The Journal of Maternal-Fetal & Neonatal Medicine

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Changes of intestinal microbiota in early life

Monica Ficara, Elisa Pietrella, Caterina Spada, Elisa Della Casa Muttini, Laura
Lucaccioni, Lorenzo Iughetti & Alberto Berardi

To cite this article: Monica Ficara, Elisa Pietrella, Caterina Spada, Elisa Della Casa Muttini,
Laura Lucaccioni, Lorenzo Iughetti & Alberto Berardi (2020) Changes of intestinal microbiota
in early life, The Journal of Maternal-Fetal & Neonatal Medicine, 33:6, 1036-1043, DOI:
10.1080/14767058.2018.1506760

To link to this article: https://doi.org/10.1080/14767058.2018.1506760

Published online: 10 Sep 2018.

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THE JOURNAL OF MATERNAL-FETAL & NEONATAL MEDICINE
2020, VOL. 33, NO. 6, 1036–1043
https://doi.org/10.1080/14767058.2018.1506760

REVIEW ARTICLE

Changes of intestinal microbiota in early life

Monica Ficaraa, Elisa Pietrellaa, Caterina Spadaa, Elisa Della Casa Muttini b, Laura Lucaccionib,
Lorenzo Iughettia,c and Alberto Berardib
a
Department of Medical and Surgical Sciences for Mothers, Children and Adults, Post Graduate School of Paediatrics, University of
Modena & Reggio Emilia, Modena, Italy; bNeonatal Intensive Care Unit, Department of Medical and Surgical Sciences for Mothers,
Children and Adults, Modena, Italy; cPediatric Unit, Department of Medical and Surgical Sciences for Mothers, Children and Adults,
University of Modena & Reggio Emilia, Modena, Italy

ABSTRACT ARTICLE HISTORY

There is an increasing evidence that the intestinal microbiota plays a pivotal role in the matur- Received 11 March 2018
ation of the immune system and in the prevention of diseases occurring during the neonatal Accepted 27 July 2018
period, childhood, and adulthood. A number of nonphysiological conditions during the perinatal
KEYWORDS
period (i.e. caesarean section, prolonged hospitalization, formula feeding, low gestational age)
Antibiotics; breastfeeding;
may negatively affect the normal development of the microbiota, leading to decreased amounts gut neonatal microbiota;
of lactobacilli and bifidobacteria and increased amounts of Clostridia. In addition, perinatal anti- necrotizing enterocoli-
biotics can cause intestinal dysbiosis that has been associated with short- and long-term dis- tis; sepsis
eases. For example, prolonged early empiric antibiotics increase the risk of necrotizing
enterocolitis (NEC) and late-onset sepsis (LOS) in preterm neonates, whereas the administration
of intrapartum antibiotic prophylaxis (IAP) has been associated with inflammatory bowel dis-
eases, obesity, and atopic conditions, such as eczema and wheezing. Promoting breastfeeding,
reducing the length of hospital stay, and reducing unnecessary antibiotic therapies are useful
strategies to counterbalance unintended effects of these conditions.

Introduction scenarios. Studies based on cultural techniques dem-

onstrated microbial invasion of the amniotic cavity in
Microbiota is the sum of microbial life present on the
spontaneous labor at term with intact membranes,
surface or inside the human body. Microbiota plays a
prelabor membranes rupture at term and preterm, or
central role in host nutrition, in the protection from
preterm labor [5]. More recent studies, based on poly-
pathogens by stimulating the synthesis of IgA, and by
merase chain reaction (PCR) techniques, have shown
influencing the development of the immune system
that microbial invasion of the amniotic cavity is
[1]. The intestinal microbiota continuously evolves from 30%–50% higher than previously reported by cultural
birth, especially during the early phases of life [2], due techniques, demonstrating even uncultivated species
to intrinsic (as the development of the immune sys- (i.e. Sneathia sanguinegens and Leptotrichia spp. from
tem) or extrinsic factors (as mode of delivery, type of the family of Fusobacteriaceae) [6]. Di Giulio et al.
nutrition, use of antibiotics, hospitalization) [3]. described a significant inverse correlation (p < .002)
Changes in the composition of microbiota have between bacterial DNA concentration in amniotic
been associated with a number of diseases occurring fluid (detected by PCR) and gestational age at
in the neonatal period (i.e. necrotizing enterocolitis delivery [7].
(NEC), sepsis), childhood, or adulthood (i.e. asthma, Ardissone et al. suggest that fetal gut microbiome
atopic dermatitis, diabetes mellitus, inflammatory derives from the swallowing of the amniotic fluid and
bowel diseases) [3,4]. infected amniotic fluid can induce an intestine-derived
inflammatory response leading to preterm labor.
Furthermore, the bacterial composition of amniotic
The development of microbiota
fluid seems more similar to the meconium micro-
For decades, neonatal gut has been considered sterile biome, suggesting than meconium reflects the
at birth, but new evidences suggest different intrauterine environment [8].

CONTACT Alberto Berardi [email protected] Unit
a Operativa di Terapia Intensiva Neonatale, Dipartimento Integrato Materno-
Infantile, Azienda Ospedaliero-Universitaria Policlinico, Via del Pozzo, 71, 41124 Modena, Italy
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
 THE JOURNAL OF MATERNAL-FETAL & NEONATAL MEDICINE 1037

During the first days of life, the gastrointestinal tract

of healthy full-term newborns is colonized by faculta-
tive anaerobic species (Escherichia coli, Enterococcus
spp, a-hemolytic streptococci, and Staphylococcus spp).
Subsequently, oxygen consumption and oligosacchar-
ides in human milk shift the microbial composition to
the predominance of anaerobic bacteria, such as
Bacteroides, Bifidobacterium, and Clostridium spp [1].
Compared to healthy full-term, preterm newborns
have a lower bacterial diversity, with a prevalence of
Figure 1. Factors impairing the physiological development of
pathogens (enterococci, Enterobacteriaceae, E. coli,
intestinal microbiota.
staphylococci, streptococci, and Clostridia), whereas
colonization by beneficial strains (i.e. bifidobacteria,
Bacteroides) is delayed [9].
In healthy, full-term, breast-fed newborns, there is a
strong association between specific microbial strains
found in the fourth day and those found in the fourth
month of life, showing how the composition of micro-
biota during early phases can affect the subsequent
development [10]. These data show that the develop-
ment of microbiota is a dynamic process, strongly
influenced by perinatal factors.

Factors influencing the composition

of microbiota
Figure 2. Effects of perinatal antimicrobial therapies. Risks
Figure 1 summarizes factors influencing the physio- associated with prolonged early empiric antibiotic therapies
logical development of intestinal microbiota. Figure 2 (VLBW neonates) and IAP (all neonates).
resumes the short-term complications of gut dysbiosis
(very low birth weight (VLBW) neonates) after pro- important step for developing sepsis [13]. The shift
longed antibiotic therapies during the first days of life from infants’ microbiota toward a more mature and
and the long-term consequences of intrapartum anti- definitive colonization occurs later in VLBW infants [12].
biotic prophylaxis (IAP; all neonates). VLBW neonates are often exposed to prenatal and/or
postnatal insults able to influence the development of
gut microbiota. Indeed, they are often delivered by
Gestational age cesarean section and fed mixed feeding. During their
Mode of delivery and feeding are main factors influenc- hospitalization, they often receive antibiotics or H2-
ing infant microbiota in full-term neonates. The gut blockers [14]. Interestingly, preterm gut colonization is
microbiota of healthy full-term newborns reaches the very dynamic. A study carried out with sequencing
characteristics of the adult-type microbiota by 3 years techniques suggests that frequent and unpredictable
of age. In contrast to full-term neonates, the develop- abruptions in the relative ratios of microbial classes
ment of gut microbiota in preterm infants is influenced occur during the development of preterm microbiota.
predominantly by the low gestational age [11,12] and However, the burden of Clostridia approximates to
microbiota seems less stable than in full-term neonates. older infants by 33–36 weeks of postconceptional age
With respect to older children, preterm and small for [15]. The gut colonization by bifidobacteria seems to
gestational age neonates have immature immune sys- occur over a given threshold of gestational age.
tem and the intestinal barrier is particularly susceptible Indeed, Butel et al. studied 52 preterm neonates (24
to external noxae. Furthermore, additional risk factors after vaginal delivery and 28 after C-section) fed with a
(such as invasive devices, surgery, prolonged hospital- standard or a fermented preterm formula, or mixed
ization) may concur to an abnormal intestinal flora, feeding. Fecal samples were collected twice a week for
where antibiotic-resistant Gram-negative bacilli pre- at least 2 weeks. A stable colonization by bifidobacteria
dominate. This promote bacterial translocation, an occurred in 18 of 52 newborns at 33.9 ± 0.8 weeks,
1038 M. FICARA ET AL.

regardless of birth weight, mode of delivery, antibiotics, Compared to spontaneous delivery, C-section reduces
and feeding. Gestational age at birth is therefore the the colonization with bifidobacteria and Bacteroides
main factor affecting the timing of gut colonization by while it increases the amounts of C. difficile [2]. Mode
bifidobacteria (p<.05) [16]. of delivery affects bacteria that will come first in con-
tact with the infant. Dominguez-Bello et al. reported
Feeding differences between the neonatal microbiota after
vaginal delivery or C-section, by comparing maternal
Human milk contains amino acids, vitamins, minerals,
(before delivery) to neonatal microbiota. After vaginal
and oligosaccharides, functioning as prebiotics.
delivery, neonatal and maternal vaginal microbiota are
Human milk oligosaccharides (HMOs) may differ
similar, with predominance of Lactobacillus, Prevotella,
among mothers and change during the different
Sneathia spp. In contrast, after C-section neonatal
stages of lactation. For example, HMO content is
microbiota is more similar to the maternal cutaneous
22–24 g/L in human colostrum and 12–13 g/L in
flora, and influenced by hospital environment. These
mature milk, respectively. HMOs also play a role in
findings underscore the important role of hospital
mineral absorption, in the development of immune
admission and suggest that exposure to hospital
system and gastrointestinal structure. Compared to
full-term, breast milk of mothers who deliver preterm environment may affect the neonatal microbiota after
have higher levels of HMOs [17]. C-section [22]. B€ackhed and coworkers studied 98
Feeding may affect the composition of gut micro- healthy full-term neonates. Maternal fecal microbiota
biota. Bifidobacteria and Clostridium difficile predominate at delivery was compared with neonatal microbiota in
in breast-fed neonates, whereas Bacteroides and early days, 4 and 12 months of life. Differences conse-
Clostridium perfrigens prevail in formula-fed infants [18]. quent to mode of delivery decreased at 4 and 12
Penders et al. studied 100 full-term neonates born after months of life although after C-section microbiota
vaginal delivery and unexposed to antibiotics. With remained more heterogeneous than after vaginal
respect to formula-fed, breast-fed neonates had lower delivery [11]. These studies suggest that the mode of
amounts of E. coli (p ¼ .004) and C. difficile (p ¼ .03) in delivery is one of the most important determinants
gut [19]. A large prospective cohort study (Koala study) of the gut microbial composition although these
enrolled 957 infants. Investigators found that newborns differences dampen over time.
colonized with C. difficile had a higher risk of eczema
(OR ¼ 1.40; 95% CI ¼ 1.02–1.91) and other atopic mani- Hospitalization
festations compared to uncolonized infants. C. difficile
could affect T regulator cell responses resulting in Preterm infants admitted to the same neonatal inten-
immune dysregulation. In addition, C. difficile might sive care unit (NICU) have comparable composition of
increase the gut permeability by facilitating microbial gut microbiota during the first weeks of life, probably
translocation and subsequent sensitization to normally because of cross-transmission [1]. Brooks et al.
harmless antigens through an increased production of sampled systematically two VLBW admitted in the
toxins [20]. Early breastfeeding has long-lasting effects same room using 16S rRNA gene sequencing techni-
on gut microbiota, and effects persist even after wean- ques. Repeated neonatal fecal and occasional surface
ing. A longitudinal study enrolled 605 infants. Feeding room samples were collected from the third day of
(human milk vs formula) and mode of delivery (vaginal life. Surface room samples and neonatal gut flora were
vs C-section) affected intestinal microbiota up to similar, supporting the idea of a circular “infant-room
6 months after weaning, whereas peripartum antibiotics colonization” that may concur to the diffusion of
had no longer effects. Weaning contributes to the genes conferring antibiotic resistance (so-called gut-
development of adult gut microbiota, increasing the associated resistome) [23,24].
expression of genes for digesting complex carbohy- The colonization with C. difficile increases in both
drates [21]. However, the interruption of breastfeeding term and preterm infant admitted to NICUs, and in
(rather than weaning) seems to be the most important neonates with prolonged hospitalization [1]. A French
factor affecting gut microbiota maturation [11]. retrospective study enrolled 76 preterm neonates.
Colonization with Clostridia was strongly associated
with a prolonged hospitalization (27.5% of cases after
Mode of delivery
1, 78.9% after 4, and 100% after 7 weeks, respectively)
Several studies suggest that C-section changes the [25]. The role of hospital environment is more relevant
normal microbial composition, reducing its diversity. for infants delivered after C-section, unexposed to
Table 1. Main changes of gut microbiota after intra- and/or postpartum antibiotics in full-term and preterm neonates.
Antibiotics and neonatal microbiota
Timing of
neonatal fecal
References SG CG Mode of delivery sampling (age) Gut microbiota and main results
Keski-Nisula 17 IAP-exposed full- 28 IAP-unexposed full- SG: vaginal CG: vaginal At birth, before IAP and membrane rupture lasting over 18 h
et al. [28] term neonates term neonates breastfeeding reduce significantly the vertical transmis-
sion of lactobacilli
Azad 85 healthy IAP-exposed full- 113 healthy IAP-unex- SG: vaginal (n ¼ 42), 3 and 12 months At age 3 months:
et al. [29] term neonates posed full- emergency CS (n ¼ 25),  decreased richness in IAP exposed with
term neonates or elective CS (n ¼ 18) vaginal delivery
CG: vaginal  decreased amounts of Bacteroides and
Parabacterioides, increase in Firmicutes
(i.e. Enterococci and Clostridia) in SG
 diversity and richness inversely associ-
ated with exclusive breastfeeding
Corvaglia 35 IAP-exposed neonates 49 IAP-unexposed neo- SG: vaginal 7 and 30 d IAP decreases Bifidobacteria at age 7 d
et al. [30] (35–37 weeks of gestation) nates (35–37 weeks CG: vaginal No effects of IAP at age 30 days. Greater
of gestation) amounts of Lactobacilli (regardless of IAP)
in breastfed newborns
Aloisio 26 healthy IAP exposed, full-term, 26 healthy IAP-unex- SG: vaginal 7d Quantitative–qualitative reduction of
et al. [31] breastfed neonates posed, full-term, CG: vaginal Bifidobacteria after IAP
breastfed neonates
Aloisio 10 healthy IAP-exposed full-term 10 healthy IAP-unexposed SG: vaginal 7d SG: Lower amounts of Actinobacteria and
et al. [32] breastfed neonates full-term CG: vaginal Bacteroides, over-representation of
breastfed, neonates Proteobacteria. Decreased microbial rich-
ness and biodiversity, predominance of
Enterobacteriaceae
Arboleya 27 VLBW neonates (mixed feed- 13 full-term breastfed SG: vaginal (n ¼ 7), or CS 24–48 h, At age 2, 10, and 30 d: SG: lower amounts
et al. [33] ing), divided in four groups neonates, without (n ¼ 20) 10, 30 and 90 d of total bacteria and Bacteroidaceae
(according to intra- and post- postpartum antibiotics, CG: vaginal At age 10 and 30 d: higher amounts of
partum antibiotics) (three IAP, 10 Enterobacteriaceae
IAP unexposed) At age 90 d: Disappearance of many
differences
Dardas 12 neonates (24–32 weeks of 15 neonates (24–32 weeks SG: 2 vaginal, 10 CS, 10 and 30 d At age 10 d:
et al. [35] gestation), exposed to of gestation), exposed CG: 13 vaginal, 2 CS CG: Firmicutes is the predominant
7–10 days of postnatal atb to 2 days of postna- phylum; SG: less diversity of microbiota
tal atb At age 30 d:
CG: predominance of Firmicutes; increase
in Actinobacteria and Proteobacteria
Greenwood 61 preterm neonates ( 32 weeks 13 preterm neonates ( SG: weeks 1, 2, and 3 Enterobacter species more represented in
et al. [36] of gestation): 32 weeks’ gestation) a) 31 CS, 17 vaginal the second and third weeks of life, with
a) 48 exposed to Atb (1–4 d) atb unexposed b) 8 CS, 5 vaginal lower diversity of microbiota in SG (b)
b) 13 exposed to Atb (5–7 d) CG: 12 CS, 1 vaginal
Zhu et al. [37] 24 preterm neonates 12 preterm neonates SG: 3 and 7 d At age 3 d: more represented Actinobacteria
(28–37 weeks of gestation): (28–37 weeks of gesta- a) 2 vaginal, 10 CS b) and Bacteroidaceae (SG)
a) 12 exposed to penicillin and tion) atb unexposed 2 vaginal, 10 CS At age 7 d: reduced diversity and increase
moxalactam (7 d) CG: 2 vaginal, 10 CS of pathogena (SG); higher amounts of
THE JOURNAL OF MATERNAL-FETAL & NEONATAL MEDICINE

b) 12 exposed to piperacillin–- Shigella–Escherichia (a) or Enterococci (b)

tazobactam (7 d)
Atb: antibiotics; CG: control group; CS: caesarean section; d: days; SG: study group sampling from oral cavity.
1039
1040 M. FICARA ET AL.

maternal vaginal flora [26]. These studies show that antibiotic therapies (7–10 days) had a lower diversity
prolonged hospitalization may inhibit the physiological of microbiota in the first days of life, whereas differen-
development of microbiota, increasing the exposition ces were no longer observed on day 30. However,
to pathogens. neonates exposed to short antibiotic therapies showed
a more uniform distribution of the various genera [35].
Greenwood et al. [36] confirmed that preterm neo-
Antimicrobial therapies and microbiota
nates exposed during the first week of life to pro-
Intrapartum antibiotic prophylaxis (IAP) prevents verti- longed (5–7 days) antibiotic course have lower
cal transmission of group B streptococcus (GBS) [27]. diversity of microbiota in the second and third weeks
Several studies have investigated how antibiotics of life and Enterobacter spp are more represented.
affect the composition of microbiota in relation to IAP More recently, Zhu et al. [37] also found no changes
or the duration of postnatal treatments. Table 1 details of intestinal flora after 3 days of antibiotics, but a
the main changes of gut microbiota after intra- and/or reduction in diversity and an increase in pathogens
postpartum antimicrobials, according to the mode of (streptococci, pseudomonas, and enterococci) after
delivery, both in full-term or preterm neonates. prolonged (at least 7 days) antimicrobial therapies.
Vertical transmission of lactobacilli is reduced after Although very heterogeneous and difficult to com-
IAP or membrane rupture. By collecting maternal vagi- pare, these studies show that antibiotics may have
nal (approximately 21 days before delivery) and neo- profound, short- and long-term effects on the nascent
natal oral specimens (at birth, before breastfeeding) microbiota (both in preterm and full-term neonates),
Keski-Nisula et al. found that IAP and membrane rup- even if some changes can be counterbalanced by
ture lasting over 18 h reduce significantly the vertical breastfeeding. Dysbiosis would affect the physiological
transmission of lactobacilli [28]. Azad and coworkers development of many systems, including the regula-
found that both richness and diversity of the intestinal tion of the immune responses. Strategies minimizing
microbiota in full-term newborn are affected by IAP unnecessary antibiotics both in preterm and full-term
exposure and mode of delivery. However, at age neonates should be promoted [38,39].
12 months, changes in intestinal flora induced by IAP
seem to be minimized by breastfeeding [29]. IAP
Intestinal microbiota, sepsis, and NEC
reduces significantly bifidobacteria of the intestinal
microbiota in the first week of life. By collecting fecal Broad spectrum antibiotics may disrupt intestinal
samples at 7 and 30 days of life, Corvaglia et al. [30] homeostasis and promote the emergence of resistant
found that the effect of IAP was transient, since bifido- pathogens. A prolonged antibiotic therapy in VLBW
bacteria normalized at age 1 month, and exclusively newborns in the first week of life is associated with
breast-fed newborns had greater amounts of increased risk of NEC, late-onset sepsis (LOS), and
Lactobacillus spp at 7 and 30 days of life, regardless of death. Kuppala et al. [40] studied a cohort of 365 pre-
IAP. Aloisio et al. confirmed a significant quantitative– term neonates (birth weight 1500, gestational age
qualitative reduction of bifidobacteria after IAP. 32 weeks) with sterile cultures, and demonstrated
Bifidobacteria would counteract GBS colonization that initial antimicrobial therapy lasting 5 or more
through their antimicrobial activity [31]. Reduced days was an independent risk factor for LOS (OR: 2.5,
microbial diversity, increased Enterobacteriaceae, and CI: 1.28–4.67) and the combination of LOS, NEC, or
decreased Actinobacteria were also found after death (OR: 2.66, CI: 1.12–6.3). With respect to healthy
IAP [32]. preterm neonates, higher amounts of coagulase-nega-
The effects of antimicrobials on intestinal flora were tive staphylococci and c-Proteobacteria (especially
also investigated in infants with lower gestational age. Enterobacter spp) and lower amounts of Firmicutes,
By comparing VLBW neonates and breastfed full-term characterized the microbiota of preterm neonates who
neonates, Arboleya et al. [33] found that VLBW neo- subsequently developed NEC [41].
nates exposed to different antibiotic regimens had From 72 h to 1 week before NEC, preterm neonates
abnormal microbiota but changes apparently were no show significantly increased amounts of Proteobacteria
longer observed at age 90 days. Preterm neonates and decreased amounts of Firmicutes [42]. Antibiotic-
exposed to IAP or to postnatal antibiotics had dependent dysbiosis results in bacterial translocation
decreased levels of intestinal microbial metabolites across the epithelial barrier by promoting TLR4-medi-
(i.e. the short-chain fatty acids acetate, propionate and ated inflammatory cascade [12]. A number of studies
butyrate) [34]. Newborns receiving prolonged address the risk of NEC according to the duration of
 THE JOURNAL OF MATERNAL-FETAL & NEONATAL MEDICINE 1041

antimicrobial therapy [43]. In a retrospective study, 124 the neonatal gut without contamination by the mater-
cases were compared with 248 controls (matched for nal surrounding skin [50].
birth weight and gestational age). The duration of anti-
biotic exposure prior to NEC was an independent risk
Conclusions
factor. The risk of NEC increased nearly 20% for each
day of antibiotic exposure (OR ¼ 1.2, CI: 1.10–1.54) [44]. In conclusion, newborns may be exposed to several
Compared to formula-fed, breast-fed preterm neonates nonphysiological conditions during the perinatal
have lower risk of NEC (OR ¼ 0.62, CI ¼ 0.51  0.77, period. These conditions impair the normal develop-
p ¼ .02) [45]. ment of intestinal microbiota, leading to decreased
amounts of beneficial bacterial strains and increased
amounts of pathogens. Although the currently avail-
Prebiotics, probiotics, and synbiotics
able data are heterogeneous and sometimes difficult
Probiotics are microorganisms with beneficial effects to compare, increasing evidence suggests the pivotal
when given in appropriate amounts. Lactobacilli, bifi- role of intestinal flora in childhood and adult diseases.
dobacteria, and streptococci prevent intestinal colon- Promoting breastfeeding, reducing the length of hos-
ization by pathogens through the down-regulation of pital stay, and minimizing unnecessary antibiotics are
proinflammatory cytokines, raised levels of IgA, or useful to prevent the adverse effects of these condi-
improved integrity of the intestinal epithelial barrier tions. Further studies should investigate which neo-
[1]. Lin et al. carried out a prospective, blind, random- nates can benefit of a pre/probiotic supplementation.
ized, multicenter trial by enrolling 474 VLBW neonates.
The risk of developing moderate to severe NEC was
Disclosure statement
significantly lower in newborns receiving probiotics
(4 of 217) with respect to untreated control group No potential conflict of interest was reported by the authors.
Dr. Alberto Berardi has received fees from these Companies:
(14 of 217; p < .02) [46]. Similarly, Braga et al. supple-
Pfizer (2015), Putnam Associates (2016) and GSK (2017).
mented human milk with probiotics in 119 VLBW neo-
nates. No cases of NEC were registered in study group
compared to four cases of NEC in control group
(112 unsupplemented VLBW neonates). Furthermore, References
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