FOCUS ON GUT MICROBIOTA

Microbial contact during pregnancy, intestinal

colonization and human disease
Samuli Rautava, Raakel Luoto, Seppo Salminen and Erika Isolauri
Abstract | Interaction with colonizing intestinal bacteria is essential for healthy intestinal and immunological
development in infancy. Advances in understanding early hostmicrobe interactions indicate that this early
microbial programming begins inutero and is substantially modulated by mode of birth, perinatal antibiotics
and breastfeeding. Furthermore, it has become evident that this stepwise microbial colonization process,
as well as immune and metabolic programming by the microbiota, might have a long-lasting influence
on the risk of not only gastrointestinal disease, but also allergic, autoimmune and metabolic disease, in
later life. Modulating early hostmicrobe interaction by maternal probiotic intervention during pregnancy
and breastfeeding offers a promising novel tool to reduce the risk of disease. In this Review, we describe
the current body of knowledge regarding perinatal microbial contact, initial intestinal colonization and its
association with human disease, as well as means of modulating early hostmicrobe interaction to reduce the
risk of disease in the child.
Rautava, S. etal. Nat. Rev. Gastroenterol. Hepatol. 9, 565576 (2012); published online 14 August 2012; doi:10.1038/nrgastro.2012.144

Introduction
Our understanding with regard to the influence of
microbes on human health has gradually expanded
from pathogens causing infectious disease to a mutu
ally beneficial interaction with indigenous micro
organisms that contribute to normal human physiology
and immune homeostasis. That gastrointestinal dis
orders, such as IBD or infectious gastroenteritis, are
associated with perturbations of gut microbiota com
position (discussed elsehwere1) is now well established.
The vital role of contact with environmental and colo
nizing bacteria in early infancy for healthy immune
and metabolic maturation beyond the gastrointestinal
tract has been brought to our attention during the past
two decades.
Advances in elucidating early hostmicrobe interac
tions identify two hitherto largely unknown areas of
interest. First, groundbreaking experimental and clini
cal studies suggest that microbial contact inutero and
during the neonatal period, modified by mode of deliv
ery and breastfeeding, exert marked effects on immune
and metabolic programming in the fetus and infant.2,3
Second, reports from clinical studies indicate that early
microbial contact has a long-term influence on intes
tinal colonization patternsthat is, microbiological
programming (Box1)and so, consequently, on the
immune responder phenotype, metabolic status and
risk of disease later in life.48 In our opinion, the concept
of early origins of human disease should, therefore, be
revised to include the multiple interactions between
maternal environmental and indigenous microbes
Competing interests
The authors declare no competing interests.
during pregnancy and birth, and their association
with mode of delivery, perinatal antibiotic exposure,
breast-milk composition and immune physiology in
the mother, fetus and infant.
In this Review, we describe the current body of knowl
edge regarding the importance of perinatal microbial
contact to health and disease in the gut, and the body as
a whole. We also suggest that modulating the complex
network of prenatal and postnatal metabolic, immuno
logical and microbiological interactions with specific
probiotics could provide a powerful tool to reduce risk
of disease in the child.
Development of intestinal microbiota
Microbes are present in all natural environments.
The fact that the most predominant species (such as
Bifidobacterium spp.) present in the indigenous intes
tinal microbiota of human infants911 are not found in
substantial quantities in the environment suggests that Department of
Pediatrics, Turku
infants acquire their commensal bacteria from other University Hospital,
humans, presumably, and most importantly, from the Kiinamyllynkatu 48,
20520 Turku, Finland
mother. Conventionally, the human fetus has been (S. Rautava).
considered sterile and microbial colonization has been Department of
thought to begin during birth and then develop under Pediatrics, University of
Turku, Kiinamyllynkatu
the influence of breastfeeding and skin-to-skin contact 48, 20520 Turku,
with the mother. Contact with the enormous bacterial Finland (R.Luoto,
E.Isolauri). Functional
load of the extrauterine world during and after birth Foods Forum, University
marks the beginning of massive bacterial colonization of of Turku, Itinen
mucosal surfaces, particularly the gastrointestinal tract. Pitkkatu 4A, 20520
Turku, Finland
Consistent with this notion, lactobacilli residing in the (S.Salminen).
birth canal need to be characterized as they might be one
Correspondence to:
important source of intestinal microbes during the first E. Isolauri
days of life.1214 erika.isolauri@utu.fi

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY VOLUME 9 | OCTOBER 2012 | 565

2012 Macmillan Publishers Limited. All rights reserved
REVIEWS
Key points
Contact with microbes begins inutero and proceeds in a stepwise manner
during birth and early infancy
Early hostmicrobe interaction is a crucial component of healthy immune and
metabolic programming
Mode of delivery, prematurity, perinatal antibiotic exposure and breastfeeding
have a major influence on early microbial contact, intestinal colonization and
subsequent risk of disease
Maternal probiotic supplementation during pregnancy and breastfeeding
has shown potential in reducing the risk of immune-mediated and metabolic
disease via modulation of early hostmicrobe interactions
Box 1 | Definitions of key concepts in early microbial colonization
Programming
Early life exposures to microbiota partially determine adult phenotypes. In the
case of human disease, programming refers to the risk of developing disease
generated at an early age and is, therefore, not strictly deterministic. The
programming factors, such as contact with specific microbes, are components of
causal complexes and therefore nonsufficient and non-necessary causal factors.
Stepwise colonization
Successive steps occur in the microbial colonization of the infant gut. Colonization
takes place in a stepwise succession of microbiota components and in major
steps at birth (delivery) and early life following delivery, during breastfeeding and
first contact with the environment, and finally during and after weaning.
Microbial contact
Direct interaction with a microbe or microbial component that results in a
response in the host. Contact with a microbe can result in an inflammatory
response with potentially detrimental consequences (as in the case of
infection) or evoke a tolerogenic immune response resulting in withholding from
inflammatory activation.
Nutrition
Breastfeeding exerts a major influence on gut coloni
zation patterns in early infancy. The gut microbiota of
breastfed infants has been reported to be dominated by
bifidobacteria, whereas formula-fed infants harbour
a more diverse microbiome.9 By contrast, Penders and
colleagues15 examined the gut microbiota composition in
50 breastfed and 50 formula-fed infants and found that,
on the species level, all infants were colonized by bifido
bacteria with no differences in prevalence or the amount
of bifidobacteria between the groups.15 The same investi
gators later analysed faecal samples obtained from >1,000
infants at 1month of age. No differences between bifido
bacterial colonization were detected between breastfed
and formula-fed infants, but the exclusively formula-fed
infants were more often colonized with Escherichia coli,
Clostridium difficile, Bacteroides spp. and lactobacilli.16
The authors concluded that vaginally born, full-term
infants who were exclusively breastfed were most likely
to have the highest numbers of intestinal bifidobacteria.
In a 2010 study, seven breastfed and seven formula-fed
infants were followed until the age of 18months; faecal
samples obtained at >20 time points over the course of
18months were analysed.10 During exclusive breastfeed
ing, bifidobacteria dominated the gut microbiota with
a >50% share of total bacterial counts whereas, over
the same time period, only ~25% of gut microbes were
bifidobacteria in formula-fed infants. The breastfed
infants in the study received breast milk until the age of
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2012 Macmillan Publishers Limited. All rights reserved
615months and harboured a more complex composi
tion of Bifidobacterium spp. than formula-fed infants.11
Moreover, the differences in faecal Bifidobacterium spp.
composition between breastfed and formula-fed infants
seemed to remain after cessation of breastfeeding.10,11
Development of enterotypes
Our knowledge of the human microbiome composition
and activity has increased rapidly. Infants have been
reported to harbour a heterogeneous microbiota that
is prone to compositional changes caused by environ
mental disturbances such as the use of antibiotics. 17,18
Infants develop a distinctive microbiota that is more
individual and open to influence by life events than
that of adults; in adults, three distinct and predomi
nant microbiota enterotypes have been proposed. 19
What underlies the formation of these enterotypes is
unknown and the early development of enterotypes in
infants is not yet clear,19,20 but the somewhat stable indi
vidual gut microbiota is thought to develop according
to modifications by dietary and environmental factors
during infancy. A stepwise developmental trend towards
an overall more adult-like profile seems probable.
The first step comprises early colonization by mainly
Firmicutes, including aerobic or facultatively anaerobic
bacteria (for example, Staphylococcus, Streptococcus and
Enterobacteriaceae). These early colonizers are then
followed by Actinobacteria as well as more anaerobic
bacteria, which typically include bifidobacteria espe
cially in breastfed infants.911 The next succession step
is the increase in the Bacteroidetes phylum of bacteria
and other anaerobes, such as Clostridia and Eubacteria,
appear after the increase in Bacteroidetes.17,18 By 1year
of age, the infant microbiota is thought to begin to con
verge toward a profile that resembles that of adults, being
established at about 23years of age.17,18 Nonetheless,
our understanding of the dynamics of gut microbiota
composition is still fragmentary and it is evident that
essential changes in physiological processes and dietary
and lifestyle exposures, which affect gut microbiota
composition, take place beyond this age.
Preterm infants
Bacterial colonization of preterm infants is an important
clinical and scientific issue as ~513% of infants are born
prematurely.21 Preterm infants are not fully developed
to cope with the massive postnatal microbial contact
and colonization that arises after birth. Furthermore,
as survival of extremely low-birth-weight or premature
infants was rare before the advent of modern neonatal
intensive care, our knowledge of the normal or physi
ological microbiota for such infants hardly exists. Factors
that can disturb normal postnatal intestinal microbial
c olonizationincluding caesarean section delivery,
broad-spectrum antibiotic use, parenteral nutrition and
delayed onset of breastfeeding, and reduced skin-to-skin
contact (discussed below)often cluster in very low-
birth-weight infants (<1.5kg) and premature infants.
To date, little is known about the compositional
development of intestinal microbiota in preterm infants.

An early study has indicated that there is delayed colo
nization by Bifidobacterium spp. as compared with
colonization in full-term infants. 22 Although not all
subsequent studies have corroborated this finding,23
it seems that the premature gut is not yet fully devel
oped for optimal bifidobacterial colonization.24 A 2011
study assessing faecal microbiota by novel pyrosequenc
ing techniques in serial samples obtained from 10 very
low-birth-weight preterm infants revealed that gut colo
nization in these preterm infants is a dynamic process
involving a large number of bacterial species, of which
~20% are currently not classified.25 Nonetheless, several
investigators have reported that the gut microbiota in
preterm infants has low diversity when compared with
that from full-term infants.26,27 Even lower diversity has
been detected in preterm infants who develop necrotiz
ing enterocolitis, a devastating immunoinflammatory
intestinal process often resulting in death or severe
impairment.26,28 Despite extensive studies of samples
obtained before the onset of necrotizing enterocolitis,
however, no single organism or distinct bacterial colo
nization patterns that would be causative or predictive
of necrotizing enterocolitis have been identified, even
though organisms such as Clostridium perfringens or
members of Enterobacteriaceae have been implicated.29,30
Immunological and metabolic maturation
Interaction with colonizing microbes is essential for
healthy immune maturation and development of tolerance.
Our core understanding of early hostmicrobe interactions
is mainly derived from innovative studies conducted using
germ-free or gnotobiotic animals. These findings should,
however, be interpreted with some caution, bearing in
mind the limits of these reductionist approaches when
considering the complex interactions between microbes,
the host and dietary and other environmental factors in the
infants gastrointestinal tract.
As early as 1997, Sudo and colleagues31 showed that
germ-free animals fail to develop immune tolerance, and
that tolerance is achieved after monocolonization with
the commensal Bifidobacterium infantis in the neonatal
period, but not if B.infantis was introduced at a later
age (after the neonatal period). Unfortunately, whether
the effect is specific to this bacterium is not certain as,
to our knowledge, data regarding other commensal
bacteria introduced in the same experimental design
have not been reported. Early microbial colonization
has subsequently been demonstrated to have persistent
effects on host immune phenotype.32 Advances in experi
mental research suggest that innate immune receptors
such as Toll-like receptors (TLRs) are key mediators of
the communication between the host and colonizing
microbes that leads to tolerance.33 Transient immune
activation and subsequent immune maturation medi
ated by TLR-dependent mechanisms can be induced in
germ-free animals by colonization with the single spe
cific commensal microbial strain, Bacteroides fragilis, or
even by administration of a specific commensal surface
molecule such as the B.fragilis surface polysaccharide
PSA.3436 In a parallel manner, stimulation of human
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2012 Macmillan Publishers Limited. All rights reserved
FOCUS ON GUT MICROBIOTA
intestinal epithelial cells with bacterial TLR ligands has
been observed to initially activate proinflammatory
pathways, but after continuous exposure lead to a state
of hyporesponsiveness.37 The importance of gut microbes
and TLR signalling to the establishment and mainte
nance of immune tolerance is further demonstrated in a
report that indicates that food allergy can be induced in
mice whose gut microbiota has been reduced using anti
biotics.38 Moreover, similar susceptibility to sensitization
to food allergens has been observed in animals in which
signalling from intestinal microbes is lacking as a result
of deficient TLR4 function.38
The wide range of intestinal physiology affected
by microbial colonization is demonstrated by a study
in which monocolonization of germ-free mice with
Bacteroides thetaiotaomicron, a prominent member
of the normal mouse gut microbiota, leads to major
changes in the expression of genes involved in nutri
ent absorption, mucosal barrier function, metabolism,
angiogenesis and maturation in intestinal epithelial
cells.39 The indigenous gut microbiota has come to be
seen as a key organ involved in host energy homeo
stasis, affecting the harvest and storage of energy from
the diet.40 Pioneering studies using experimental animal
models have provided evidence of metabolic activity of
the gut microbiota that facilitates the extraction of calo
ries from ingested dietary substances and their storage
in host adipose tissue for later use.40,41 Following obser
vations of characteristic alterations in the gut microbiota
composition of obese compared with lean mice,42 the
transferable nature of the obese phenotype by trans
plantation of obese microbiota to germ-free mice has
been recognized. 43 Collectively, these experimental
data suggest that early interaction with indigenous
microbes is essential for healthy immunological and
metabolic programming.
Microbial programming of host health
Epidemiological and intervention studies have high
lighted analogous microbial influences in diverse clini
cal conditions. Allergic disease and obesity, as well as
chronic inflammatory diseases such as coeliac disease
and IBD, share common environmental risk factors and
immunological mechanisms, and frequently coexist in
the same individual (reviewed in detail elsewhere44,45).
Consequently, early microbial contact has been sug
gested to be involved in the initiation and perpetua
tion of aberrant immune activation and responsiveness
central to the pathogenesis of allergic, autoimmune and
metabolic disease. Failure to maintain immune tolerance
towards indigenous intestinal microbes is a key element
of IBD.46 To take an example, caesarean section delivery
seems to increase the risk of coeliac disease,47,48 type1
diabetes mellitus49,50 and asthma,51,52 as compared with
vaginal delivery. These conditions, generally classified as
diseases associated with excessive or aberrant Thelper
(TH)1 and TH2 immune responsiveness, again, have been
associated with deviations in the compositional develop
ment of gut microbiota,1,44,5355 typified as a less diverse
microbiota specifically lacking bifidobacterial species
REVIEWS
Transfer of
bacteria
Placenta and
amniotic fluid
Mode of
delivery
Breast milk
Maternal gut
Infant gut
Figure 1 | The gut microbiota programmes host healthfrom mother to infant. The
fetus comes into contact with microbes originating in the maternal gut through the
placenta and amniotic fluid. A massive inoculum of maternal bacteria is
encountered during vaginal delivery, which is also associated with increased
maternal intestinal permeability and translocation of gut bacteria into breast milk.
Breastfeeding not only modulates neonatal bacterial colonization and immune
maturation, but is a direct source of maternal bacteria. After birth, skin-to-skin
contact and nursing ensures direct transfer of maternal bacteria to the infant to
enhance healthy immune and metabolic maturation.
with simultaneous increases in specific clostridia.1,5456
Immunoinflammatory diseases mediated by pathological
TH1 and TH2 responses thus seem to have common roots
in early life, with perturbations in the intestinal microbial
colonization process leading to failure to establish and
maintain tolerance to environmental, dietary, microbial
or self antigens.
In a similar fashion, alterations in gut microbiota
composition have been detected between obese and lean
individuals.42,43,56 Obesity is associated with alterations
in the relative amounts of Bacteroidetes and Firmicutes,
and animal studies have demonstrated that a 20%
increase in Firmicutes and a corresponding decrease in
Bacteroidetes were associated with an increased energy
harvest.57 Early microbial colonization patterns might
not only be associated with, but also have a causal role,
in the development of obesity and metabolic disease,
as alterations in gut microbiota have been detected
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2012 Macmillan Publishers Limited. All rights reserved
during infancy in individuals who later become over
weight.6 Moreover, the indigenous microbiomes of obese
individuals are enriched in gene categories involved
in carbohydrate and lipid metabolism, 42,56 and some
experimental data indicate that gut microbes directly
influence energy harvest and storage by fermenting non
digestible carbohydrates, activating digestive enzymes
and modulating adipocyte function (for example, by
inducing the expression of fasting-induced adipocyte
factor).40 Finally, gut microbiota composition has been
suggested to be an instrumental component in initiating
the low-grade inflammatory state and, as a consequence,
insulin resistance associated with metabolic diseases par
ticularly among individuals predisposed to obesity.58,59
Hence, although the key cause of overweight in humans
is indubitably an imbalance between energy intake and
expenditure, it would seem that a combination of dietary,
environmental and genetic factors work in concert to
define the individual microbial composition, which
could favour either an obese or lean phenotype. Taken
together, it is evident that the nutritional status, immune
response and mode of delivery collectively interact with
each other, and all these elements are in turn influenced
by the gut microbiota (Figure1).
Early sources of microbial contact
During the past decade, it has become evident that
environmental influences during fetal development can
have long-lasting health consequences. Prenatal pro
gramming of human physiology as a result of nutritional
deficiency and intrauterine growth restriction has been
extensively investigated. In parallel, research advances
suggest that the intimate interaction between the host
and environmental and indigenous microbes begins
inutero. Maternal immune cells have been reported to
cross the placenta and modulate immune responses in
the human fetus.60 In addition, maternal immunological
and metabolic state during pregnancy can lead to inflam
matory immune activation in the placenta.61 This state
also modulates the initial microbial inoculum for the
newborn and breast-milk composition, thereby affect
ing the stepwise microbial colonization in early infancy.62
The complex interactions between microbes, inflamma
tory responses, dietary factors and immunological and
metabolic programming that influence the risk of disease
could thus partially have their origins in fetomaternal
hostmicrobe interactions (Figure1).
Microbial contact during pregnancy
From the point of view of evolutionary biology, the
dogma of the microbiologically and immunologically
naive fetal life seems bizarre, as mammals have evolved
in a world already inhabited by an enormous variety of
microbes. Perhaps more appropriately, it can be argued
that the intrauterine environment, which is protected
by maternal barriers against pathogenic invaders, offers
the embryo and fetus a controlled environment in which
there is limited and selective exposure to microbes
during the vulnerable periods of organogenesis and
early development.

It has gradually become evident that maternal micro
bial exposure during pregnancy can have implications
for infant health. Data from epidemiological studies
indicate that maternal exposure to farm animals, which
presumably results in higher maternal microbial contact
during pregnancy, protects the infant from develop
ing eczema, an immune-mediated skin condition.2
Moreover, decreased occurrence of eczema is associated
with altered expression of TLR genes at birth, which
suggests that maternal microbial exposure modulates
fetal immune physiology and points to a critical role
for innate immune function in the fetoplacental unit.2
In line with this notion, maternal mucosal contact with
Acinetobacter lwoffiiF78, a bacterium isolated from
cowsheds, modulates TLR gene expression in the pla
centa and decreases the risk of developing asthma in the
offspring in an experimental mouse model.3
Microbial contact inutero
In addition to the indirect modulation of fetoplacental
immune responses through maternal exposure to bac
teria, accumulating evidence points to direct intra
uterine contact with microbes or microbial components.
Bacteria are often present in the nonpregnant uterus.63
Live bacteria thought to originate both from the skin
and the gastrointestinal tract have also been discov
ered in nearly half of placentas obtained after extremely
premature delivery at 2328 weeks of gestation. 64
Chorionamnionitis is a major cause of premature birth,
accounting for ~2540% premature deliveries.21 Other
known risk factors for preterm birth include mater
nal IBD, bacterial vaginosis and periodontitis, 21,65 all
of which are associated with perturbations of mucosal
microbiota and inflammation. Even though periodon
tal disease associated with premature delivery has not
been observed to be related to intra-amniotic bacterial
translocation or inflammation,66 some data indicate that
streptococci, Porphyromonas gingivalis or Fusobacterium
nucleatum detetcted in amniotic fluid might have
oral origins.67,68
Bacteria in the placenta and amniotic membranes
can lead to premature birth, fetal demise and severe
neonatal and maternal infection. Invasion of the amni
otic cavity by bacteria such as Ureaplasma urealyticum
or Mycoplasma hominis markedly increases the risk of
premature rupture of membranes and preterm birth.69
Furthermore, vertical transfer of U.urealyticum fre
quently occurs70 and has been associated with increased
risk of bronchopulmonary dysplasia71 and necrotizing
enterocolitis in preterm infants.72 The presence of bac
teria in the placenta has also been related to an increased
risk of cerebral palsy in extremely preterm infants.73
These clinically important associations mean that
research efforts to understand bacterial presence in the
placenta and amniotic fluid have mostly concentrated
on women with imminent preterm delivery caused by
preterm premature rupture of membranes or preterm
labour. Of note, these conditions can be caused by, or
result in, bacterial translocation in the amniotic sack
and, therefore, determining the direction of causality
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2012 Macmillan Publishers Limited. All rights reserved
FOCUS ON GUT MICROBIOTA
or ruling out contamination by maternal bacteria is not
possible. Interestingly, however, live bacteria have also
been detected in 25% of placentas after caesarean section
delivery necessitated by pre-eclampsia at 2328weeks of
gestation.64 In line with this observation, bacteria have
been cultured in the chorionamnion of 15% of women
after caesarean section without rupture of membranes.74
Thus, data exist to suggest that traces of microbes are
detectable in the fetoplacental unit from healthy, term
pregnancies.7578 DNA from intestinal bacteria such as
bifidobacteria and lactobacilli has been found in term
placentas after both vaginal and caesarean section deliv
ery.75 In a similar fashion, microbial DNA is present at
term in fetal membranes without signs of inflamma
tion.76 Our data confirm these findings by demonstrating
the presence of bacterial DNAmost often belonging
to the common gut bacteria Lactobacillus spp. and
Bifidobacterium spp.in all placentas and 43% amniotic
fluid samples obtained after 29 sterile elective caesarean
section deliveries at term without signs of infection,
rupture of membranes or onset of labour.77 The pres
ence of bacterial DNA in infant meconium suggesting
prenatal origin has also been reported.78
Prenatal immune effects
At which point or how bacteria gain entry into the
amniotic cavity is not currently known, but entry could
take place by ascension from the birth canal, haemato
geneously through the placenta or by retrograde spread
via the Fallopian tubes.21 Data indicate that bacterial
DNA is frequently detected in the amniotic fluid as early
as midtrimester and is not always associated with adverse
outcome or inflammation.79 The consequences of this
asymptomatic prenatal microbial contact are only now
starting to be elucidated.
Bacteria in the placenta have been reported to induce
a systemic immune response in neonates after preterm
delivery.80 Moreover, the response induced seems to be
species specific with, for example, lactobacilli resulting
in a reduced inflammatory response in the infant.80 We
have observed that DNA from nonpathogenic bacteria
detected in the placenta is associated with alterations
in TLR-related innate immune gene expression in the
fetal gut.77 Maternal obesity, on the other hand, has
been observed to modulate innate microbial pattern-
recognition receptor gene expression in the placenta in
an experimental sheep model81 and is known to be asso
ciated with increased inflammatory mediator expression
in the placenta.61 Collectively, these emerging new data
suggest that manipulation of maternal microbial expo
sure during pregnancy might provide a novel means to
modulate early immune development and to reduce the
risk of immunoinflammatory or metabolic disease both
in the mother and the child (Figure2).
Antibiotics in the perinatal period
The use of antimicrobial agents is known to cause per
turbations in indigenous microbiota, particularly those
in the gut. Prenatal maternal antibiotics are used to
treat infections such as chorionamnionitis and also
REVIEWS

Delivery Extrauterine enviroment

Maternal factors Mode of delivery: Breastfeeding:
Maternal nutrition Microbiological factors Vaginal Exclusive breastfeeding
Metabolic state Enviromental and Assisted vaginal Formula feeding
Immune state indigenous microbes Caesarian section Microbes
Immune
and
Genotype Fetal life Birth Early infancy
metabolic
phenotype
Early programming
Figure 2 | Early programming of immune and metabolic phenotype. During pregnancy and early infancy, early programming
of immune and metabolic phenotypes occurs as a result of environmental and indigenous microbes that are modulated by
maternal state, mode of delivery and feeding practices. The intensity of the bold arrow reflects the window of opportunity to
modify the programming process to reduce the risk of disease in later life.

prophylactically to prevent infections in the neonate.
After birth, a large proportion of neonates are subjected
to antibiotic treatment for suspected sepsis. Whilst these
practices are life-saving in the case of disease, exposure
to antibiotics in the perinatal period could also disturb
healthy hostmicrobe interactions. In line with this
notion, broad-spectrum maternal perinatal antibiotic
therapy has been associated with an increased risk of
necrotizing enterocolitis 82,83 and cerebral palsy in the
infant.84 Postnatally, prolonged empiric antibiotic admin
istration for suspected sepsis when infection was not later
confirmed also reportedly increases the risk of death and
necrotizing enterocolitis in the preterm infant,85,86 but the
effects on long-tem outcomes have, to our knowledge,
not been reported. Antibiotic use in the first year of life
has also been associated with increased risk of IBD in
childhood.87 These potentially detrimental effects of peri
natal antibiotic use highlight the importance of microbial
contact during birth and in the neonatal period.
Breast milk modulates hostmicrobe crosstalk
During pregnancy, the fetus swallows only amniotic
fluid, which passes through the gastrointestinal tract,
whereas nutrition is provided parenterally by the mother
via the placenta and umbilical cord. The newborn infant
starts to feed during the first hours of life and the naive
gut has to rapidly adjust to nutrient uptake and contact
with a myriad of foreign antigens whilst simultaneously
being colonized by microorganisms. From a physiologi
cal and evolutionary point of view, breast milk serves
as the sole source of nutrition during the first months
of life and could also be a source of colonizing bacteria
for the infant gut. In addition, breast milk provides the
infant with important protection against disease during
the vulnerable neonatal period. The beneficial health
effects of breastfeeding range from a reduced risk of
necrotizing enterocolitis and infections of the gastro
intestinal and respiratory tracts in infancy to improved
cognitive development and decreased occurrence of
coeliac disease, type2 diabetes mellitus, obesity, hyper
cholesterolaemia and asthma in later life.88 Whereas
protection from infectious disease during breastfeed
ing can be explained by passive immunoprotection by
molecules such as IgA antibodies and oligosaccharides
in breast milk, the long-term health effects suggest active
programming of infant physiology, which could be medi
ated by modulation of infant immune responses and gut
microbiota composition.88
Due to its unique protein and oligosaccharide compo
nents, breast milk modulates the neonatal gut microbiota
composition by promoting the growth of bifidobacteria.88
Breast milk might also be a source of colonizing bacte
ria to the newborn infant (Table1).89,90 In addition to
providing a distinct inoculum for the stepwise micro
bial colonization process through physical contact with
maternal vaginal and intestinal bacteria, vaginal delivery
is associated with stress and hormonal changes during
labour, which influence maternal gut permeability
and neonatal immune physiology.91 Interestingly, the
importance of delivery mode on both gut microbiota
composition and infant immune responses is detectable
throughout the first year of life.92,93
We have observed that the microbial composition of
human breast milk is different after vaginal or caesar
ean section delivery.94 Milk samples from mothers who
had undergone elective, but not nonelective, caesarian
section contained a different and less diverse bacte
rial community than milk samples from mothers who
had undergone vaginal delivery. Mothers giving birth
by elective caesarean section had a marked composi
tional shift in the microbiotapresenting decreased
levels of Leuconostocaceae and increased levels of
Carnobacteriaceae, among otherscompared with
those who had a vaginal delivery.94 These observations
might be explained by a hypothesis according to which
the lactating mother introduces her gut microbiota to the
newborn infant. This theory has been proposed on the
basis of observations indicating that there is increased
bacterial translocation in the maternal gut during preg
nancy and lactation, resulting in the presence of bacteria
within dendritic cells in the mammary gland in experi
mental animals.95 In humans, maternal peripheral blood
mononuclear cells and cells in breast milk have also
been shown to contain bacteria and bacterial DNA.95 In
accordance with this hypothesis, the number and type
of bifidobacteria in maternal faeces and breast milk cor
relate with those in infant faeces,90,96 and breast milk and
infant faeces consistently share strains of staphylococci,
bifidobacteria and lactobacilli.97 The link between the
maternal gut and the mammary gland is also suggested
by a study showing that maternal consumption of the
probiotic Lactobacillus reuteri is associated with recovery
of live L.reuteri in breast milk.98
Bioactive molecules in breast milk could substan
tially modulate interactions between the host immune
system and the developing gut microbiota. For instance,

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 FOCUS ON GUT MICROBIOTA

Table 1 | Characterization of bacteria present in breast milk

Type of bacteria, species or class Characterization methods Reference
Culture method Molecular method
Streptococcus spp., Staphylococcus spp., Bacillus Yes NA Bjrkstn etal. (1980)137
spp., Escherichia coli and Enterobacter spp.
Staphylococcus epidermidis, Enterococcus faecalis, Yes PCR, DGGE Martin etal. (2003)138
Streptococcus mitis, Propionibacterium acnes and
Staphylococcus lugdunensis
Staphylococcus aureus, Enterococcus fecalis, Yes, cultured PCR and/or RFLP Heikkil & Saris (2003)139
Streptococcus mitis, Lactobacillus rhamnosus, in MRS broth
L.crispatus and Leuconostoc mesentroides
Lactococcus lactis Yes PCR Beasley & Saris (2004)140
Lactic acid bacteria, Lactococcus lactis, NA Identification by PCRDGGE Martn etal. (2007)141
Leuconostoc citreum, Pseudomonas spp. and
Propionibacterium spp.
Bifidobacterium longum, B.breve, B.adolescentis NA qPCR Gueimonde etal. (2007)89
and B.animalis
Staphylococcus epidermidis, Enterococcus spp., Yes Identification by PCRDGGE Delgado etal. (2008)142
Streptococcusmitis and other Streptococcus spp.,
Corynebacterium spp., Klebsiellaoxytoca and
unculturable bacteria
Staphylococcus epidermidis, Enterococcus faecalis, Yes Genetic profiling Jimnez etal. (2008)143
Streptococcus mitis, Propionibacterium acnes and
Staphylococcus lugdunensis
Bifidobacterium breve, B.longum, B.adolescentis, NA qPCR Martn etal. (2009)144
B.bifidum and B.dentium
Carnobacterium spp., Veillonella spp., Leptotrichia NA Pyrosequencing and qPCR Hunt etal. (2011)145
spp., Prevotella spp., Lactobacillus spp. and
Bifidobacterium spp.
Staphylococcus spp., Lactobacillus spp. and Yes PCR, RAPD, PFGE, and/or Martn etal. (2012)97
Bifidobacterium spp. MLST genotyping
Staphylococcus spp., Streptococcus spp., Weisella NA Pyrosequencing and qPCR Cabrera Rubio etal.
spp., Leuconostoc spp., Veillonella spp., (2012)94
Leptotrichia spp. and Prevotella spp.
Abbreviations: DGGE, denaturing gradient gel electrophoresis; MLST, multilocus sequence type; NA, not applicable; PFGE, pulsed field gel electrophoresis;
qPCR, quantitative PCR; RAPD, random amplified polymeric DNA; RFLP, restriction fragment length polymorphism.

transforming growth factor (TGF)2, one of the most
comprehensively investigated immunomodulatory
factors in breast milk, is known to be necessary for
breast-milk-induced immune tolerance.99 Experimental
studies indicate that TGF2 at a concentration cor
responding to that in breast milk considerably alters
inflammatory responses, and that exposure to TGF2
induces immune maturation in the immature human
gut.100,101 The interactions between breast milk TGF2,
gut microbes and infant immune development are
further demonstrated by the observation that mater
nal consumption of the probiotic L.rhamnosusGG
during pregnancy and breastfeeding increases the con
centration of TGF2 in breast milk and is associated
with a reduced risk of atopic eczema in the infant.102
On the other hand, administration of the probiotic
Bifidobacterium breve to preterm infants modulates
their responsiveness to TGF, although the clinical
effects of this response are unknown. 103 On the basis
of these collective data, we hypothesize that breast
milk might function as a vehicle to introduce maternal
gut microbiota to the infant in a tolerogenic immune
milieu (Figure3).
Successful reprogramming by probiotics
The association between early microbial contact and
subsequent development of disease has spawned inter
est in the modulation of early hostmicrobe interaction
as a means to reduce the risk of disease. The potentially
harmful effects of caesarean sections (discussed earlier)
have prompted caution when choosing the optimal mode
of delivery. Data from well-conducted clinical studies
have brought to attention the adverse effects of perinatal
or prolonged neonatal empirical antibiotic administra
tion.8287 In addition to these potential changes in clinical
practice, novel interventions aiming to modulate early
hostmicrobe interactions have been introduced and
assessed in clinical trials.
Prebiotics are nondigestible food ingredients, the
purpose of which is to selectively promote the growth of
indigenous intestinal bacteria thought to be beneficial
to health. Accordingly, consumption of prebiotic oligo
saccharides reportedly increases the amount of faecal
bifidobacteria in infants.104107 Published data regard
ing clinical efficacy of prebiotics in infancy are still
sparse. Administration of a combination of prebiotic
galacto-oligosaccharides and fructo-oligosaccharides

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REVIEWS
Maternal gut Breast milk
Microbes TGF-2
Increased
responsiveness
Colonization Maturation Tolerance Inflammation
Infant gut
Figure 3 | Interactions between indigenous microbes and TGF2 in breast milk.
TGF2 is essential for breast-milk-induced tolerance towards environmental
antigens, attenuates excessive inflammatory responses in the neonatal gut and
induces gut maturation and production of IgA antibodies. These processes are
also dependent on the indigenous gut microbiota. According to a novel hypothesis,
maternal gut bacteria are actively transported into the mammary gland and
secreted in breast milk and are thus transferred to the infant in a tolerogenic
immune milieu with TGF2. Moreover, maternal supplementation with probiotic
lactobacilli is associated with increased levels of TGF2 in the breast milk and
bifidobacteria in the infant gut optimize responsiveness to breast milk TGF2.
Abbreviation: TGF, transforming growth factor.
(90% galacto-oligosaccharides and 10% fructo-
oligosaccharides as defined in the infant formula direc
tive in Europe) in infancy has been shown to modulate
antibody production and reduce the incidence of aller
gic outcomes such as eczema.108,109 Subsequently, a larger
multicentre trial of >800 infants demonstrated that oligo
saccharide-enriched infant formula might reduce the risk
of eczema in low-risk infants.110 However, oligofructose-
enriched infant cereal has failed to decrease the incidence
of diarrhoeal disease in a developing country setting 111
and both inulin and fructo-oligosaccharides have been
deemed to have no influence on the risk of antibiotic-
associated diarrhoea in children.112 It might be argued
that prebiotics only are effective if an appropriate com
position of bifidobacteria, which is amenable for specific
prebiotic enhancement, resides in the target gut.
The justification for the synbiotic approach, therefore,
lies in providing both the beneficial microbial stimulus
(probiotics) and their growth factors (prebiotics), thus
mimicking the net effect of breastfeeding. Our current
knowledge of the optimal prebiotic component for each
specific probiotic is fragmentary at best. Nonetheless,
published data are available indicating that supplemen
tation with synbiotics is associated with reduced inci
dence acute diarrhoeal disease,113,114 whereas data on the
effects of synbiotic combinations on the risk of eczema
have been conflicting.115,116 Dissecting the contribution of
the prebiotic and probiotic elements or their synergistic
influence in these studies is difficult.
The most substantial body of clinical evidence to date
regarding a reduction in the risk of disease in humans
572 | OCTOBER 2012 | VOLUME 9  www.nature.com/nrgastro
2012 Macmillan Publishers Limited. All rights reserved
within the context of modulating early microbial contact
relates to the use of specific probiotics. Even though this
approach is still experimental and the long-term effects
of modulating early microbial contactsome of which
might not be beneficialremain largely unknown,
specific probiotics can be considered a paradigmatic
example of an intervention aiming to modulate host
microbe interaction. Perinatal probiotic intervention has
been well tolerated and might influence early microbiota
composition and activity, but no interference with the
long-term composition or quantity of gut microbiota has
yet been reported. Although permanent establishment or
colonization by probiotics has not been observed,117120
the clinical health effects of probiotic intervention have
been shown to be long-lasting, consistent with the notion
of perinatal microbial programming.121123
Data suggesting that specific probiotics administered
in early infancy might enhance healthy immune matura
tion and reduce the risk of eczema in high-risk infants
are accumulating,115,121128 even though not all studies
have generated positive results.129131 On the basis of
published reports, it seems that probiotic intervention is
most effective if commenced prenatally.115,121,124128 One
putative mechanism for the protective effect postnatally
has been suggested to be increased concentration of
TGF2 in breast milk of women receiving probiotics.102
These observations emphasize both prenatal and post
natal interaction between maternal gut microbes and
infant immune system.
The discovery of the importance of prenatal micro
bial contact to fetal and infant development opens up
intriguing new possibilities for probiotic intervention
via the pregnant woman. In line with this notion, we
have observed altered innate immune gene expression
in the placenta and fetal gut after prenatal maternal
probiotic intervention.77 Perhaps more importantly, we
have reported that maternal supplementation with the
combination of the probiotics L.rhamnosusGG and
Bifidobacterium lactisBb12 from the first trimester of
pregnancy to the end of exclusive breastfeeding reduces
the risk of atopic sensitization in high-risk infants.131
Surprisingly, a statistically significant reduction of ges
tational diabetes mellitus was observed in women receiv
ing probiotics as compared with those on placebo in the
same study. 132 Furthermore, probiotic supplementa
tion resulted in consistently improved plasma glucose
concentrations and insulin sensitivity not only during
pregnancy, but also 12months postpartum among meta
bolically healthy women.133 The same study has provided
clinical evidence that probiotic consumption reduces the
risk of maternal central adiposity over the 6month post
partum period.134 Maternal obesity and glucose intoler
ance are known risk factors for overweight and obesity in
the offspring and, consistent with this evidence, we have
demonstrated that perinatal probiotic intervention mod
erates excessive weight gain especially among children
who later became overweight during the first years of
life, the effect being most pronounced at 4years of age.135
To our knowledge, no published trials have assessed
the efficacy of probiotics in the primary prevention of
 FOCUS ON GUT MICROBIOTA

gastrointestinal disease (such as IBD or coeliac disease)
in infants.
Conclusions
Our life-long coexistence with microbes seems to
begin earlier than conventionally thought (Figure1).
Microbial contact during embryogenesis and fetal life is
selectively buffered by maternal protective mechanisms.
Nonetheless, environmental and maternal indigenous
microbes, together with maternal nutritional and meta
bolic state, exert direct and indirect effects on the fetus,
which might be reflected in later health and disease.
Vaginal delivery seems not only to provide substantial
bacterial exposure, but also primes healthy hostmicrobe
interactions and immune development with hitherto
poorly understood effects on infant immunology and
microbial composition of breast milk. Establishment
of the indigenous microbiota in the perinatal period
and early infancy might be considered the last phase of
organogenesis, affecting not only gastrointestinal, but
also systemic, physiology. Similarly, breastfeeding can
be viewed as an extension of intrauterine maternal guid
ance and protection that facilitates survival and healthy
programming in the microbial world during early life.
The various components and functions of breast milk
underscore the complex interactions between contact
with microbes, diet and immune maturation, including
establishment and maintenance of tolerance towards
dietary antigens and indigenous microbiota.
Emerging understanding of the importance of micro
bial contact during the fragile periods of fetal life,
delivery and infancy to healthy immune and metabolic
programming creates new opportunities to improve
infant health and reduce the risk of disease in later life.
The notion of perinatal origins of human disease sug
gests an early window of opportunity (Figure2) to influ
ence disease risk and necessitates prenatal and perinatal
intervention to achieve the desired effects. Consistent
with this hypothesis, accumulating evidence indicates
that modulation of early microbial contact during preg
nancy and breastfeeding with maternal probiotic sup
plementation could be effective in reducing the risk of
immunoinflammatory and metabolic disease.
Review criteria
This Review article was created to give the reader a
comprehensive, but critical, view on the current state-of-
the-art on hostmicrobe interaction at the critical stage
of maturation including prenatal, perinatal and early
postnatal microbial exposure. The literature selection
was based on the personal experience of the authors
representing the different areas of the topic: immunology,
microbiology, food science, clinical paediatrics and
neonatology. The expertise of the authors derives from
pioneer research work in the field, as well as expert
activities with probiotics and health messages related to
functional foods.

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