal
infection), and functional Treg lymphocytes with
plasticity for either secreting IL-17 or suppressing
activation of the immune system, depending on the
external stimuli [20]. Polysaccharide A (PSA), a
specific symbiosis factor produced by the commensal Bacteroides fragilis, uses TLR-2 receptor activation
to activate Foxp3 Treg cells to promote tolerance
in a mouse model. PSA-lacking B. fragilis cannot
promote this response, clearly demonstrating how
specific bacterial products of symbionts manipulate
host immune responses [21]. Butyrate, a short-chain
fatty acid (SCFA) produced by commensal microorganisms during starch fermentation, has also been
&&

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Preterm infants
Neonates born preterm are exposed to potentially
detrimental factors in the development of the
healthy microbiome, including prelabour rupture
of membranes, caesarean delivery, maternal and
neonatal antibiotic intervention, and lack of
breast-feeding. Previous studies, although limited,
showed reduced diversity and relative instability
[31] (Fig. 2). Recently Arboleya et al. [32 ] studied
21 preterm infants (32 weeks mean gestation) and
20 FTVDBF infants: the aim was to establish how
differently 18 microbial groups were represented,
and SCFAs were also measured reflecting microbiome metabolic activity. Increased facultative
anaerobic microorganisms including Enterobacteriaceae, Enterococcaceae, and Lactobacillus and
fewer anaerobes, including Bifidobacterium, Bacteroidetes, and Atopobium, were seen in the preterm
cohort. The authors hypothesize that this delay in
aerobic establishment may delay immune maturation in preterm neonates. Further work in an
entirely preterm cohort exploring the development
of the gut microbiota from multiple births also
&

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The neonatal bowel microbiome Berrington et al.

(a)
Diversity

Healthy preterm

Diseased preterm

(b)

Age

not seen in healthy controls [35]. A potential

signature seen more frequently in NEC was identified as grouping to the Enterobacteriaceae family,
but did not match any sequence at greater than
97%. Likewise, a case report of a single preterm
infant (25 weeks gestation) who developed lethal
NEC on day 48 with a dramatic increase in Proteobacteria, specifically Klebsiella, 2 days before. Proteobacteria contributed greater than 97% of phyla
compared with less than 50% before. Ileal fluid and
mucosal samples also identified the same pattern
[36 ]. Jenke [37 ] identified an increase in E. coli
preceding NEC in comparison both to earlier
samples from the same individual and to healthy
preterm infants. Fecal inflammatory markers were
also studied: S100A12 and hBD2 were seen to correlate with total bacterial counts and E. coli, linking
microbiomic changes and host immune responses.
Two potentially distinct patterns of dysbiosis were
identified by Morrow et al. [38 ] in 11 NEC infants:
an early presentation (n 4) dominated by Firmicutes, and a later presentation dominated by Proteobacteria. In our own cohort of infants less than
32 weeks gestation, distinct microbiomic differences
between NEC and/or LOS compared with healthy
infants were observed [33 ]. Detailed examination of
twins discordant for NEC demonstrated a reduction
in diversity and increasing dominance of Escherichia
(since identified as E. coli) preceding NEC [39 ].
Preterm infants, whose exposure to lipopolysaccharide (LPS) from nosocomial bacteria is modified by
neonatal intensive care practices, may be less tolerant when they do meet these bacteria and mount a
resultant excessive inflammatory response. Interestingly, a Danish group did not identify microbiomic
shifts in association with NEC using molecular
analysis, although the timing of the sampling in
relation to NEC development in this cohort is not
described, and is clearly key [40]. This group also
analyzed small intestine specimens resected surgically in infants with NEC, demonstrating technical
feasibility, but no specific profiles were identified
associated with NEC [43].
&

Diversity
Healthy term

Healthy preterm

Age

&&

&&

FIGURE 2. Schematic diagram to demonstrate the typical

patterns in diversity in (a) healthy preterm infants compared
with preterm infants diagnosed with disease and (b) healthy
term infants compared with healthy preterm infants.

&

&

found Bifidobacterium abundance to be low [33 ].

Lower levels of naturally occurring probiotic species
in preterm infants are important, given the possibility of supplementing these for the prevention of
NEC [34].

SPECIFIC INFECTIOUS DISEASES

Dysbiosis appears pivotal to both LOS and NEC
(Fig. 2). Patterns are beginning to emerge that
appear associated, but causality is not proven.

Necrotizing enterocolitis
NEC, an important neonatal disorder, is increasingly recognized as a spectrum of disease culminating in a final common pathway.
Bacterial elements
Is there a signature microbial pattern associated
with NEC? If so, does it predate NEC onset to allow
early diagnosis or modification of practice to
improve outcomes?
Recent studies addressing these questions are
shown in Table 1, highlighting populations, molecular techniques employed, and key findings. A
pyrosequencing study of nine preterm infants with
NEC sampled before NEC diagnosis and within 72 h
of diagnosis identified a bloom in Proteobacteria
and a decrease in Firmicutes between sampling,

&

Nonbacterial elements
Increasingly the role of nonbacterial organisms is
being recognized in disease etiology.
Archaea
Palmer [25] identified archaea in infants gut, but
considerably less frequently and more variably than
fungi or bacteria, appearing only transiently in the
first few weeks of life. In inflammatory bowel disease
(IBD), the presence of methanogenic archaea in the
human gut was indicative of a healthy microbiota,
with reduced methanogen presence in individuals

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Paediatric and neonatal infections

Table 1. Recent microbiomic analysis in preterm infants with necrotizing enterocolitis or sepsis
Population gestational
age (weeks) (n) of
whom illness (n)
<32 (38) NEC (7) LOS
(13)

Technique
DGGE

Sampling
strategy

Key findings

Author [ref]

Opportunistic surveillance
study, n 99

Enterococci associated with

health

Stewart et al. [33 ]

&

Enterobacter associated with NEC

Staphylococcus associated with
LOS
<32 (18) NEC (9)

454 Flex chemistry

310 days before NEC

and within 72 h of NEC
onset

Bloom in Proteobacteria and a

reduction of Firmicutes in
NEC

Mai et al. [35]

Gammaproteobacteriacea family
signature for NEC
25 (1) NEC (1)

Pyrosequencing
(bTEFAP)

Weekly

Decreased diversity and

Proteobacteria (specifically
Klebsiella) dominant 2 days
before NEC

Tran et al. [36 ]

<27 (68) NEC (12)

qPCR

Alternate days for 4 weeks

Increase in E. coli before NEC

Jenke et al. [37 ]

&

&&

Associated increase in fecal

s100A12 and hBD2
<29 (32) NEC (11)

Pyrosequencing

Day 49 and day 1016

urine for metabolomics
(NMR)

Two patterns of dysbiosis noted

associated with NEC:
Firmicute dominant
(Staphylococcus and
Enterococcus)
Proteobacteria dominant
(Enterobacteriaceae)

Morrow et al. [38 ]

&&

(Urinary alanine increased in

group 1)
<32 (32) NEC (7) LOS
(13)

DGGE 16S rRNA

and 28S rRNA

First and weekly

Sphingomonas associated with

NEC

Stewart et al. [39 ]

<30 (163) NEC (21)

DGGE

Three samples (day 05,

10, and 30)

No differences between NEC and

non-NEC identified but no data
on sample timing relating NEC

Smith et al. [40]

Pretem (6) LOS (2), 2 well,

2 culture negative

Pyrosequencing

First, weekly, and at sepsis

evaluation

Increased Firmicutes
(Staphylococcus) in LOS

Madan et al. [41 ]

<32 (28) LOS (10)

Pyrosequencing

Weekly

Lower Bifidobacteria in cases

Mai et al. [42 ]

NEC (24)

FISH

Surgical resection
specimens

Proteobacteria (49%) Firmicutes

(30%)

Smith et al. [43]

&

&

Postulate healthy microbiome

&

DGGE, denaturing gradient gel electrophoresis; FISH, fluoresecent in-situ hybridization; LOS, late onset sepsis; NEC, necrotizing enterocolitis.

with IBD [44]: their absence from neonates may thus

be important and mark dysbiosis.
Viruses
Viruses may contribute to NEC by direct host viral
infection or by phage infection of a microbiomic
constituent. Torovirus [45] and astrovirus [46] have
been previously linked to NEC onset, but recently
there has been a resurgence of interest in cytomegalovirus (CMV). CMV was reported in association
with fulminant lethal NEC in a 25-week-gestation
infant: post-mortem inclusion bodies prompted
recognition of CMV in the intestine [36 ]. An
&

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associated bloom in Proteobacteria was noted:

CMV may have contributed to direct mucosal injury
(seen in patients with IBD and CMV) or have promoted a microbiomic shift that heralded NEC. Deep
sequencing of viral RNAs and DNAs revealed that
many viruses in the infant gut are bacteriophages,
although less diversity is seen in infants [47]. Phages
may influence microbiome composition by infecting and lysing specific bacterial hosts, allowing
another bacterial strain the opportunity to become
abundant [48]. Although identified as present in the
neonatal gut they have not been studied specifically
in the context of NEC or LOS [47].
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The neonatal bowel microbiome Berrington et al.

Fungial
LaTuga et al. [47] extended microbiomic analysis
beyond the bacterial elements in their study of
11 infants less than 1000 g in their first month, five
of whom had coagulase-negative staphylococci
infection and two of whom developed NEC (but
only one within the sampling window). They identified fungal species including several Candida
species despite nystatin prophylaxis, a finding confirmed by Stewart et al. [49 ] who identified 12 fungal
species (all nonviable) in their cohort who received
fluconazole prophylaxis. The role of fungi in NEC
pathogenesis requires further elucidation.
&

INFECTION
There are many parallels between LOS and NEC. Gut
dysbiosis with a preponderance of Proteobacteria
and Firmicutes abundance has been noted in
neonates with LOS. Altered barrier and immune
function and imbalance between pro- and antiinflammatory responses have also been implicated.
Mai et al. [42 ] analyzed 10 preterminfants with LOS
and 18 preterm controls, showing association
between microbiomic signatures in the two weeks
before diagnosis of LOS but interestingly not at
diagnosis. A high-resolution study of six preterm
infants showed differences in microbial patterns
in the first weeks of life in infants at high risk
&

of sepsis: less diversity and a preponderance of

staphylococci [41 ]. Archaea, viruses, and fungi in
the gut may also influence LOS, but as with NEC,
studies to date do not adequately analyze these
domains in the pathogenesis of sepsis.
&

THERAPEUTIC INTERVENTIONS
Interventions that affect the microbiome affect
health: prolonged early postnatal antibiotics
increase the risk of NEC [50], oral antibiotics appear
to reduce it [51], probiotics can reduce NEC [52],
lactoferrin appears to reduce LOS and possibly NEC
[53,54]; however, mechanistic understanding for
these findings is lacking.
However, clinical interventions could theoretically be manipulated and tailored to engineer a gut
microbiota reflective of an FTVDBF infant. Breast
milk contains complex immune-protective and
growth factors, bioactive immune-modulatory cells
and other immunonutrients including amino acids,
fatty acids, lysozyme, lactoferrin, minerals, and
metals such as zinc, and prebiotic oligosaccharides
as well as live bacteria. It seems unlikely that artificial supplements could ever replicate the complexity of own mothers breast milk. Manufacturing
complex combination supplements is attractive,
but a better understanding is needed before this
would be feasible. Few clinical trials in neonates

Is dysbiosis of the microbiome cause or effect of NEC and LOS?

What is the contribution of the host genetic background to
alterations to the microbiome in disease states?
Can aspects of clinical care be manipulated to engineer a gut
microbiota reflective of a FTVDBF infant?
Can the host immune response be selectively enhanced to promote
the growth of organisms seen in health states rather than in disease
pathophysiology?
Can probiotic use, dose, strain, and duration be realistically
optimized given the number of options and babies available to
study?
What is the role of prebiotics either instead of probiotics or in
combination (synbiotics)?
Can intelligent use of antibiotics help harness the microbiome to
promote health?
What role do non-bacterial members have individually and in
community interactions with other microorganisms?
How are vast amount of data best presented, analysed and made
publicly available? Can data from different studies be meaningfully
meta-analysed or visually presented?

FIGURE 3. Key unanswered questions and challenges FTVDBF, full-term vaginally delivered breast-fed; LOS, late-onset sepsis;
NEC, necrotizing enterocolitis.
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Paediatric and neonatal infections

have attempted mechanistic evaluations: few probiotic studies assess the microbiome it is hypothesized but unproven that probiotics result in a
reduction in the growth of potential pathogens
including enterobacteria, enterococci, and clostridia
[55]. A serendipitous increase in Lactobacillus spp. in
preterm infants was associated with 1% lactulose
added to feeds, potentially attributable to its prebiotic effects [56]. Lactoferrin, a molecule with
many mechanistic effects on the microbiome, has
not been subject to detailed exploration of the
effects on the preterm infant microbiome [57].

CONCLUSION
Understanding the neonatal microbiome may hold
the key to preventing LOS and NEC. Although
patterns are emerging, the causal pathway is
unclear. Where interventions do reduce morbidities (e.g., probiotics), the mechanisms are poorly
understood, and key questions remain unanswered
(Fig. 3). Mechanistic evaluation in the context of
controlled clinical trials would help elucidate this,
and may provide insight that allows improved prevention, diagnosis, and treatment. The inclusion of
microbiomic assessment, including bacterial and
nonbacterial elements and noninvasive sample
collection, in future clinical trials of microbiomic
modulation deserves further attention. This could
include trials of feeding, probiotics, and enteral
or immunonutrient supplements. Key unknowns
about host response should also be addressed. Little
is known about gut immune tissue responses to NEC
in humans, yet many infants have resection of gut
as part of their treatment, and immunohistochemistry techniques now allow elucidation of these
aspects.
Acknowledgements
None.
Conflicts of interest
There are no conflicts of interest.

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manipulation or supplementation of bacterial products.

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