Advances in Experimental Medicine and Biology 1125

Advances in Microbiology, Infectious Diseases and Public Health

Stefano Guandalini
Flavia Indrio Editors

Probiotics and Child

Gastrointestinal
Health
Advances in Microbiology, Infectious
Diseases and Public Health Volume 10
Advances in Experimental Medicine
and Biology

Volume 1125

Advances in Microbiology, Infectious Diseases

and Public Health

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Stefano Guandalini • Flavia Indrio
Editors

Probiotics and Child

Gastrointestinal Health
Advances in Microbiology,
Infectious Diseases and Public Health
Volume 10
Editors
Stefano Guandalini Flavia Indrio
Department of Pediatrics Department of Pediatrics, Ospedale
University of Chicago Giovanni XXI
Chicago, IL, USA University of Bari Aldo Moro
Amendola, Bari, Italy

ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISSN 2365-2675 ISSN 2365-2683 (electronic)
Advances in Microbiology, Infectious Diseases and Public Health
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Foreword

We have seen this happening so many times in science and medicine: some-
thing new appears on the scene, gains momentum, reaches a top of consensus,
and then starts losing its appeal. The pendulum is swinging. Is this occurring
now also for probiotics? After enjoying a tremendous rise of popularity both
in the general public and in the scientific community for close to two decades,
very recently much less enthusiastic opinions and even vocal skepticism have
been expressed by some parts of the scientific community, based on data
showing lack of efficacy of probiotics in fields considered “strong.” Will also
the general public begin swinging in the opposite direction? At what point of
the curve will the pendulum finally stop to point at an honest, objective,
unbiased assessment?
This book aims at providing a fair, rigorously evidence-based answer to
some of the most relevant questions currently open for the potential use of
probiotics in pediatric gastrointestinal health, ranging from NEC to colic, food
allergy to celiac disease, IBD to liver disease, acute diarrhea to the ever-so-
common functional disorders, to Clostridium difficile infections.
We are grateful to all the authors – all well-respected investigators and
recognized authorities in their area – for the effort they have put in generating
rigorous analyses and syntheses of the currently available evidence and are
confident the reader will find valuable information throughout this book.

Chicago, USA Stefano Guandalini

Amendola, Italy Flavia Indrio

v
Contents

Part I The Newborn

Shaping Microbiota During the First 1000 Days of Life . . . . . . . . 3
Marta Selma-Royo, Maria Tarrazó, Izaskun García-Mantrana,
Carlos Gómez-Gallego, Seppo Salminen,
and Maria Carmen Collado
Necrotizing Enterocolitis and the Preterm Infant Microbiome . . . . 25
Jillian R. Baranowski and Erika C. Claud
Can Postbiotics Represent a New Strategy for NEC? . . . . . . . . . . 37
Fabio Mosca, Maria Lorella Gianni, and Maria Rescigno

Part II The Infant

Preventing and Treating Colic . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Flavia Indrio, Vanessa Nadia Dargenio, Paola Giordano,
and Ruggiero Francavilla
Targeting Food Allergy with Probiotics . . . . . . . . . . . . . . . . . . . . . 57
Lorella Paparo, Rita Nocerino, Carmen Di Scala, Giusy Della Gatta,
Margherita Di Costanzo, Aniello Buono, Cristina Bruno,
and Roberto Berni Canani
Use of Probiotics to Prevent Celiac Disease and IBD
in Pediatrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Gloria Serena and Alessio Fasano

Part III The Child

Fighting Fatty Liver Diseases with Nutritional
Interventions, Probiotics, Symbiotics, and Fecal Microbiota
Transplantation (FMT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Valerio Nobili, Antonella Mosca, Tommaso Alterio,
Sabrina Cardile, and Lorenza Putignani
Probiotics in the Treatment of Inflammatory Bowel Disease . . . . . 101
Stefano Guandalini and Naire Sansotta

vii
viii Contents

Acute Infectious Diarrhea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Andrea Lo Vecchio, Vittoria Buccigrossi, Maria Cristina Fedele,
and Alfredo Guarino
Probiotics in Functional Gastrointestinal Disorders . . . . . . . . . . . . 121
Iva Hojsak
Clostridium difficile Colitis Prevention and Treatment . . . . . . . . . . 139
Meltem Dinleyici and Yvan Vandenplas

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
 Part I
The Newborn
Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2019) 1125: 3–24
https://doi.org/10.1007/5584_2018_312
# Springer Nature Switzerland AG 2019
Published online: 26 January 2019

Shaping Microbiota During the First 1000

Days of Life

Marta Selma-Royo, Maria Tarrazó, Izaskun García-Mantrana,

Carlos Gómez-Gallego, Seppo Salminen,
and Maria Carmen Collado

Abstract early microbiota exposure within the male and

The data obtained in prior studies suggest that the female reproductive tracts at the point of
early microbial exposition begins prior to conception and during gestation, focusing on
conception and gestation. Given that the host- the potential impact on infant development
microbe interaction is shaped by the immune during the first 1000 days of life. Furthermore,
system response, it is important to understand we conclude that some dietary strategies
the key immune system-microbiota including specific probiotics could become
relationship during the period from conception potentially valuable tools to modulate the gut
to the first years of life. The present work microbiota during this early critical window of
summarizes the available evidence concerning opportunity for targeted health outcomes
throughout the entire lifespan.
M. Selma-Royo and I. García-Mantrana
Department of Biotechnology, Institute of Agrochemistry Keywords
and Food Technology, Spanish National Research Council Diet · Gestation · Health · Meconium ·
(IATA-CSIC), Valencia, Spain Microbiota · Placenta · Probiotics · Semen ·
M. Tarrazó Vagina
Service of Obstetrics and Gynecology, Hospital
Universtario Doctor Peset, Valencia, Spain
C. Gómez-Gallego 1 Perinatal Microbial
Functional Foods Forum, University of Turku, Turku,
Finland
Environment and Its Relevance
for Human Health
Institute of Public Health and Clinical Nutrition,
University of Eastern Finland, Kuopio, Finland
The human microbiome is defined as the complex
S. Salminen
communities of microorganisms that live on, as
Functional Foods Forum, University of Turku, Turku,
Finland well as in, the human body, and it is principally
composed of bacteria but also of viruses, fungi,
M. C. Collado (*)
Department of Biotechnology, Institute of Agrochemistry archaea and bacteriophages (Milani et al. 2017;
and Food Technology, Spanish National Research Council Perez-Muñoz et al. 2017). The microbiome is
(IATA-CSIC), Valencia, Spain present in different sites of the human body,
Functional Foods Forum, University of Turku, Turku, including the skin, oral, nasopharyngeal and
Finland genito-urinary tract (Rautava et al. 2012; Milani
e-mail: [email protected]

3
4 M. Selma-Royo et al.

et al. 2017). The differential microbiome the foetus and, later, the infant. Yet, despite the
compositions depend on the host’s genetics as growing knowledge base, the different prenatal
well as the host’s environmental characteristics, factors that can affect the maternal microbiota
including humidity, pH, nutrients and oxygen and, consequently, the foetal microbiota remain
levels (Milani et al. 2017). unclear. The present review, therefore, aims to
The microbiome is responsible for numerous describe the available evidence concerning early
essential functions within the human body, microbial contact during the first 1000 days of life
including assisting with digestion and metabo- (Fig. 1). The review will also provide an overview
lism, the production of vitamins, maintaining the of the theory regarding the maternal-foetal-neo-
gut barrier and regulating the development of the natal microbiota and, additionally, the potential
immune system (Rautava et al. 2012; Gensollen offered by the application of dietary interventions
et al. 2016). with probiotics during this critical window of
Microbiome dysbiosis has previously been opportunity.
associated with an increased risk of
non-communicable diseases (NCDs), such as
asthma, obesity, diabetes and autoimmune 2 Preconception Microbial
conditions (e.g. Crohn’s disease), all of which Environment: Reproductive
are characterized by the over-responsiveness of Microbiotas
the immune system, which in turn leads to an
increasing pro-inflammatory status (Clemente In healthy non-pregnant women, the vaginal
et al. 2012; Koleva et al. 2015; Gensollen et al. microbiota is a complex ecosystem populated by
2016; Tamburini et al. 2016). more than 200 bacterial species, with Lactobaci-
The first 1000 days of life (i.e. from gestation llus spp. representing the dominant species,
until the first 2 years of life) are crucial for both followed by other less abundant bacteria such as
the colonization and the establishment of pioneer Prevotella, Streptococcus, Bacteroides and
microorganisms within the human body and, Veillonella (Aagaard et al. 2012; Mendling
additionally, for the development and maturation 2016). Based on the composition of the Lactoba-
of the immune system. Hence, this period is con- cillus, five types of microbial communities that
sidered to represent a ‘window of opportunity’ differ in terms of both their composition and their
during which any event will have a pivotal impact abundance have been described (Ravel et al.
on the metabolic, immunological and 2011). These communities can be clustered into
microbiological programming that affects later five groups, which are known as the ‘community
human health (Agosti et al. 2017). Any alterations state types’ (CSTs), with each group hosting a
and disruption to the step-wise neonatal specific bacteria: Lactobacillus (L.) crispatus
microbiota colonization have the potential to (CSTI), L. gasseri (CSTII), L. iners (CSTIII),
increase the risk and predisposition of individuals diverse group (CSTIV; exhibiting a lower pres-
to developing diseases in the short and long term ence of Lactobacillus spp.) and L. jensenii
(Verdu et al. 2016). However, recent contradic- (CSTV) (Ravel et al. 2011). The prevalence of
tory scientific evidence suggests that the first the different CSTs differs according to the
microbial contact may actually take place prior individual’s geographical location and ethnic ori-
to birth and, further, that such contact might play gin. For example, a higher abundance of CSTIV
an important role in the development of the foe- among African-American and Hispanic women
tus. This evidence has contributed to our chang- has been reported in the USA (Stout et al.
ing perspective on the sterile environment and the 2017). The Lactobacillus genus plays an impor-
origins of microbiota acquisition (Collado et al. tant role in both the maintenance of a low pH and
2016; Perez-Muñoz et al. 2017). Nevertheless, the secretion of metabolites in order to prevent
the maternal microbiota represents the most rele- pathogenic colonization in the vagina (Ravel et al.
vant prenatal and postnatal microbial source for 2011; Aagaard et al. 2012). A growing body of
Shaping Microbiota During the First 1000 Days of Life 5

Fig. 1 Early microbial contact from conception to birth

Overview of microbes present in male and female reproductive tracts, in the maternal-foetal-neonatal microbiota as
placenta, amniotic fluid and meconium. Microbes are present before egg fertilization and would play a key role during
conception. Furthermore, microbiota changes during pregnancy would support the foetal development, and accumulating
data suggest the presence of microbes in placenta and amniotic fluid although the biological impact is still uncovered

scientific evidence has demonstrated the potential et al. 2017; Miles et al. 2017; Chen et al. 2017;
use of the Lactobacillus species as biomarkers of Benner et al. 2018). The endometrial microbiota
vaginal health (Petrova et al. 2015). However, the is characterized by a high amount of Lactobacil-
vaginal microbiota does not remain static. Indeed, lus, followed by Gardnerella, Prevotella,
temporal dynamics have been found in the vagi- Atopobium and Sneathia, which have also been
nal microbiota over a 2-week period (Gajer et al. identified in the vagina (Moreno et al. 2016;
2012), whereby some communities changed and Moreno and Franasiak 2017). However, it
others remained relatively stable, depending on appears that the endometrial bacteria population
the CST. Recent studies have highlighted the differs somewhat from that in the vagina, which
need to analyse the host factors that affect the suggests that the two microbiotas are related, but
vaginal microbiota, since little is currently not identical (Franasiak and Scott 2017). Further-
known about the impact it has on the different more, the fallopian tubes are known to be
bacterial communities or the short- and long-term colonized by bacteria such as Lactobacillus,
impacts on the individual’s overall health status Staphylococcus, Enterococcus, Prevotella and
(Witkin 2018). Propionibacterium, with Pseudomonas being
The female reproductive system contains bac- the identified genus (Pelzer et al. 2018). Impor-
teria that have an impact on women’s health also tantly, recent reviews have highlighted the link
outside of the vagina (Chen et al. 2017; Younes between commensal bacteria in the uterus, fertil-
et al. 2018). Several studies have demonstrated ity and pregnancy complications (Prince et al.
that the uterus harbours a specific endometrial 2014; Franasiak and Scott 2017; Moreno and
microbiota that has been linked to both reproduc- Franasiak 2017; Power et al. 2017).
tive and uterine health (Moreno et al. 2016; Tao
6 M. Selma-Royo et al.

Yet, very little research has previously been so promote correct growth (Wahlqvist et al.
conducted concerning the male microbiota 2015). Gestational physiological changes, such
identified in the reproductive tract. Recent studies as endocrine, immunological and metabolic
have demonstrated the presence of microbiota in alterations, favour a pro-inflammatory status,
semen, with the Firmicutes representing the most which is reflected in specific shifts seen in the
predominant phyla, followed by the maternal microbiota at different body sites,
Bacteroidetes, Proteobacteria and Actinobacteria, including the vagina, gut and oral cavity
of which Lactobacillus, Prevotella, Gillisia, (Nuriel-Ohayon et al. 2016).
Corynebacterium and Gardnerella are the most
common genera (Mändar et al. 2015). It has been
hypothesized that the predominance of 3.1 Vaginal and Uterine Microbiota
Gardnerella vaginalis in the vaginal microbiota
is related to inflammation challenges in males, During pregnancy, microbial diversity within the
which could be related with colonization/infec- vaginal microbiota decreases, while members of
tion from the vagina (Mändar et al. 2017). It has Lactobacillus species increase, potentially
further been suggested that a link exists between reinforcing their protective function (Romero
the male and female reproductive microbiomes et al. 2014; MacIntyre et al. 2015). The uterine
that has an effect on fertility, reproduction, health microbes may prove beneficial to the foetus and
and gestation. In fact, the majority of prior semen newborn favouring tolerance towards organisms
microbiome studies have focused on the issue of which enhance postnatal well-being such as the
infertility, highlighting differences in the micro- Lactobacillus genus (Sisti et al. 2016). However,
bial profiles and diversity (Hou et al. 2013; Weng there is accumulating evidence of dysbiosis
et al. 2014; Monteiro et al. 2018). For example, a occurring in the vaginal microbiome during preg-
recent study reported different seminal nancy leading to an increased risk of preterm birth
microbiomes in patients with obstructive or (Bretelle et al. 2015), with such disturbances hav-
non-obstructive azoospermia, thereby showing ing been found to occur as early as during the first
an increase in the Bacteroidetes and Firmicutes trimester (Haque et al. 2017). It has further been
as well as a decrease in the Proteobacteria and demonstrated that women whose medical history
Actinobacteria when compared to healthy men includes repeated urinary tract infections exhibit
(Chen et al. 2018). Taken together, the uterine an increased risk of preterm delivery. Differences
and seminal microbiomes appear to be highly in the vaginal microbiota in terms of the compo-
relevant for reproductive medicine, with the evi- sition, stability and diversity have been observed
dence suggesting the potential of specific between full-term deliveries and preterm
microbiota profiles as biomarkers in assisting in deliveries (Nuriel-Ohayon et al. 2016). Bacterial
the development of new tools for diagnosing and communities characterized by high levels of
treating infertility. Atopobium, Gardnerella and Ureaplasma as
In this scenario, the human reproduction takes well as lower levels of Lactobacillus spp. or a
place under ‘microbial environment’, and those higher presence of Candida albicans have been
microbes must have greater attention due to their found to be correlated with preterm birth
relevance from preconception onwards. (DiGiulio et al. 2010; Hyman et al. 2014; Bretelle
et al. 2015; Farr et al. 2015).
In addition, the seminal microbiota may inter-
3 Maternal Microbiota During fere with both conception and preterm delivery. It
Pregnancy has been reported that a higher presence of typical
seminal bacteria, such as L. iners, during preg-
During pregnancy, the maternal physiology and nancy is associated with preterm deliveries,
metabolism are adjusted in order to afford the whereas the dominance of L. crispatus, another
foetus an optimal intrauterine environment and common bacterial species in semen, has been
Shaping Microbiota During the First 1000 Days of Life 7

identified as protective (Bennett 2017). In any has a vital impact on both the maternal and infant
case, the detection of abnormalities within the microbiotas (Mandal et al. 2016; Barrett et al.
vaginal microbiome during gestation could pro- 2018; Lundgren et al. 2018). Human studies
vide new insights on potential microbial suggests that changes in the female gut
biomarkers for predicting the likelihood of pre- microbiota during pregnancy are vulnerable to
term delivery. modulation by the maternal pre-gestational body
max index (BMI), as well as to weight gain over
gestation (Collado et al. 2008, 2010; Santacruz
3.2 Gut Microbiota et al. 2010). A lower presence of Bifidobacterium
spp. has been observed in overweight and obese
While during the first trimester of gestation the mothers, as well as in mothers who gained exces-
composition of the gut microbiota remains stable sive weight during pregnancy, when compared to
and resembles pre-pregnancy microbiota, from lean mothers or to those mothers who maintained
the end of the first trimester onwards, its compo- a weight gain in keeping with recommendations
sition changes radically (Magon and Kumar (Collado et al. 2008). Another study reported
2012). Indeed, it has been reported that the gut similar shifts according to weight status during
microbiota changes during pregnancy to reflect a pregnancy, and lower levels of Bacteroides spp.,
more pro-inflammatory profile in a manner simi- along with higher abundances of Staphylococcus
lar to the changes observed in the case of diabetes spp. and Escherichia coli, were identified in over-
or metabolic syndrome (Koren et al. 2012). weight pregnant women (Santacruz et al. 2010).
However, this inflammatory status is implicated Such changes in the maternal gut microbiota
in metabolic adaptations to gestation, and it could be associated with differences in the intes-
contributes to a healthy pregnancy, meaning that tinal colonization process in neonates born via
it is beneficial for the development of the vaginal delivery (Mueller et al. 2016).
offspring (Koren et al. 2012). The changes are
mainly observed in terms of an increase in the
abundance of Actinobacteria and Proteobacteria 3.3 Oral Microbiota
phyla, along with a decrease in the butyrate-
producing bacteria and a decline in the diversity Pregnancy also induces changes in the oral
of the gut bacteria (Koren et al. 2012). Yet, other microbiota (Adriaens et al. 2009; Borgo et al.
studies have reported no significant microbial 2014). In fact, significant differences have been
changes in the gut during pregnancy (DiGiulio reported when comparing the abundances of sev-
et al. 2015), which indicates that further research eral species in the oral cavities of pregnant and
is needed to clarify the impact of pregnancy on non-pregnant women. The oral microbiota during
the gut microbiota. In addition, it has been widely gestation is characterized by an increase in the
demonstrated that antibiotic exposure during viable bacterial counts, along with higher levels
pregnancy alters the mother’s bacterial ecosystem of pathogenic bacteria, such as Porphyromonas
and, consequently, that of the offspring gingivalis, Aggregatibacter actinomycetem-
(Stokholm et al. 2014). Antibiotic use during comitans and Candida, which validates the grow-
gestation increases the vaginal colonization by ing body of evidence proving the link between oral
the Staphylococcus species, and potential micro- infections and periodontal disease associated with
bial shifts in other areas than the gut could be pregnancy complications, which includes preterm
linked to an increased risk of allergies and obesity delivery (Offenbacher et al. 2006; Zi et al. 2015;
(Kuperman and Koren 2016). Fujiwara et al. 2017). These data suggest that the
Furthermore, the maternal microbiota is oral microbiota also plays a key role in the mater-
influenced during pregnancy by both the mother’s nal and neonatal outcomes, including pregnancy
diet and nutritional status. It has recently been complications, risk of preterm birth and early
reported that the maternal diet during pregnancy foetal/neonatal microbial colonization.
8 M. Selma-Royo et al.

Thus, further research concerning the associa- Moreover, the above-mentioned issues give
tion between microbial markers and pregnancy rise to several questions concerning the origin
outcomes could prove instrumental in preventing and presence of these bacteria, as well as their
undesirable microbial changes during pregnancy, role in infant health in terms of both immunolog-
which may be linked to pregnancy complications. ical development and colonization process. The
most commonly discussed hypothesis suggests a
potential vertical ascendant route from the vagina
to the foetus or a haematogenous route (from the
4 Prenatal Microbial Exposition:
oral, gut and other areas through the blood to the
In Utero Colonization?
placenta, including the gut-foetal route and oral-
foetoplacental route) (Kuperman and Koren
Advances in molecular and sequencing technologies
2016). It is important to note that recent studies
have permitted the study of the human microbiota
have suggested the presence of a ‘blood
in areas previously considered sterile, including
microbiome’ in healthy individuals (Païssé et al.
the placenta (Aagaard et al. 2014), amniotic fluid
2016). Furthermore, it has been reported that the
(DiGiulio 2012; Combs et al. 2014) meconium
presence of foetal DNA (Liao et al. 2014) and
(Moles et al. 2013; Collado et al. 2016; Nagpal
placenta-derived exosomes (Lai et al. 2018) in the
et al. 2016; Shi et al. 2018) and foetal membranes
maternal circulation actually evidences their
(Steel et al. 2005; Parnell et al. 2017; Lannon et al.
potential association with placenta health,
2018). Some such studies have been able to isolate
preeclampsia and the risk of preterm birth
and identify bacteria from those areas (Moles et al.
(Taglauer et al. 2014; Seval et al. 2015; Van
2013; Combs et al. 2014; Collado et al. 2016).
Boeckel et al. 2018).
However, the ‘in utero colonization hypothesis’
Prior studies concerning the placental
remains subject to debate (Perez-Muñoz et al.
microbiome have hypothesized that the oral
2017). Despite a number of studies conducted in
microbiota could represent the main source of
the field in recent years, it is not yet fully clear
the bacteria observed in the placenta tissue.
whether the detection of microbes in such areas
Indeed, it has been observed that the composition
would represent a specific microbiota niche
of the placental microbiome could be more
(e.g. placenta microbiota) and, additionally, what
related to that of the oral microbiota than the
their biological relevance would be (Tables 1 and
composition of the vagina or gut microbiota
2). Other studies have suggested that there is no
(Nuriel-Ohayon et al. 2016). Such observations
evidence of a placenta microbiome and/or early
are supported by the relation that has been
microbial contact (Lauder et al. 2016; Leon et al.
identified between inflammatory oral disease,
2018; Rehbinder et al. 2018). The critical issue
principally periodontitis, and complicated preg-
concerns the low amount of DNA and, further, the
nancy outcomes, such as preterm delivery. Prince
potential bias stemming from the contaminant
et al. observed an incremental increase in the oral
DNA, as well as its detection and filtering (Lauder
commensal bacteria in the placental microbiome
et al. 2016; de Goffau MC et al. 2018), which
of preterm subjects with chorioamnionitis (Prince
increase the risk of false positive results. In this
et al. 2016). This suggests the particular relevance
sense, it has been a significant which increases the
of Fusobacterium nucleatum, which is a common
risk of false positive results. For this reason, a
oral bacterium found in the placenta, in relation to
significant amount of effort have been expended
negative pregnancy outcomes (Vander Haar et al.
in identifying new techniques that should reduce
2018), including preterm birth (Doyle et al.
the amount of false positive results with regard to
2014), neonatal sepsis (Wang et al. 2013) and
the microbiota analysis and also facilitate the con-
hypertension (Barak et al. 2007). The proposed
taminant detection in samples with a low micro-
oral-foetoplacental route has been demonstrated
bial biomass (Avershina et al. 2018; Goffau et al.
in animal models (Fardini et al. 2010). On the
2018; Davis et al. 2017).
other hand, it has been demonstrated in animal
Shaping Microbiota During the First 1000 Days of Life 9

Table 1 Recent studies on the placental microbiome

Relevant findings and/or Author/
Birth Population Disease Methods author conclusions References
Preterm n ¼ 1083- Preterm birth Culturing  " bacterial colonization Onderdonk
(<28 weeks) ELGAN related to the preterm et al. (2008a)
cohort delivery and gestational age
Preterm n ¼ 1365- Preterm birth Culturing; PCR  50% microbial detection Onderdonk
(23–27 weeks) ELGAN in placental chorionic plate et al. (2008b)
cohort
Term and n ¼ 195 Preterm birth Morphological  Intracellular bacteria in Stout et al.
preterm techniques 27% of total studied (2013)
(imaging) placenta
 In preterm birth this
colonization rise to 54%
Term n¼8 Healthy Culturing Intracellular bacteria in Cao and
trophoblast. Mysorekar
(2014)
Term and Term Preterm birth 16S rRNA gene  Streptococcus, Doyle et al.
Preterm n ¼ 10 sequencing Microbacterium, (2014)
Preterm (pyrosequencing Rhodococcus and
n ¼ 14 454) Corynebacterium in term
placenta
"Fusobacterium,
Streptococcus,
Mycoplasma, Aerococcus,
Gardnerella and
Ureaplasma in preterm
birth
Term and n ¼ 320 Healthy (PTB* 16S rRNA gene "Proteobacteria in placenta Aagaard et al.
Preterm included) sequencing microbiome. Escherichia (2014)
Metagenomics coli as the most abundant
 Species from oral
microbiome were
commonly detected
Term N ¼ 110 Preeclampsia 16S rRNA gene No bacteria were detected Amarasekara
sequencing in normotensive women et al. (2015)
(Illumina) and 12.7% from women with
PCR preeclampsia were positive
Organisms detected:
Bacillus, Listeria,
Salmonella, Escherichia,
Klebsiella
Term n¼2 Healthy (mode of 16S rRNA gene  Meconium microbiome Dong et al.
birth) sequencing resemble placenta (2015)
microbiome
 Micrococcineae as the
most abundant
Term and N ¼ 237 Healthy (PTB* 16S rRNA gene  Excess gestation weight Antony et al.
preterm included) sequencing gain related to placental (2015)
microbiome changes
among preterm birth
Term N ¼ 24 Low birth weight 16S rRNA gene  Placental microbiome Zheng et al.
sequencing linked to low birth weight (2015)
 "Firmicutes in placenta
microbiome, Enterococcus
as the most abundant genus
(continued)
10 M. Selma-Royo et al.

Table 1 (continued)
Relevant findings and/or Author/
Birth Population Disease Methods author conclusions References
Term N ¼ 22 GDM 16S rRNA gene  #Pseudomonadales order Bassols et al.
sequencing and Acinetobacter genus in (2016)
GDM* placenta
Term N ¼ 15 Healthy Culture; DGGE  #Richness and diversity Collado et al.
Elective 16S rRNA gene and "Proteobacteria in (2016)
C-section sequencing placenta microbiome
 Shared features between
placenta and amniotic fluid
and meconium
Term n¼6 Healthy 16S rRNA gene  Placental and negative Lauder et al.
sequencing and controls were (2016)
RT-PCR indistinguishable
Preterm Cross- Chorioamnionitis/ Metagenomics  Spontaneous PTV Prince et al.
sectional funisitis (Illumina) placental samples harbour (2016)
6 cohorts placental microbiome that
(n ¼ 9–15 differs by severity of
subjects/ chorioamnionitis
cohort)  In infection, " urogenital
and oral commensal
bacteria
Term N ¼ 20 Macrosomia 16S rRNA gene  Distinct placental Zheng et al.
sequencing microbiome associated to (2017b)
(Illumina) macrosomic foetus
Term N ¼ 20 GDM 16S rRNA gene  Distinct placental Zheng et al.
sequencing microbiome is present in (2017a)
(Illumina) GDM
 Placental microbiota is
composed of four dominant
phyla: Proteobacteria,
Bacteroidetes,
Actinobacteria and
Firmicutes
Term N ¼ 27 Healthy Culturing and  Mycobacteria were Dimova et al.
RT-PCR present in term placenta (2017)
Term N ¼ 1097 Rural Malawi 16S rRNA gene  Bacterial DNA was Doyle et al.
sequencing and detected in 46.8% of (2017)
RT-PCR placental samples. Bacterial
load was 4.8 log10 of 16S
rRNA gene copies/ul
Term N ¼ 57 Heathy (mode of 16S rRNA gene  Microbiota exhibit Parnell et al.
birth) sequencing and spatially distinct profile (2017)
RT-PCR depending on the location
within the placenta
Term N ¼ 37 Obesity 16S rRNA gene  Placental microbiome Gomez-
sequencing resemblance oral Arango et al.
microbiome (2017a, b)
Term n ¼ 329 Healthy 16S rRNA gene  Bacterial DNA was found Tuominen
sequencing in 49% of placental samples et al. (2018)
 Firmicutes and
Proteobacteria the most
abundant phyla
 Placental microbiome
related with HPV infection
*GDM gestational diabetes mellitus, PTB preterm birth
Shaping Microbiota During the First 1000 Days of Life 11

Table 2 Recent studies on the amniotic fluid microbiome

Author/
Birth Population Methods Relevant findings and/or author conclusions References
Preterm N ¼ 166 Culturing  Microbial prevalence, diversity and abundance DiGiulio
Preterm labour Specific PCR were correlated with host inflammation and with et al. (2008)
with intact RT-PCR gestational and neonatal outcomes
membranes  Positive PCR was associated with histologic
chorioamnionitis
Term and N ¼ 44 Culture  A total of 31 species (15 nonredundant) were Wang et al.
preterm 16S rRNA gene identified (2013)
PCR  Escherichia coli and Fusobacterium nucleatum
as the most prevalent
Term N ¼ 15 Culture; DGGE  Amniotic fluid: # richness and diversity and Collado
16S rRNA gene "Proteobacteria et al. (2016)
sequencing "Enterobacter and Escherichia/Shigella
 Shared features amniotic fluid and in infant
meconium
Term and N ¼ 79 16S rRNA gene  In chorioamnionitis, " bacterial abundance and Urushiyama
preterm sequencing; # diversity et al. (2017)
Chorioamnionitis digital PCR  AF microbiome profile as a tool to predict
chorioamnionitis
Term and N ¼ 248 16S rRNA gene Species richness of the amniotic fluid samples Wang et al.
preterm sequencing was comparable to the maternal oral and (2018)
GDM (Illumina) intestinal communities
Species differentially abundant in amniotic fluid
depends on the GDM status of the mother
(e.g. Corynebacterium or Anoxybacillus)
Term N ¼ 24 Culture . No evidence of amniotic fluid microbiota Rehbinder
PreventADALL Digital PCR  Bacterial DNA in amniotic fluid from deliveries et al. (2018)
16S rRNA gene with intact membranes comparable to controls
sequencing  50% bacteria in ruptured membranes
(Illumina)
Term N ¼ 24 16S rRNA gene  No evidence of amniotic fluid microbiota Lim et al.
sequencing; (2018)
qPCR

models that microbial translocation from the gas- Lactobacillus strains containing lux genes to the
trointestinal (GI) tract is augmented during preg- mammary tissue and milk, as well as in the kid-
nancy (Donnet-Hughes et al. 2010). This ney, liver and spleen (de Andrés et al. 2018).
translocation may be mediated by the complex Amniotic fluid is particularly important with
bacteria-immune system relations that are, in regard to studying the effect of the microbiota on
turn, mediated by the dendritic cells in the GI the neonate’s health, since it is continually
tract (Rescigno et al. 2001). Studies conducted swallowed by the foetus and, thus, creates a con-
in animal models have reported that orally tact with the foetal gut throughout gestation. In
administered Enterococcus faecium was found medical practice, it is thought that a bacterium
in both the placenta tissue and meconium within the amniotic fluid represents an unhealthy
(Jiménez et al. 2008). A similar model reported condition due to causing an amniotic cavity infec-
the physiological translocation of ingested tion, namely, chorioamnionitis, which is the
12 M. Selma-Royo et al.

cause of the approximately 25% of preterm births to those found in the placenta, principally the
(Romero et al. 2014). Indeed, some authors have Proteobacteria, especially the Enterobacter and
reported that the bacterial composition of the Shigella genera, and Propionibacterium species.
amniotic fluid in chorioamnionitis patients could However, little is currently known about the role
be used as a biomarker of the stage of inflamma- of these bacteria during the maternal-foetal inter-
tion (Urushiyama et al. 2017). Due to the associ- phase in terms of foetus development and infant
ation between amniotic fluid bacteria and preterm health in the early years of life.
birth, it has been suggested that the study of the The presence of bacterial DNA in the placenta
amniotic fluid microbiome in such cases could and amniotic fluid suggested by some authors has
represent an essential tool in terms of preventing opened the door to systematic studies concerning
preterm birth or even studying the possible the importance of the pregnancy period to coloni-
molecular mechanisms by which specific bacte- zation process, suggesting that this could be
rial populations could trigger negative outcomes initiated in utero. A growing body of data suggest
in pregnancy (Urushiyama et al. 2017). A differ- the presence of bacteria in the meconium, the first
ent microbial population has previously been neonatal stool that represents in utero life, as
found in the amniotic fluid of preterm mothers, determined by culture-dependent and culture-
featuring the notable presence of Ureaplasma and independent methodologies (Gosalbes et al.
Mycoplasma species (DiGiulio et al. 2008, 2010; 2013; Hansen et al. 2015; Jiménez et al. 2008),
Combs et al. 2014). At the same time, Prince et al. which suggests microbial colonization prior to
observed a different placental microbiome in pre- birth. However, some authors have suggested
term mothers with severe chorioamnionitis, in that neonatal colonization could start during
this case dominated by Ureaplasma and uterus contraction and membrane rupture
Fusobacterium, as well as term infants with the (Rehbinder et al. 2018), thereby explaining the
same disease (Prince et al. 2016). As mentioned presence of bacteria in the meconium. A recent
in the case of the placenta, some studies have study reported that infants born via elective
detected bacterial DNA in the amniotic fluid of caesarean section showed a similar meconium
healthy mothers (DiGiulio et al. 2008). However, microbiota profile to those born via vaginal deliv-
the hypothesis concerning the bacterial presence ery and non-elective caesarean section (Chu et al.
in the amniotic cavity of healthy individuals 2017). The meconium microbiota is composed of
remains controversial, partially due to contradic- species from the Proteobacteria and Firmicutes
tory prior observations (Rehbinder et al. 2018; phyla, such as the Enterobacteriaceae and
Lim et al. 2018). There have also been studies Enterococcaceae families (Moles et al. 2013;
that identified no microorganisms in the amniotic Hansen et al. 2015), which are typically present
fluid. In their analysis, Rehbinder et al. concluded in the microbiota during adulthood to a greater
that the concentrations of prokaryotic DNA in the degree than during infancy, reflecting the mater-
amniotic fluid stemming from intact membranes nal origin of such bacteria. In addition, other
were comparable to those found in negative genera were also found, including
controls. Indeed, it was only in the amniotic Bifidobacterium, Bacteroides and Prevotella
fluid obtained from ruptured membranes that (Hansen et al. 2015). Indeed, it has been observed
was possible to perform the anaerobic isolation that the meconium microbiota shares more
of bacteria by means of anaerobic culturing similarities with the amniotic fluid than with the
(Rehbinder et al. 2018). However, other studies maternal vaginal or oral microbiota (Ardissone
have found bacterial DNA in the amniotic fluid et al. 2014), which may reflect the origin of the
obtained from healthy and term pregnancies with- meconium bacteria. Recent animal studies have
out clinical or anatomopathological infection also noted the microbes shared between the foetal
symptoms (Aagaard et al. 2014; Collado et al. gut and the placenta (Martinez et al. 2018).
2016). The microbial populations detected in A growing body of evidence has highlighted
‘healthy’ amniotic fluid should be very similar the impact of different maternal factors on the
Shaping Microbiota During the First 1000 Days of Life 13

meconium microbiota, including diabetes products and metabolites can reach the systemic
(Hu et al. 2013), atopic disease (Gosalbes et al. circulation and hence arrive in different places.
2013), maternal diet (Ma et al. 2014; Chu et al. Therefore, the metabolites derived from the
2016), BMI (Mueller et al. 2016) and perinatal maternal microbiota may cross the placenta and
antibiotic use (Mueller et al. 2015; Gosalbes et al. be present in both the amniotic fluid and breast
2016). Briefly put, the meconium of infants born milk (Gomez de Agüero et al. 2016). These
to mothers with pre-gestational type 2 diabetes observations highlight the potential impact of
mellitus harboured an enrichment of bacteria nor- microbes and their metabolites during gestation
mally observed in adult diabetic patients (Hu et al. and, further, suggest the need to increase our
2013). A high-fat maternal diet influences the knowledge in order to promote infant health.
meconium microbiota with regard to a notable
depletion of the Bacteroides genus (Chu et al.
2016). Nogacka et al. showed that intrapartum 5 Postnatal Bacterial Exposition:
antibiotic use could alter the intestinal microbiota From Birth to the First Years
during the first week of life, including a lower of Life
presence of Actinobacteria and an incremental
increase in the Proteobacteria and Firmicutes A growing body of evidence has demonstrated
proportions (Nogacka et al. 2017, 2018). Indeed, the vertical bacterial transmission from mothers
another study reported that antibiotics to their children (Dominguez-Bello et al. 2010;
administered during delivery could also modify Asnicar et al. 2017; Duranti et al. 2017). During
the oral microbiome in neonates, thereby birth, the neonate encounters an immense quan-
resulting in a higher abundance of families from tity and diversity of microbes, as well as other
the Proteobacteria phylum following the antibi- organisms from the vagina and maternal gut, in
otic treatment (Gomez-Arango et al. 2017a, b). addition to those from the environment. Neonatal
This could have important consequences in terms colonization is a sensitive and dynamic step-wise
of child development. Relatedly, Mueller et al. process that is particularly relevant to health. The
observed that children who were exposed to anti- process could be influenced by maternal and neo-
biotic treatment during the second and third natal factors, including the mode of delivery,
trimesters of gestation had an 84% higher risk of prematurity and early breastfeeding practices
developing obesity (Mueller et al. 2015). (Rodríguez et al. 2015; Gomez-Gallego et al.
However, it remains unclear how long the 2016). The neonatal gut microbiota is
meconium shifts would be maintained for during characterized by a low microbial diversity and
the early years of life. Additionally, the potential richness as well as a relative higher abundance
effects on the immune system, metabolism and of Proteobacteria and Actinobacteria phyla. Sub-
health outcomes are currently unknown. More- sequently, the gut microbiota evolves, and it
over, the molecular mechanisms by which the becomes more diverse, resembling the adult com-
placenta, amniotic fluid and meconium position by between 2 and 5 years of age
microbiota affect immune system development (Rodríguez et al. 2015).
are not yet fully understood despite the growing It has previously been demonstrated that
body of evidence concerning the immunomodu- neonates born vaginally have a microbiota resem-
latory activity of the gut microbiota in disease and bling the vaginal microbiome, enriched with Lacto-
health during both childhood and adult life (Nash bacillus and Prevotella species (Dominguez-Bello
et al. 2017; Dzidic et al. 2018). et al. 2010), although other bacteria, such as the
A recent study showed that the microbial Enterobacteriaceae family, including Escherichia
metabolites in the foetal environment can have a or Klebsiella, are also present. Neonates born via
major impact on neonatal development and caesarean section presented a distinct microbiota
immune system maturation (Gomez de Agüero similar to the oral, skin and environmental
et al. 2016). It has been established that microbial microbiota dominated by Staphylococcus,
14 M. Selma-Royo et al.

Corynebacterium and Propionibacterium 6 Tools to Modulate

(Dominguez-Bello et al. 2010). The maternal gut the Microbiota During Early
also appears to be an important source of early Life: From Conception
colonizing bacteria, since 72% of gut bacteria in to 2 years of Life
vaginally delivered newborns have been found to
be of maternal intestinal origin as compared to 41% Diet is a key factor in terms of shaping our gut
in subjects born via caesarean section (Bäckhed microbiota (Graf et al. 2015). Dietary and
et al. 2015). Several prior studies have reported nutritional counselling, as well as nutritional sup-
that infants born via caesarean section exhibited plementation, are hence recommended in order to
delayed colonization of the Bacteroides genus as achieve an adequate nutritional status during the
well as reduced diversity (Jakobsson et al. 2014). preconception and gestational stages (Scholl
After birth, breastmilk both supports the micro- 2008). It has recently been reported that the
bial gut colonization and drives the immune system maternal diet during pregnancy has a key impact
maturation (Riskin et al. 2012; He et al. 2014; on both the maternal and infant microbiota
Turfkruyer and Verhasselt 2015; Cacho and (Mandal et al. 2016; Barrett et al. 2018; Lundgren
Lawrence 2017). Differences between breastfed et al. 2018). However, it remains to be established
and formula-fed neonatal microbiota have been which foods, food components, nutrients and
reported (Guaraldi and Salvatori 2012; Madan other dietary compounds affect the microbiota
et al. 2016; Pannaraj et al. 2017), although recent during the perinatal period (García-Mantrana
studies have shown that the cessation of et al. 2016). Taken together, these observations
breastfeeding, rather the introductions of comple- explain the increasing research interest in perina-
mentary and solid food, had a key impact on the tal dietary interventions, as well as in the use of
infant microbiome (Bäckhed et al. 2015). probiotics, prebiotics and postbiotics and
Traditionally, human milk was considered to synbiotics during pregnancy in order to modulate
be sterile, although an increasing amount of evi- the microbiota and promote a ‘favourable micro-
dence has shown the presence of Staphylococcus, bial vertical transmission’. There are some
Streptococcus and certain Proteobacteria (Pseu- indications of the protective role of probiotics
domonas and Acinetobacter) in breast milk when administered during gestation in relation
samples. Moreover, Bifidobacterium and Lacto- to vaginal infections, maternal and infant weight
bacillus have also frequently been identified and gain, preeclampsia, gestational diabetes mellitus,
isolated (Gomez-Gallego et al. 2016). A recent lactational mastitis and, also, allergic diseases.
study showed that the infant gut derives 27.7% of
its bacteria from breast milk and 10.3% from the
areola skin (Pannaraj et al. 2017), thereby
highlighting the potential impact of breastfeeding 6.1 Impact of Probiotics on Fertility
on the infant gut microbiota. In addition, shared
microbial and viral species between breast milk Vaginal infections affect the Lactobacillus equi-
and the infant gut have been identified (Martín librium, favouring the growth of other bacteria
et al. 2009; Pannaraj et al. 2018). that could have an effect on fertility
Other factors have also been widely studied in (Mastromarino et al. 2014). Hence, supplementa-
this regard, such as antibiotics, gestational age, host tion with Lactobacillus should help to re-establish
genetics and other environmental factors, including the equilibrium. An in vitro fertilization (IVF)
geographical location, rural areas, siblings, pet own- study involving healthy women showed
ership, etc. (Rodríguez et al. 2015; Milani et al. promising results following vaginal colonization
2017; Chong et al. 2018). These factors, individu- with L. crispatus (Sirota et al. 2014). However, a
ally or in combination, have been shown to exert an pilot study showed that a vaginal probiotic with
impact on the microbiota establishment and L. acidophilus, Bifidobacterium bifidum and
immune system maturation (Dzidic et al. 2018). B. longum at the time of egg retrieval for IVF
Shaping Microbiota During the First 1000 Days of Life 15

was not linked to either an increase in the Lacto- vulvovaginal candidiasis (VVC) and bacterial
bacillus levels in the vagina or improved preg- vaginosis (BV). These problems have been linked
nancy outcomes (Gilboa et al. 2005). to adverse maternal-neonatal pregnancy
Differences in the semen microbiota composi- outcomes, including preterm birth risk and foetal
tion have been reported in cases of infertility and growth restriction. Probiotics have been proposed
assisted reproductive medicine (Moretti et al. 2009). as an attractive prophylactic alternative for
Several studies involving the use of probiotics in treating BV and UTIs (Stapleton et al. 2011;
animal models have shown the potentially benefi- Homayouni et al. 2014), either by maintaining
cial effect of probiotics in terms of semen or restoring the vaginal microbiota or preventing
characteristics and quality parameters (Dardmeh pathogens from colonizing the urinary tract.
et al. 2017; Inatomi and Otomaru 2018). In humans, Prior studies have shown the potential thera-
however, the knowledge in this regard needs to be peutic effect of a probiotic combination, namely,
expanded. A recent study showed the effect of Lactobacillus acidophilus, Bifidobacterium lactis
Lactobacillus rhamnosus CECT8361 and and Bifidobacterium longum, on Group B Strep-
Bifidobacterium longum CECT7347 supplementa- tococcus (GBS) colonization in pregnant women
tion over a 6-week period in terms of the improve- (Hanson et al. 2014). Recently, a clinical study
ment of sperm quality parameters and motility, as reported that supplementation with L. rhamnosus
well as the decrease of DNA fragmentation and GR-1 and L. reuteri RC-14 proved effective in
reactive oxygen species (ROS) levels, in treating GBS infection in pregnant women
asthenozoospermic males (Valcarce et al. 2017). (Ho et al. 2016). Further, a recent pilot study
Another study reported the impact of synbiotics involving 36 GBS-positive women reported a
(Flortec, Lactobacillus paracasei B21060 and significant increase in the vaginal commensals
arabinogalactan, fructooligosaccharides and having an impact on vaginal GBS during preg-
l-glutamine) on the semen quality and quantity of nancy (Olsen et al. 2018).
patients with oligoasthenoteratospermia (Maretti
and Cavallini 2017). Preeclampsia A recent study involving 70,149
pregnancies found that the consumption of
probiotics (Lactobacillus acidophilus [LA-5],
6.2 Probiotics During Gestation Bifidobacterium lactis [Bb12] and Lactobacillus
rhamnosus GG) during late pregnancy signifi-
Overall, the administration of probiotics during cantly reduced the risk of preeclampsia
pregnancy would offer an interesting alternative (Nordqvist et al. 2018). This study also
for influencing the maternal-foetal health risks highlighted the relevance of ‘timing’ (i.e. when
experienced during pregnancy. In addition to no the probiotic intervention was performed), since
incremental improvement being seen in terms of no effect was observed when the probiotics were
adverse pregnancy outcomes following probiotic consumed prior to, or during early, pregnancy.
intervention, randomized probiotic trials during
pregnancy remain scarce. Those studies that do Gestational Diabetes Mellitus (GDM)
exist have mostly focused on the risk of preterm L. rhamnosus GG and B. lactis BB12 combined
birth and the need to reduce genital infections and with dietary counselling have been shown to pre-
pathogenic invasion, gestational diabetes mellitus vent GDM, reduce plasma glucose concentrations
(GDM) and preeclampsia (Reid et al. 2013; Jarde and improve insulin sensitivity (Laitinen et al.
et al. 2018), which are occasionally considered to 2009; Okesene-Gafa et al. 2018). Another probi-
be secondary health outcomes in clinical trials. otic strain, namely, the L. rhamnosus HN001
strain, was able to reduce the prevalence of
Genito-Urinary Tract Infections and Pathogen GDM, particularly among older women and
Invasion Pregnant women often suffer from those with previous GDM (Wickens et al. 2017).
recurrent urinary tract infections (UTIs),
16 M. Selma-Royo et al.

A recent meta-analysis of 4 high-quality (Goldenberg et al. 2015, 2017; Szajewska et al.

randomized controlled trials (RCTs) involving 2016), (ii) allergy prevention (Chang et al. 2016;
288 participants (Taylor et al. 2017) showed that Huang et al. 2017; Garcia-Larsen et al. 2018), (iii)
probiotics were able to significantly reduce the the prevention of necrotizing enterocolitis and
HOMA-IR level, although no effect was observed late-onset sepsis in preterm neonates (van den
in terms of reducing the fasting glucose and Akker et al. 2018; Patel and Underwood 2018)
LDL-cholesterol levels in women with GDM. A and (iv) the prevention of infant colic (Sung and
recent systematic review including more than Cabana 2017; Sung et al. 2018).
30 studies conducted during pregnancy showed However, a recent systematic review
the potential effect of probiotic intervention on highlighted the potential benefits of inactivated
the improvement of glycaemic control, the reduc- probiotic bacteria, postbiotics, that provide a
tion of VDL cholesterol and triglycerides and the safe alternative during the very early and sensitive
decrease of inflammatory markers (Dallanora period of life, as well as in sensitive populations,
et al. 2018). such as preterm neonates (Deshpande et al.
2018).
Preterm Risk While the use of probiotics has Furthermore, another example of the postnatal
been shown to represent a good alternative for use of probiotics is related to mastitis, including
the treatment of vaginal infections during preg- infectious mastitis and Staphylococcus mastitis
nancy, there is currently insufficient data (Angelopoulou et al. 2018). The probiotics used
concerning the impact on preterm birth (Othman in this case belong to the Lactobacillus genus
et al. 2007). However, a recent study reported that (L. salivarius, L. gasseri and L. fermentum),
a probiotic combination during late pregnancy which has also been identified and isolated from
was able to reduce the risk of preterm birth healthy human breast milk samples.
(Nordqvist et al. 2018). Furthermore, as men-
tioned in this review, the oral microbiota during
pregnancy has been linked to the risk of preterm
7 Conclusions
birth, indicating that the potential use of
prebiotics (xylitol) (Gueimonde et al. 2016) and
Microbes and their human hosts have co-evolved
probiotics (Toiviainen et al. 2015; Schlagenhauf
over the last couple of hundred thousand years,
et al. 2016) could be useful, although further
and microbes are strongly associated with human
scientific evidence during gestation is needed.
health. Prior to conception, male and female
reproductive microbes play a key role in fertility,
and they can have an impact during conception.
6.3 Probiotics During the Postnatal Changes in the maternal microbes are seen during
Period: From Birth to 2 Years pregnancy in parallel to the experienced physio-
of Life logical and hormonal changes, and they are
required to enhance immune tolerance during
Extensive data and several studies reporting the pregnancy. Microbes have been identified in
findings of RCTs, systematic reviews and meta- known sterile areas, such as the uterus, endome-
analyses are available in the literature. Evidence trium, fallopian tubes and semen, as well as in the
concerning the beneficial effect of specific maternal-foetal interphase, such as the placenta
probiotics, such as Saccharomyces boulardii, and amniotic fluid, and in the breast tissue and
Lactobacillus rhamnosus GG (LGG), Lactobacil- breast milk. All these data suggest the pivotal
lus reuteri and Bifidobacterium animalis lactis impact of early microbial exposition at concep-
(Bb12), has been reported. The main targets of tion and during gestation, although further evi-
probiotic use during early infancy have been dence derived from well-designed studies
(i) diarrhoea and infection prevention controlling for potential contamination and
Shaping Microbiota During the First 1000 Days of Life 17

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Acknowledgements Authors would like to acknowledge Bäckhed F, Roswall J, Peng Y et al (2015) Dynamics and
the European Research Council (ERC) under the European stabilization of the human gut microbiome during the
Union’s Horizon 2020 research and innovation program first year of life. Cell Host Microbe 17:690–703.
(ERC Starting Grant, project no. 639226). MSR would https://doi.org/10.1016/j.chom.2015.04.004
acknowledge the support from ACIF program of Barak S, Oettinger-Barak O, Machtei EE et al (2007)
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# Springer Nature Switzerland AG 2019
Published online: 26 January 2019

Necrotizing Enterocolitis and the Preterm

Infant Microbiome

Jillian R. Baranowski and Erika C. Claud

Abstract also be considered, including breastfeeding,

Bacterial colonization patterns in preterm prebiotics, and targeting the maternal
infants differ from those of their term microbiome.
counterparts due to maternal microbial diver-
sity, delivery mode, feeding methods, antibi- Keywords
otic use, and exposure to commensal Gastrointestinal microbiome · Necrotizing
microbiota and pathogens in the neonatal enterocolitis · Preterm birth · Probiotics · Very
intensive care unit (NICU). Early gut low birth weight infant
microbiome dysbiosis predisposes neonates
to necrotizing enterocolitis (NEC), a
devastating intestinal disease with high mor- 1 Introduction
bidity and mortality. Though mechanisms of
NEC pathogenesis are not fully understood, Necrotizing enterocolitis (NEC) is a devastating
the microbiome is a promising therapy target infant disease common in neonatal intensive care
for prevention and treatment. Direct adminis- units (NICUs) across the globe. Characterized by
tration of probiotics to preterm infants has ischemic bowel necrosis, NEC has long been linked
been shown to reduce the incidence of NEC, to microbial dysbiosis in premature infants (Claud
but is not without risk. The immature immune and Walker 2001), and NEC risk factors continue to
systems of preterm infants leave them vulner- be actively studied in hopes of developing improved
able to even beneficial bacteria. Further therapies for prevention and treatment. Given its
research is required to investigate both short- important role in NEC pathogenesis, the
term and long-term effects of probiotic admin- microbiome presents a promising target for therapy.
istration to preterm infants. Other methods of This chapter will first offer a broad overview of the
altering the preterm infant microbiome must human microbiome, the development of the infant
microbiome, and specific factors impacting preterm
infant colonization. From there, the focus will shift
J. R. Baranowski
to an overview of NEC and the role of the preterm
Pritzker School of Medicine, University of Chicago,
Chicago, IL, USA microbiome in developing NEC. Finally, research
e-mail: [email protected] investigating probiotic administration to preterm
E. C. Claud (*) infants for prevention of NEC will be critically
Department of Pediatrics, The University of Chicago reviewed and alternatives for altering the
Pritzker School of Medicine, Chicago, IL, USA microbiome will be proposed.
e-mail: [email protected]

25
26 J. R. Baranowski and E. C. Claud

2 The Human Microbiome on delivery mode, feeding methods, environmen-

tal factors, early use of antibiotics, and potentially
The human microbiota is a collective term for all even intrauterine transmission (Perez-Muñoz
organisms colonizing the human gut, skin, mouth, et al. 2017).
and urogenital tract, outnumbering human Mode of delivery has consistently been found
somatic cells but comprising less than 1% of to influence early patterns of colonization. The
total body mass on average (Sender et al. 2016). vaginal microbiome shifts toward predominance
The microbial community is home to thousands of Lactobacillus spp. during pregnancy, which
of unique bacterial species that play a critical role may provide defense against pathogenic bacteria
in maintaining homeostasis (Eckburg et al. 2005), through maintenance of a low pH (Romero et al.
including development and maintenance of mul- 2014). Infants born vaginally are colonized most
tiple organ systems, such as the gut, endocrine readily by the mother’s vaginal microbiota, while
system, lungs, liver, bone, and brain. Microbial the microbiomes of infants born by cesarean sec-
colonization begins around the time of birth and tion most resemble microbiota found on their
stabilizes by around 3 years of age, when a mothers’ skin in early days of life (Dominguez-
toddler’s diet begins to resemble that of adult Bello et al. 2010). Additionally, infants delivered
counterparts (Bergström et al. 2014). Dysbiosis via cesarean section were found to have less gut
early in life, or a shift toward pathogenic imbal- microbial resemblance to their mothers in the first
ance of microbes, has been implicated in a num- year of life (Bäckhed et al. 2015) and a notable
ber of disease states including food sensitivities, lack in diversity of Bacteroides species that was
allergy (Azad et al. 2015), type I diabetes, auto- present in most vaginally delivered babies
immune disorders (Kostic et al. 2015), and obe- (Yassour et al. 2016).
sity (Tilg and Kaser 2011). Feeding method and diet also impact
The microbiome presents a unique target for microbiome development in multiple ways.
therapy, because it can be altered quickly and Formula-fed infants have been found to exhibit
reversibly in most cases. Direct administration of an overrepresentation of less favorable bacteria
probiotics, live organisms that confer a health species, such as Clostridium difficile, when com-
benefit to the host, is just one example of how pared to exclusively breastfed infants (Azad et al.
the microbiome can be manipulated to improve 2013). Breastfeeding promotes development of a
health outcomes. Fecal transplantation, prebiotic microbiome that more closely resembles that of
supplementation, and restriction of prophylactic the mother, with contributions both from the
antibiotics are also potential targets for altering contents of breast milk and through contact with
the microbiome. Alteration of maternal the areolar skin (Pannaraj et al. 2017). Human
microbiome during pregnancy and breastfeeding breast milk contains diverse genera of bacteria
also presents a promising therapy target in infants. with up to 70–88% found to be shared between
maternal milk and infant fecal samples (Murphy
et al. 2017). Maternal diet may also affect vertical
transmission of microbiota through breastfeeding.
3 Basics of the Infant One study found differences between South
Microbiome Asian and Caucasian infant cohorts at 1 year of
age, with South Asians exhibiting higher abun-
Rapid bacterial colonization of the infant intesti- dance of Lactobacillales and Caucasians having
nal tract begins around the time of birth, with higher abundance of Clostridiales, with
microbes transferred from the mother and the differences attributed to maternal and infant diet
environment during delivery and shortly thereaf- variation (Stearns et al. 2017).
ter (Bäckhed et al. 2015). Multiple factors con- Environmental factors and treatment with
tribute to early microbial colonization, including antibiotics also play a major role in early coloniza-
vertical transmission during delivery dependent tion. One study found a correlation between the
Necrotizing Enterocolitis and the Preterm Infant Microbiome 27

number of older siblings at home and greater infant the preterm infant. Preterm and low birth weight
microbial diversity, specifically from the phyla (LBW) infants exhibit lower microbial diversity
Firmicutes and Bacteroidetes (Laursen et al. across multiple body sites when compared to
2015). Infants treated with antibiotics are found to age-matched full-term infants (Costello et al.
have less bacterial diversity, in both species and 2013). One metagenomic study found that the
strain, sometimes with a single strain dominating a same founding bacterial populations colonized
species (Yassour et al. 2016). Maternal treatment preterm infant skin, mouth, and gut,
with antibiotics prior to delivery has also been hypothesizing that the overlap across sites
associated with a decrease in infant microbial diver- contributes to overall lower diversity (Olm et al.
sity, specifically lacking Bifidobacterium species, a 2017). This lack of diversity may allow for path-
genus generally regarded as favorable (Aloisio ogenic organisms to grow unchecked and more
et al. 2014). easily produce the conditions which lead to
With all these factors in mind, what should be disease.
considered a “healthy” infant microbiome? There Other factors contributing to lower diversity
is great variation even among healthy infant and dysbiosis in preterm infants include the
microbiomes, but generally high levels of diver- determinants listed previously that are known to
sity are considered protective, because diversity impact early colonization, such as delivery mode,
prevents overgrowth of any single dominant feeding method, and use of antibiotics. According
organism (Wang et al. 2009). Dysbiosis occurs to the Centers for Disease Control and Prevention
when unfavorable microbes become dominant (CDC) in 2016, the rate of cesarean birth for all
and increase the risk of disease. Many different live births was 31.9% (Martin et al. 2018). How-
bacteria species have been implicated in a variety ever, from 2007 to 2016 (or in the period between
of disease pathologies, as will be discussed in 2007 to 2016), the rate of cesarean delivery in
later sections. preterm births (<37 weeks’ gestation) was 47.1%
Early microbial colonization has been tied to (CDC 2018). As discussed previously, cesarean
development of proper anatomic structures and birth may lead to less favorable patterns of early
physiologic responses, most notably immunologic, colonization when compared to vaginally born
metabolic, and neurologic. Decreased colonization infants. Preterm and LBW infants are also less
with Lactobacillus and Bifidobacterium spp. in likely to breastfeed due to decreased ability to
infancy is associated with greater risk of develop- suck and swallow (Dodrill et al. 2008), further
ing allergy later in life (Sjögren et al. 2009). Addi- inhibiting the trajectory of protective coloniza-
tionally, decreased Bifidobacterium spp. along tion. Furthermore, an estimated 60–75% of
with increased Staphylococcus aureus in infancy mothers of preterm neonates are treated with
is associated with childhood obesity (Kalliomäki antibiotics during pregnancy (Stoll et al. 2015)
et al. 2008). Commensal microbiota also play a as compared to the overall rate of antibiotic treat-
role in proper brain development. Gnotobiotic ment during pregnancy which is around 37%
mouse studies have found that alteration of the (Stokholm et al. 2013; Broe et al. 2014).
microbiome leads to altered expression of insulin- Antibiotics represent the most common class of
like growth factor 1 (IGF-1) (Lu et al. 2018) which medications prescribed in the NICU (Clark et al.
is associated with perinatal growth. Low IGF-1 is 2006), despite great variability in prescribing
associated with poor growth and cognitive practices. The extensive treatment of both
impairment (Walenkamp et al. 2005). mothers and preterm infants with antibiotics has
the potential to reduce microbial diversity, lead-
ing to emergence of dominant pathogenic strains.
4 Preterm Bacterial Colonization Another factor affecting the preterm infant
Patterns microbiome is the vaginal microbiome of the
mother. Studies of the vaginal microbiome have
With this broad understanding of how the found that preterm delivery is associated with
microbiome develops early in life, now consider decreased microbial diversity during pregnancy
28 J. R. Baranowski and E. C. Claud

(Hyman et al. 2014; Stout et al. 2017). This 5 The Microbiome and NEC
finding is not only significant for its possible
future applications in predicting and preventing Necrotizing enterocolitis is ischemia of the gas-
preterm birth, but it has implications for the trointestinal (GI) tract with subsequent necrosis of
important role that vaginal microbes play as the the intestines and characteristic pneumatosis
first source of colonization for an infant. It stands intestinalis (Bell et al. 1978; Walsh and Kliegman
to reason that even if a preterm infant is delivered 1986). NEC primarily affects premature infants,
vaginally, they may not be exposed to as rich a first presenting with nonspecific symptoms and a
vaginal microbiome as their term counterparts. distended abdomen (Neu 2014). Martin Bell and
Most preterm infants will spend their first days associates from Washington University School of
of life in the NICU, so it is also important to Medicine and St. Louis Children’s Hospital first
examine the unique environmental factors that proposed three stages and staging criteria for
contribute to dysbiosis. For example, common NEC in 1978. Stage I is the “suspect” stage,
surfaces that infants come in contact with in the characterized by GI manifestations and abdomi-
NICU, such as positive pressure airway nal distension on radiograph. Stage II, the “defi-
machines, ventilators, incubators, and even nite” stage, is marked by GI bleeding and
pacifiers, have been found to harbor Streptococ- multiple radiographic findings including signifi-
cus, Staphylococcus, Neisseria, and Enterobac- cant intestinal distention, ileus, edema,
teriaceae communities (Bokulich et al. 2013). pneumatosis intestinalis, and portal vein gas.
There is evidence to suggest that these colonies Finally, Stage III is the advanced stage of the
serve as reservoirs for infant gut colonization disease, progressing to rapid deterioration,
(Brooks et al. 2014), contributing to early bowel necrosis, septic shock, GI hemorrhage,
dysbiosis and nosocomial infections. Common and pneumoperitoneum on radiograph. Stage III
NICU treatments may also contribute to early requires operative intervention for resection of
dysbiosis. In an evaluation of preterm fecal necrotic intestine (Bell et al. 1978). Although
microbiota, one study found that Bacilli, the criteria have been periodically modified and
Gammaproteobacteria, and Clostridia were the despite criticism that they are outdated, use of
most abundant taxa present, with a fairly consis- Bell’s staging criteria remains standard practice
tent pattern first dominated by Bacilli, then (Gregory et al. 2011; Gordon et al. 2012).
Gammaproteobacteria, and finally Clostridia NEC affects up to 13% of preterm neonates in
(Grier et al. 2017). The study looked at the pro- developed countries with an increasing incidence
gression of gut microbiota by post-menstrual age rate over time, likely reflecting the increasing
and a variety of other factors that preterm survival rate of extremely preterm infants (Stoll
neonates face. Most notably, they demonstrated et al. 2015). Mortality rates are highest in the least
that treatment with histamine receptor blockers mature infants, with overall mortality between
(H2-blockers) or proton pump inhibitors (PPI), 20% and 30% (Fitzgibbons et al. 2009).
both of which act to reduce production of stom- Although systemic bacterial infection is not
ach acid, correlated with increased abundance of common in NEC (Clark et al. 2012), microbial
Proteobacteria, Actinobacteria, and dysbiosis and inflammation are thought to play a
Bacteroidetes, while treatment with antibiotics critical role in the pathogenesis of NEC, though
correlated to decreased abundance of Firmicutes the mechanisms are not fully understood. A
and increased abundance of Proteobacteria developing hypothesis is that NEC may have
(Grier et al. 2017). This highlights the under- several unique pathologies that all converge to a
standing that the microbiome of preterm neonates common inflammatory pathway with microbiome
is susceptible to shifts related to common dysbiosis underlying them all. Many risk factors
treatments encountered in the NICU. have been investigated extensively.
Necrotizing Enterocolitis and the Preterm Infant Microbiome 29

Rates of NEC are higher in infants delivered appears to be relative abundance and emergence
via cesarean section, in those who are “small for of a single dominant species (Wang et al. 2009).
gestational age,” and in neonates with bacterial Many supporting studies have found an increased
infections. Cardiac malformations, GI relative abundance of bacteria from the phylum
malformations, and chromosomal abnormalities Proteobacteria in the stool of infants who
are also associated with higher rates of NEC develop NEC, with others more specifically
(Ahle et al. 2018). Additionally, treatment with reporting increased abundance of Gammaproteo-
H2-blockers is positively associated with inci- bacteria (Pammi et al. 2017), such as
dence of NEC (Bilali et al. 2013). On the other Cronobacter sakazakii, Klebsiella sp., and
hand, breast milk intake by premature infants has Escherichia coli (Coggins et al. 2015). From the
been found to be protective against NEC, with a phylum Firmicutes, coagulase-negative
dose-related reduction in risk of developing and staphylococci (CoNS) are often found in the
dying from NEC (Meinzen-Derr et al. 2009). stool of infants with NEC (Stewart et al. 2012)
Consistent with these risks and protective and may take advantage of the reduced intestinal
factors, lack of bacterial diversity contributes to barrier function as a result of parenteral nutrition
development of NEC. One study investigating (Kansagra et al. 2003). Clostridium spp. and
microbiome diversity using the Shannon diversity associated toxins are also often found in abun-
index (abundance and distribution of microbes dance in the stool of neonates with NEC and have
present) found that preterm neonates who went been associated with more severe outcomes and
on to develop NEC had lower diversity scores faster progression of NEC (Kosloske et al. 1978;
than their healthy preterm counterparts, although Kosloske and Ulrich 1980), despite being part of
all preterm infants demonstrate low overall diver- the commensal microbiome of preterm neonates
sity. The NEC group infants were individually (Ferraris et al. 2012). Though not the focus of this
found to have dominant Gammaproteobacteria discussion on probiotics, it is worth mentioning
genera, representing more than 50% of their bac- that other nonbacterial microbes may also play a
terial colonization. This was not found to be true role (both protective and pathogenic) in develop-
in the healthy controls. In considering possible ment of NEC, specifically virus-associated NEC
mechanisms, infants in the NEC group were and Candida (Coggins et al. 2015).
found to have more days of antibiotic therapy Considering the extensive list of microbes and
prior to developing NEC, which likely broad range of results collected across multiple
contributed to this relative lack of diversity as studies, it is unlikely that a single pathogen will
compared to control (Wang et al. 2009). Another be isolated that is entirely responsible for NEC.
multisite, retrospective cohort study found an More important is understanding the elements of
association between prolonged antibiotic therapy the microbiome that may help develop the host
and NEC, including that odds of NEC increased immune system and provide protection against
with each additional day of antibiotic treatment NEC and other diseases.
(Cotten et al. 2009). These findings are consistent
with the hypothesis that reducing microbial diver-
sity puts babies at greater risk for pathogenic
6 Administration of Probiotics
colonization and NEC.
to At-Risk Infants
While live bacteria appear to be required for
to Prevent NEC
the pathogenesis of NEC (White et al. 2017),
specific organisms have not been isolated with
It stands to reason that if microbial dysbiosis is
consistency. That said, many organisms have
responsible, at least in part, for developing NEC,
been implicated in NEC, mainly from the phyla
altering a preterm infant’s microbiome to favor
Firmicutes and Proteobacteria, but the cited
healthy diversity may prevent NEC. Prophylactic
organisms are also found in healthy infants. The
administration of probiotics to preterm infants to
key difference in determining pathogenesis
30 J. R. Baranowski and E. C. Claud

protect against NEC is certainly a promising area increased toll-interacting protein (TOLLIP) and
of study, but it remains controversial. single Ig IL-1-related receptor (SIGIRR), respon-
In the 2017 meta-analysis by Chang et al., sible for moderating inflammation and homeosta-
randomized control trials (RCT) were compared sis in the colonic epithelium, respectively
to investigate the efficacy of single-strain versus (Wu et al. 2017).
multistrain probiotics. They found that multistrain Despite the evidence suggesting that probiotic
probiotics led to a significant reduction in NEC treatment can reduce the incidence of NEC, fur-
incidence, while single-strain probiotics exhibited ther clinical testing should be conducted before
borderline effects (Lactobacillus species) or no concrete recommendations are formulated. While
benefit at all (Bifidobacterium species) in reduc- many RCTs and retrospective cohort studies have
ing NEC or mortality (Chang et al. 2017). Single- been performed to study the effects of probiotic
strain probiotics do not increase microbiome administration on incidence of NEC, many
diversity, so it is not surprising that they were finding beneficial effects of probiotics, the medi-
not found to confer benefit. In a recent RCT cal evidence is yet insufficient. Primary concerns
conducted at a tertiary care unit in Turkey since include that most studies are underpowered,
this latest meta-analysis, the multistrain hypothe- probiotics of different bacterial strains are diffi-
sis was supported. The study administered a com- cult to compare in meta-analysis, there is lack of
mercially prepared, combined prebiotic long-term outcome measures, and there remain
(fructooligosaccharides and galactooligosac- significant studies that suggest probiotics confer
charides) and probiotic (Lactobacillus no protection against NEC or sepsis (Neu 2014).
rhamnosus, Lactobacillus casei, Lactobacillus
plantarum, and Bifidobacterium animalis) to pre-
mature infants in high doses and compared NEC
7 Risks of Administering
rates and mortality rates to control (no pre- or
Probiotics to Premature Infants
probiotics). The study found significantly lower
rates of NEC and mortality in the probiotics group
Administering probiotics to premature and very
(Güney-Varal et al. 2017). This evidence suggests
low birth weight (VLBW) infants is not without
that combined prebiotic-probiotic therapy with
risks. These infants are already at higher risk for
multistrain probiotics may lead to more favorable
many adverse health outcomes; therefore
outcomes than single-strain probiotic treatments
colonizing them with even nonpathogenic bugs
alone. As of yet, controlled studies have not been
should be done with great care. Among the major
performed to compare across multistrain probi-
concerns for prophylactic administration of
otic therapies to evaluate which combinations
probiotics are the risk of infections and sepsis,
are most effective in reducing NEC and related
inconsistency among probiotics of different spe-
morbidities and mortality.
cies and strains, and lack of regulation by the
Single-strain probiotic treatments should not
Food and Drug Administration (FDA) or other
be completely disregarded, however, as some
agencies to ensure the product safety and efficacy
studies have reported some benefit. Using a neo-
of commercial products.
natal rat model with induced NEC, it was
Premature and VLBW infants have weak and
observed that rats supplemented with
immature immune systems (Melville and Moss
Bifidobacterium adolescentis exhibited increased
2013). This is due to multiple factors, including
survival rates and decreased intestinal injury due
less circulating maternal IgG which only begins
to NEC, suggesting a protective effect. The study
to transfer transplacentally around 32 weeks ges-
aimed to investigate possible mechanisms and
tational age (van den Berg et al. 2011), comple-
found that the probiotic treatment decreased
ment protein deficiency (McGreal et al. 2012),
expression of toll-like receptor 4 (TLR4) and
and reduction in neutrophils and monocytes
Necrotizing Enterocolitis and the Preterm Infant Microbiome 31

(Carr and Modi 1997). With these deficiencies, population subgroups (Saldanha 2008). However
even favorable bacteria can easily grow when considering use of probiotics to prevent or
unchecked, increasing the risk for infection and treat disease in vulnerable populations such as
sepsis. One case report described how prophylac- preterm infants, the need for greater scrutiny is
tic administration of Bifidobacterium breve apparent. In 2014, a premature infant died of
BBG-01 led to sepsis in a neonate, marked by mucormycosis, a rare fungal infection generally
systemic edema, elevated inflammatory markers, only affecting immunocompromised individuals.
and positive blood cultures for Bifidobacterium The source of the infection was traced to a probi-
breve BBG-01 (Ohishi et al. 2010). Although this otic dietary supplement given to the infant in the
only represents one isolated case, it should be hospital which was contaminated with Rhizopus
enough cause for consideration and weighing of oryzae (Vallabhaneni et al. 2015).
the risks and benefits of probiotic prophylaxis. It is also worth mentioning that there are few
Premature and VLBW infants may have studies investigating the long-term effects of pro-
compromised defenses to keep even commensal biotic supplementation in neonates. Given at such
bacteria in check; thus it is important to exercise a young age, the administration of probiotics may
caution when administering probiotics to this vul- have irreversible effects on the GI tract, immune
nerable population. system, metabolism (Yazdankhah et al. 2008),
Furthermore, with so many species and strains and brain development (Lu et al. 2018). Though
to choose from, broad, nonspecific these long-term effects may be presumed to be
recommendations about probiotic use may be of favorable, it is important that more long-term
little value. Each strain or combination of strains studies follow infants throughout childhood and
may have unique bioactivity, thus altering the adolescence to quantify the lasting effects and
benefits and risks of administering to a premature ensure the safety of the therapy. Although not
infant. Even within products of the same strain or directly applicable to this discussion of NEC
different batches of the same product, there may and probiotic administration to infants, a recent
be natural variation that could impact infant allergy study did follow-up with RCT subjects
outcomes. This represents a challenge for regula- 4 years post trial of a combined probiotic and
tion and manufacturing consistency. peanut oral immunotherapy. The follow-up
Whether probiotics should be treated as food study found that the positive effects of the therapy
and dietary supplements or drugs for regulatory were maintained and no long-term side effects
purposes is not clear cut. For healthy individuals, were reported (Hsiao et al. 2017). This sort of
probiotics as food make sense. For most people, follow-up ought to be conducted for preterm
probiotics are primarily ingested through infants subjected to probiotic treatment to assess
fermented food, often without full knowledge of the long-term effects.
their presence. As such, most probiotics are While probiotics are a promising therapy that
marketed as dietary supplements, regulated by may have broad applications in healthcare, there
the FDA’s Center for Food Safety and Applied is need for more rigorous testing and regulation to
Nutrition, and only requiring that the FDA be ensure that our most vulnerable populations are
notified of the intent to sell. Apart from notifica- protected. The recommendation proposed here is
tion, the only stipulation is that manufacturers that only probiotics which have undergone FDA
follow the Current Good Manufacturing Practice drug testing and approval for the intended popu-
requirements (2010) for dietary supplements, as lation should be used among NICU patients for
opposed to the more rigorous testing and approval the prevention of NEC. Probiotics that are cur-
process required for products marketed as drugs rently regulated as dietary supplements should
(Venugopalan et al. 2010). In regulating health only be used in healthy populations. This will
claims of dietary supplements, the FDA only help ensure the safety and efficacy of probiotic
requires studies to be conducted on “healthy” use in NICUs among vulnerable patients.
32 J. R. Baranowski and E. C. Claud

8 Alternatives for Altering antibiotic administration to preterm infants is

the Infant Microbiome known to decrease intestinal bacteria diversity
and lead to dysbiosis (Greenwood et al. 2014).
Direct administration of probiotics to premature However, with the risk of neonatal sepsis, a life-
infants is not the only therapy option for altering threatening condition, physicians are compelled
the early microbiome. Other options include to administer prophylactic antibiotics to prevent
administration of prebiotics, breastfeeding, limiting infection given any major risk factor, such as
prophylactic antibiotics, and targeting the maternal premature rupture of membranes or maternal
microbiome with probiotics and prebiotics. fever (WHO 2013). Further investigation and evi-
Prebiotics are indigestible dietary supplements dence are required to weigh the risks of suspected
intended to stimulate growth of commensal sepsis against adverse events associated with
microbes (Sohn and Underwood 2017). In antibiotics, with the aim of developing stricter
humans, these are most often oligosaccharide guidelines and more judicious use of antibiotics
prebiotics such as fructooligosaccharides (FOS) in NICUs.
and galactooligosaccharides (GOS). Prebiotics Finally, the mother’s microbiome represents a
can be supplemented in infant formula to promote promising target for altering the neonatal
growth of Lactobacillus and Bifidobacterium. microbiome. Manipulating the microbiome of
Research is promising for prebiotic use as treat- pregnant mice via gastric gavage with human
ment and prevention of infant colic (Giovannini fecal homogenate was found to confer the same
et al. 2014). While prebiotic supplementation in microbial profile to the pups (Lu et al. 2015). With
preterm infants has been found to alter the fecal this understanding, there are multiple ways of
microbiome and confer some benefit (Westerbeek modifying the pregnant microbiome to consider,
et al. 2013), there is not sufficient evidence to such as prebiotic and probiotic supplementation.
suggest that prebiotics decrease incidence of When supplemented during pregnancy, FOS and
NEC in human studies (Srinivasjois et al. 2013). GOS prebiotics have been found to increase mater-
It is also worth mentioning that the risk of con- nal Bifidobacterium spp., though this benefit is not
tamination exists for prebiotic supplements, necessarily conferred to the neonate (Jinno et al.
necessitating similar testing and regulation as 2017). Prenatal probiotic ingestion has been found
with probiotics to ensure that the vulnerable pre- to be beneficial for mothers, with a potential pro-
term population is not put at risk. tective role against preeclampsia (Brantsæter et al.
Human breast milk contains human milk 2011), gestational diabetes (Dolatkhah et al. 2015),
oligosaccharides (HMOs) which serve a prebiotic and vaginal infection (Ya et al. 2010). This benefit
function (Sohn and Underwood 2017) and shape is likely conferred to the infant by fostering a
the microbial community of breastfed infants healthier pregnancy, although studies are still
(De Leoz et al. 2014). However, breast milk limited. However, there is evidence that probiotics
from mothers of preterm infants differs from administered during pregnancy may reduce the
those delivering at term and may confer less incidence of atopic dermatitis (Zuccotti et al.
benefit to preterm infants. Animal studies have 2015), eczema, and rhinoconjunctivitis in the off-
isolated specific HMOs that may be protective spring (Bertelsen et al. 2014). Improved regulation
against NEC (Autran et al. 2016; Good et al. is still recommended for probiotics targeted for
2016). In the future, therapies could be developed prenatal administration, but the risks are less
involving breast milk or breast milk-like severe when administered to the mother versus a
supplements that select for these protective preterm infant, given the maturity of the mother’s
HMOs and ensure that the infant receives the immune system.
maximum benefit. With the many potential therapeutic targets for
Another potential target for shifting the pre- altering the microbiome of preterm infants, it is
term infant microbiome toward healthy diversity important to continue investigating means
is to limit antibiotic use in NICUs. Early empiric beyond just direct administration of probiotics to
Necrotizing Enterocolitis and the Preterm Infant Microbiome 33

preterm infants. Prebiotic supplementation, likely be the most effective, and thus
breastfeeding, antibiotic restriction, and altering recommendations for infants should reflect this
the maternal microbial community all hold prom- balance.
ise in preventing and improving NEC outcomes.

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Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2019) 1125: 37–45
https://doi.org/10.1007/5584_2018_314
# Springer Nature Switzerland AG 2019
Published online: 18 January 2019

Can Postbiotics Represent a New Strategy

for NEC?

Fabio Mosca, Maria Lorella Gianni, and Maria Rescigno

Abstract cause infection. However, data from trials

Increasing evidence indicates that many of the investigating the efficacy of postbiotics for
health beneficial effects associated with the the prevention of necrotizing enterocolitis in
establishment of a symbiotic gut microbiota preterm infants are needed, and issues regard-
are driven by bacterial metabolic by-products. ing their optimal regimen and start and dura-
The term “postbiotics” indicates any solu- tion of treatment need to be addressed.
ble factor resulting from the metabolic activity
of a live bacteria or any released molecule Keywords
capable of providing health benefits through Microbiota · Necrotizing enterocolitis ·
a direct or indirect mechanism. Postbiotics · Preventive strategy · Signaling
Alterations in preterm gut colonization
associated with the intestinal barrier immatu-
rity and the increased reactivity of the intesti-
nal mucosa to colonizing bacteria have been Abbreviations
implicated in the pathogenesis of necrotizing NEC Necrotizing enterocolitis
enterocolitis. Recent advances in the compre- TLRs Toll-like receptors
hension of the postbiotic biological effects and
related mechanisms, some of them reviewed
here, indicate that postbiotics may be a
promising effective preventive strategy against 1 Introduction
necrotizing enterocolitis while avoiding the
risk of administering live microorganisms to The human microbiota represents a highly com-
preterm infants that could translocate and plex echo-system involved in several important
functions modulating health and disease. The
most abundant bacterial cohorts are present in
F. Mosca (*) and M. L. Gianni the intestine, comprising a total of 1014 bacteria;
Fondazione I.R.C.C.S. Ca’ Granda Ospedale Maggiore particularly commensals bacteria contribute to the
Policlinico, Neonatal Intensive Care Unit, Department of development of the immune function and the
Clinical Science and Community Health, University of
Milan, Milan, Italy augmentation of the barrier against pathogens in
e-mail: [email protected]; [email protected] addition to exerting numerous metabolic
M. Rescigno (*) functions (Chong et al. 2018) (Table 1).
Humanitas University, Pieve Emanuele, Milan, Italy
e-mail: [email protected]

37
38 F. Mosca et al.

Table 1 Main functions associated with gut microbiota

Modulation of gene expression
Development of mucus and barrier function
Immune function development
Metabolism
Microbial production of nutrients (i.e., vitamins, amino acids, short-chain fatty acids)
Modulation of neural signaling (gut-brain axis)
Modulation of immune response locally and outside the local environment (i.e., gut-lung axis)
Digestion of complex macromolecules

Increasing evidence suggests that epigenetic taken into consideration that dividing the micro-
changes may be induced by the colonizing bial community into symbionts and pathobionts
microbiota indicating the presence of an may not be always so simple since innocent
epigenome-microbiome cross talk (Lu and strains may become potentially pathogenic when
Claud 2018). Moreover, recent data, mainly an impairment of the immune system coexists
derived from studies conducted using lactobacilli (Tsilingiri and Rescigno 2013).
strains, indicate that many of the beneficial
biological effects associated with the gut
microbiota are driven by bacterial metabolic 2 Necrotizing Enterocolitis:
by-products (Aguilar-Toaláa et al. 2018). Epidemiology and Pathogenic
Many factors affect the development of the Factors
infant gut microbiota including mode of feeding
and delivery, gestational age, prenatal maternal Necrotizing enterocolitis (NEC) primarily affects
exposures and nutritional status, antibiotic expo- premature newborns, occurring in 7–10% of very
sure, hospitalization, and host genetics. Although low birth weight infants, from 2 weeks to
the womb has been traditionally considered ster- 2 months of age, with an incidence inversely
ile, recent data indicate that colonization starts related to gestational age (Patel et al. 2015). It is
during fetal life (see also chapter 1). Gut associated with a mortality as high as 30%, and
microbiome appears to develop within the first survivors face an increased risk of serious health
3 years of life (Agostoni and Kim 2015) even complications including short gut syndrome and
though the recent finding that gut microbiome of neurological sequelae (Neu and Walker 2011).
preadolescent children presents compositional Moreover, the economic burden of NEC is very
and functional characteristics different from high accounting for up to one billion dollars in the
those of healthy adults indicates that gut United States annually (Hodzic et al. 2017).
microbiome development may occur within a There is agreement on the multifactorial path-
more prolonged period (Hollister et al. 2015). ogenesis of the disease that develops due to sev-
Intestinal homeostasis can be achieved eral and overlapping risk factors and/or stressors
through the establishment of a symbiotic relation- causing severe inflammation and intestinal injury
ship between the colonizing bacteria and the (Table 2). However, the exact pathophysiology of
intestinal epithelial and associated lymphoid tis- the disease has not been fully elucidated yet.
sue that, in turn, mediates the beneficial effects for Hence, there is need for developing novel preven-
the mammalian host and promotes tolerance in tive therapies.
the gut. On the contrary, inadequate colonization Preterm infants are characterized by a lack of
leading to a dysbiosis of intestinal microbiota, microbial diversity and an increased risk of path-
characterized by a preponderance of pathobionts, ogenic colonization. It has also been reported that
induces a more pronounced pro-inflammatory there may be a gestational threshold for coloniza-
response and an increased tendency toward an tion with Bifidobacterium species and anaerobic
immune dysfunction. However, it has to be species that are mostly represented from 33 weeks
Can Postbiotics Represent a New Strategy for NEC? 39

Table 2 Main factors implicated in NEC development

Intestinal dysbiosis
Relative abundances of Proteobacteria (Pammi et al. 2017)
Decreased relative abundances of Firmicutes and Bacteroidetes (Pammi et al. 2017)
Immature host defense mechanisms
Immaturity of the intestinal barrier
Underdevelopment of adaptative and innate immunity
Impaired intestinal motility
Imbalanced microvascular tone
Exaggerated pro-inflammatory response
Elevated expression of the Toll-like receptor 4
Impaired nitric oxide production
Formula feeding

of gestation (La Rosa et al. 2014). Accordingly, the expression of pattern recognition receptors.
data indicate that dysbiosis, particularly an Toll-like receptors (TLRs) are expressed by intes-
increased concentration of Gram-negative facul- tinal epithelial cells and immune cells of the lam-
tative bacteria and a decrease in obligate ina propria and greatly contribute to the
anaerobes, may play a critical role in the patho- modulation of epithelial cell proliferation, IgA
genesis of NEC; however, no specific microbial production, maintenance of cell junctions, and
pathogen has been identified as etiologic agent antimicrobial peptide expression. The role of
responsible for the development of the disease TLR4 in the pathogenesis of NEC has been
(Warner et al. 2016). Moreover, the intestinal widely investigated. Specifically, TLR4
barrier, which protects against potentially harmful recognizes lipopolysaccharides and activates
organisms and includes several components such nuclear factor-κB, leading to a pro-inflammatory
as the mucus coating, a layer of enterocytes cascade. As a result, intestinal epithelium repair is
strictly connected by a system of intracellular prevented, whereas enterocyte apoptosis is
and membrane-spanning proteins and an induced. Thus, the TLR4-mediated bacterial sig-
associated immune cells network, is still imma- naling leads to mucosal injury and enables bacte-
ture in preterm infants and, hence, contributes to ria to translocate into the circulation. Interaction
their increased susceptibility to NEC develop- between bacteria and TRL4 on the lining of the
ment (Isani et al. 2018). Within this context, the mesenteric blood vessels causes, in turn, a
role played by the immune cells has been decreased nitric oxide production with an
highlighted by several studies; specifically, associated severe vasoconstriction and reduced
when NEC develops, an imbalance among intestinal perfusion (Hodzic et al. 2017; Hackam
immune cells develops leading to an exaggerated and Caplan 2018). Remarkably, TLR4 is
pro-inflammatory state. Intestinal regulatory T expressed at high levels in the preterm infant.
cells (Tregs), which are already present from the During the first days of life, TLR4 signaling in
23rd week of gestation, play an important role in intestinal epithelial cells leads to establishment of
downregulating the pro-inflammatory response tolerance which facilitates microbiota coloniza-
and promoting the establishment of intestinal tion and contrasts inflammatory response (Lotz
homeostasis. Experimental models have et al. 2006). This control is probably more diffi-
demonstrated that NEC development is cult to be established in preterm infants.
characterized by a reduction in Tregs and an Formula feeding has also been associated with
increase in Th17, which, in turn, are associated a higher risk of NEC, with a risk ratio of 2.77
with intestinal injury and increased enterocyte (95% confidence interval 1.40–5.46) (Quigley
apoptosis (Hodzic et al. 2017). Moreover, an et al. 2007), whereas breastfeeding exerts a
important aspect of the innate immune system is dose-dependent protective effect (Patel and Kim
40 F. Mosca et al.

2018). It has been suggested that formula feeding capable of providing health benefits through a
may contribute to the alteration of gut microbiota direct or indirect mechanism (Tsilingiri and
in preterm infants, with overgrowth of bacteria Rescigno 2013; Aguilar-Toaláa et al. 2018).
and mucosal translocation. Moreover, postbiotics offer a safety advantage
NEC presents a rapid progression from early over probiotics since they decrease the risk for
clinical presentation to extensive intestinal necro- microbial translocation and infection and prevent
sis with need for surgical intervention and resec- potential detrimental effects shown by probiotics
tion of necrotic bowel. Hence, the effectiveness of within an already pro-inflammatory context
potential therapeutic treatments is limited to sup- (Tsilingiri et al. 2012). Other side effects reported
portive care at diagnosis, whereas research efforts in association with probiotic use are diffusion of
have focused on the identification of preventive antibiotic resistance gene and prevention of nor-
measures for NEC. mal colonization of other microflora. Further, we
do not know what will be in the long run the effect
of early colonization with predominant strains of
3 Preventive NEC Strategies probiotics both on microbiota maturation and
immune system development (Klaenhammer
Promotion and support of human milk feeding et al. 2012).
have been strongly advocated as the most effec- Increasing evidence indicates that postbiotics
tive strategy of NEC prevention. The exact exert different bioactive effects, including modu-
mechanisms responsible for the protective effect lation of immune function, anti-inflammatory
of human milk have not been fully elucidated but response, and antimicrobial activity, even though
include the provision of immunoglobulins, a clear insight into the exact mechanisms of
growth factors such as the epidermal growth fac- action has not been provided yet (Table 3).
tor (EGF) and the heparin-binding EGF, Postbiotics comprise several different molecules
L-arginine, nitrate/nitrite, L-glutamine, that can be classified according to their composi-
lactoferrin, and oligosaccharides. Moreover, tion into lipids, proteins, carbohydrates, vitamins/
human milk transfers microbes to the infant via cofactors, organic acids, and complex molecules
an entero-mammary pathway further contributing such as peptidoglycan-derived muropeptides and
to the shaping of the infant’s microbiota and lipoteichoic acids. Postbiotics are mainly
immune response (Maffei and Schanler 2017). obtained from Lactobacillus and Bifidobacterium
Administration of probiotics has been pro- strains; however, some authors have investigated
posed as a preventive strategy in decreasing the Streptococcus and Faecalibacterium species as a
risk of developing necrotizing enterocolitis in
preterm infants (Patel and Underwood 2018).
Table 3 Main effects exerted by postbiotics
Probiotics are defined as live microorganisms
(Aguilar-Toaláa et al. 2018)
that confer a health benefit to the host when
Local effects on the gut epithelium
ingested in adequate amounts likewise commen-
Immunomodulating
sal bacteria do (Hill et al. 2014). Recent data,
Anti-inflammatory
however, indicate that not all the health-
Antimicrobial
promoting effects are directly related to the via- Enhancement of the intestinal barrier function
bility status of the bacteria. Hence, new terms Systemic effects on organ/tissue
such as “postbiotics” have been developed Antioxidant
which denote that bacterial viability is not always Hypocholesterolemic
required for inducing health beneficial effects, Antihypertensive
offering a new scenario within the field of func- Antiobesogenic
tional foods. Postbiotic has been defined as any Antiproliferative
soluble factor resulting from the metabolic activ- Anxiolytic
ity of a live bacteria or any released molecule Antidepressants
Can Postbiotics Represent a New Strategy for NEC? 41

source of postbiotics (Tsilingiri and Rescigno different set of postbiotics, characterized by spe-
2013; Aguilar-Toaláa et al. 2018). cific biological activities, may be produced
according to the type of probiotic strains
employed and the fermentation process used
4 Postbiotic Effects on Intestinal (Tsilingiri and Rescigno 2013; Aguilar-Toaláa
Barrier Function et al. 2018).
and Homeostasis

Intestinal epithelial cells are the first cells coming 5 Effect on Shaping Microbiota
into contact with microbes in the gut lumen. They
provide physical barriers against the invasion of Postbiotics have been reported to contribute to the
intestinal microorganisms that include the mucus restoration of intestinal homeostasis through the
layer that covers the intestinal mucosa, the production of bacteriocins, an abundant and het-
glycocalyx on the microvilli of absorptive intesti- erogeneous group of ribosomally synthesized
nal epithelial cells, and the tight cell junctions. In antimicrobial peptides that exert bacteriostatic or
particular, mucus is mainly comprised by mucins bactericidal effects. It has been demonstrated that
that are secreted by goblet cells and form a gel-like bacteriocin production is affected by medium
structure. The inner layer, which is firmly adherent composition and may be enhanced by the optimal
to the intestinal epithelium, does not contain combination of carbon and nitrogen sources.
microbes and acts as a protective barrier against Moreover, bacteriocins selectively compete with
bacterial adhesion and invasion of underlying epi- specific bacterial strains, shaping the microbiota
thelial cells. On the contrary, the outer layer is composition and affecting the immune system
inhabited by a large number of intestinal microbes function (Aijaz and Sreeja 2017; Aguilar-Toaláa
and is formed by proteolytic and glycosidic degra- et al. 2018). Several other postbiotic compounds
dation of highly polymerized gel-like MUC2, the such as enzymes, small molecules, and organic
major secretory mucin, by the host or bacteria. acids have been shown to inhibit the growth of
From a historical perspective, it’s worth pathogens. Reuterin is a strong antipathogenic
remembering that the intestinal epithelial cells for molecule produced by Lactobacillus reuteri and
many years have been merely regarded as a physi- has been proposed to mediate, at least partially,
cal separator between the external world and the the health benefits ascribed to this bacterium
immune cells located in the lamina propria and as a (Cicenia et al. 2014). Specifically, it has been
semipermeable membrane through which nutrients demonstrated that reuterin inhibits bacterial
would be absorbed (Vancamelbeke and Vermeire growth inducing oxidative stress in cells and
2017). However, the key role played by intestinal contributing to the fine tuning of a healthy gut
epithelial cells in modulating the gut immune microbiota (Schaefer et al. 2010).
response has now increasingly emerged. Indeed it Bacterial exopolysaccharides have been shown
has become clear that intestinal epithelial cells to mediate some of the beneficial effect associated
mediate the delivery of signals between the gut with probiotics such as bifidobacteria. Exopolysac-
and the host immune cells through the production charides are repeating mono- or oligosaccharide
of mediators, including cytokines, chemokines, subunit linked by different glycosidic linkages,
and antimicrobial peptides, in order to prevent the and, as a result, the biological activity of these
overgrowth of potentially pathogenic bacteria and molecules may be different according to their
maintain a tolerance to commensal bacteria structural diversity. Bifidobacterial cell surface-
(Vancamelbeke and Vermeire 2017). associated exopolysaccharides appear to affect
In this context increasing evidence indicate microbiota composition since they serve as fer-
that many postbiotic components are able to mentable substrate for other bacteria. Moreover,
enhance the intestinal barrier function. It has to the bacterial exopolysaccharide expressed by a
be taken into consideration, however, that Bifidobacterium breve strain has been shown to
42 F. Mosca et al.

reduce colonization levels of the gut pathogen enterocytes, fermented milk with Lactobacillus
Citrobacter rodentium, probably through the abil- paracasei CBA L74 promotes cell growth and
ity of forming biofilms, whereas the lack of expres- differentiation, tight junction proteins and mucin
sion of surface exopolysaccharides has been 2 expression, and mucus layer thickness with a
associated with higher levels of the pathogen (Fan- dose-dependent relationship.
ning et al. 2012).

7 Immunomodulation Activity
6 Enhancement of Intestinal
Barrier Mileti et al. (2009) were among the first to dem-
onstrate that most of the anti-inflammatory activ-
The restoration of mucosal gut barrier integrity ity exerted by Lactobacillus paracasei B21060
has been associated with the provision of Lacto- was associated to the metabolites released in cul-
bacillus rhamnosus GG supernatants. Wang et al. ture, which downregulated the production of
(2012) in a mouse model have shown a reduction IL-12p70 and increased production of IL-10.
of intestinal permeability following alcohol- Schiavi et al. (2016) reported that the
induced damage trough recovery of normal levels Bifidobacterium longum 35624-associated
of tight junction proteins and factors involved in exopolysaccharide affects host immune response
mucin production following treatment of Lacto- by reducing the secretion of cytokine produced by
bacillus rhamnosus GG supernatants. Butyrate, a human dendritic cells, whereas the absence of this
short-chain fatty acid mainly produced by anaer- molecule is associated with a severe tissue dam-
obic fermentation of undigested carbohydrates, age through a strong Th-17 response to commen-
has been reported to stimulate MUC2 mucin sal bacteria. These findings confirm previous data
secretion by increasing expression of MUC2 and underline the critical role played by specific
gene in a human colonocyte cell line, indicating exopolysaccharides in modulating the host
that it contributes to the enhancement of protec- microbiome interplay.
tion against luminal agents (Canani et al. 2011). Consistently with these findings, Tsilingiri et al.
Cultures supernatant of Saccharomyces boulardii (2012) have suggested that postbiotics may repre-
have also been reported to promote wound sent a potential treatment of inflammatory bowel
healing of epithelial cells by α2β1 integrin colla- disease. Specifically, it has been demonstrated that
gen receptors activation and, as a result, to Lactobacillus paracasei B21060 culture superna-
strengthen barrier function (Giorgetti et al. tant protects against Salmonella invasion by reduc-
2015). Mice dextran sodium sulfate-induced coli- ing NFkB activation in a human organ culture
tis has been prevented by probiotic-derived system and can downregulate pro-inflammatory
polyphosphate by activation of integrin-p38 pathways active in inflammatory bowel disease
MAPK pathway and inhibition of F-actin and tissue (Tsilingiri et al. 2012; Zagato et al. 2014).
E-cadherin degradation that are protective against Of note, the authors reported that some probiotics
oxidant-induced intestinal permeability induced a local inflammatory response on healthy
(Tsilingiri and Rescigno 2013; Giorgetti et al. tissues, resembling the response induced by Salmo-
2015). Moreover, p40, a Lactobacillus nella; these results suggest that administration of
rhamnosus GG-derived soluble protein, has probiotics may not always exert beneficial effects.
been identified as one of the main mediators in Moreover, all tested probiotic strains, including
improving injury and inflammation and reducing L. paracasei, resulted to be detrimental when
apoptosis by transactivation of the epidermal co-incubated with the intestinal mucosal explants
growth factor receptor in intestinal epithelial from patients affected by intestinal inflammatory
cells (Tsilingiri and Rescigno 2013). bowel disease (Tsilingiri et al. 2012). Among
Paparo et al. (Paparo et al. 2018) found that, identified postbiotics, lactocepin, a caseinolytic ser-
through a direct interaction with human ine protease produced by Lactobacillus casei and
Can Postbiotics Represent a New Strategy for NEC? 43

paracasei, and S-layer protein A and polysaccha- Nocerino et al. (2017) have reported that the
ride A have been shown to contribute to these daily consumption of cow’s milk and rice
protective mechanisms by modulating pro- and fermented with Lactobacillus paracasei CBA
anti-inflammatory cytokine production (von L74 protects young children from infectious
Schillde et al. 2012; Konstantinov et al. 2008). diseases and is associated with increased alpha-
Protection against Yersinia enterocolitica infection defensin, beta-defensin, cathelicidin LL-37, and
through the inhibition of the NFkB activation and secretory IgA levels. The anti-infective and
the associated pro-inflammatory cytokine produc- immunomodulatory effects demonstrated with
tion have been observed in human epithelial cells the use of cow’s milk fermented with Lactobacil-
when Lactobacillus fermentum supernatants were lus paracasei CBA L74 have been confirmed in a
added (Frick et al. 2007). subsequent clinical trial (Corsello et al. 2017).
Haileselassie et al. (2016) investigated the Moreover, Paparo et al. (2018) found that,
immunomodulatory effect of Lactobacillus reuteri through a direct interaction with human
DSM 17938 cell-free supernatant on the retinoic enterocytes, fermented milk with Lactobacillus
acid-driven mucosal-like dendritic cells response paracasei CBA L74 stimulates the synthesis of
and their subsequent effect on regulatory T cells. beta-defensin and cathelicidin, in association with
When treated with Lactobacillus reuteri DSM a modulation of Toll-like receptor pathway.
17938 cell-free supernatant, retinoic acid induced
an increased production of anti-inflammatory IL-10
and a decreased expression of several
8 Conclusions
inflammatory-associated genes such as NFκB1,
RELB, and TNF. Accordingly, Sokol et al. (2008)
Recent advances in the comprehension of the
showed a decreased IL-8 concentrations and the
postbiotic biological effects and related
abolishment of NFκB activation in Caco2 cells
mechanisms indicate that postbiotics are a
when exposed to the supernatant fraction of
promising effective preventive strategy against
Faecalibacterium prausnitzii. Moreover, the
NEC while avoiding the risk of administering
authors demonstrated that the cell-free supernatant
live microorganisms to preterm infants that
added to a colitis mouse model could shift the
could translocate and cause infection. However,
immune response toward an anti-inflammatory pat-
no data from trials of postbiotic use for the pre-
tern characterized by an increase in IL-10 and a
vention of NEC in preterm infants are currently
decrease in IL-12. A similar activity was observed
available. Furthermore, issues regarding the opti-
by feeding mice with a cow’s milk fermented with
mal postbiotic regimen and the start and duration
Lactobacillus paracasei CBA L74 (Zagato et al.
of treatment need to be addressed.
2014). In this case, the authors also demonstrated
that the same effect was not observed when the live
bacterium was administered, suggesting that
postbiotics may be more effective because they References
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 Part II
The Infant
Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2019) 1125: 49–56
https://doi.org/10.1007/5584_2018_315
# Springer Nature Switzerland AG 2019
Published online: 18 January 2019

Preventing and Treating Colic

Flavia Indrio, Vanessa Nadia Dargenio, Paola Giordano,

and Ruggiero Francavilla

Abstract causes intestinal hyperperistalsis due to

Colic is a common and distressing functional increase in serotonin secretion and motilin
gastrointestinal disorder during infancy. It is a receptor expression.
behavioral phenomenon in infants aged 1–4 Probiotics may play a crucial part in the
months involving prolonged inconsolable manipulation of the microbiota. Probiotic
crying and agitated status with multifactorial administration is likely to maintain intestinal
etiology. Colic can be considered as a benign, homeostasis through the modulation of perme-
self-limited process because the baby nor- ability and peristalsis, influencing the
mally grows and feeds even with transient gut-brain axis and inhibiting hypersensitivity.
irritable mood. Nevertheless, infantile colic This is a decisive field in the development of
is a common difficulty causing anxiety during preventive and therapeutic strategies for infan-
parenthood and a recurrent reason for them to tile colic. However, further studies are needed
seek medical help, especially if it is the first for each specific formulation in order to better
child. The causes of colic can be classified as characterize pharmacodynamic and pharmaco-
non-gastrointestinal or gastrointestinal. The kinetic properties and to evaluate their appli-
former includes altered feeding techniques, cation as a possible preventive strategy if
modified child-parent relationship, immatu- administered early during infancy against the
rity of central nervous system, behavioral eti- later development of pain-related FGIDs.
ology, and maternal smoking or nicotine
replacement therapy. Instead, the latter Keywords
involves inadequate production of lactase Enteric microbiota · Gut-brain axis · Infant
enzyme, cow’s milk protein intolerance, colic · Lactobacillus · Probiotics
alteration of intestinal microbiota, gastroin-
testinal immaturity, or inflammation which
1 Introduction
F. Indrio (*)
Crying is believed to be a natural part of the first
Department of Pediatrics, Section of Gastroenterology and
Nutrition, “Aldo Moro” University of Bari, Ospedale months of life signaling basic requests that may
Pediatrico Giovanni XXIII, Bari, Italy require the presence of the parents, but a weak
e-mail: [email protected] sign of what the infant actually needs (Brazelton
V. N. Dargenio, P. Giordano, and R. Francavilla 1962; Illingworth 1955). Even though crying is
Department of Pediatrics, Ospedale Pediatrico Giovanni thought to be a physiological behavior, an
XXIII, “Aldo Moro” University of Bari, Bari, Italy

49
50 F. Indrio et al.

estimated 5–40% of babies cry unreasonably and without evidence of infant failure to thrive, fever
inconsolably in association with bouts of irritabil- or ill health” (Zeevenhooven et al. 2017). By way
ity and gas emissions (Anabrees et al. 2013; of explanation, the measurement of infant crying
Lucassen et al. 2001; Steutel et al. 2014). Colic has not been cited. Nevertheless, the cutoff of 3 h
is a common and distressing functional gastroin- should be added to the abovementioned
testinal disorder (FGID) during infancy. It is a requirements for medical research studies. This
behavioral phenomenon in infants aged 1–- recent definition can be considered a consequence
4 months involving prolonged inconsolable cry- of the high interest highlighted by pediatricians in
ing and agitated status with multifactorial identifying this condition, to investigate and pro-
etiology. In fact, colic are considered as a normal vide appropriate management.
variant in crying curve (Barr 1990). It is included
as differential diagnosis made after an accurate
evaluation of the infant who cry excessively and a 3 Epidemiology
recurrent issue in pediatric outpatient clinic. Colic
can be considered as a benign, self-limited pro- The infant colic prevalence rates change consid-
cess because the baby normally grows and feeds erably according to the criteria selected for the
even with transient irritable mood (Garrison and study, technique of data collection, type of study
Christakis 2000). Nevertheless, infantile colic is a chosen, and the population being analyzed in the
common difficulty causing anxiety during parent- study. Recently, in a systematic review, the prev-
hood and a recurrent reason for them to seek alence was described in the range from 2% to
medical help, especially if it is the first child, 73%, with a median of 17,7% (Vandenplas et al.
while the challenge for pediatricians consists in 2015). Relying instead on Rome III criteria, in
the appropriate recognition of the pathology and different studies the prevalence is ranging from
adequate treatment. 5.9% to 10.4% (Chogle et al. 2016). The studies
suggest equal incidence in males and females and
no association with formula feeding or
2 Infant Colic breastfeeding, prematurity or full-term infants,
social class, or season of the year (Clifford et al.
Morris Wessel was the first to introduce the defi- 2002; Lehtonen and Korvenranta 1995; Lucassen
nition of colic in infancy (Wessel et al. 1954) in et al. 2001). The typical peak is at about 6 weeks
his classic 1954 article, and it is also described as of age and can be associated with important care-
“the rule of three”: healthy infants who cry for giver blame and frustration. Besides, colic have
almost 3 h per day, for more than 3 days per week, been related to shaken baby syndrome and post-
and occurring over 3 weeks. During these partum depression (Barr et al. 2006; Radesky
paroxysms of irritability, the infant can be et al. 2013).
described as gassy, often with legs flexed, and,
furthermore, this behavior is not alleviated by
usual caregiver interventions. Later, Wessel 4 Etiology and Role
criteria were adapted because of the unrealistic of Microbiota
waiting time to reach a diagnosis, and, recently,
the international association for FGIDs, known as Despite decades of research, the etiology of infant
the Rome Foundation, has produced a new gen- colic remains unknown. More than one theory
eral agreement to define colic. According to this have been advanced to give a satisfactory expla-
consensus, colic are diagnosed using the follow- nation of its mechanism; nonetheless the patho-
ing criteria: “recurrent and prolonged periods of genesis of colic has not conclusively been
infant fussing, crying or irritability reported by described. It is from the archaic Greek “κoλικóς,
parents that occur without obvious cause and kolikos” that takes its origin the term “colic,” in
cannot be prevented or resolved by caregivers, reference to the colon, but it has not been
Preventing and Treating Colic 51

demonstrated yet that the intestine is the origin of (TLRs), various cytokines and chemokines are
malaise in colicky infants. For this reason, the released from cells to start the inflammatory
causes of colic can be classified as response. Since the episodes of infantile colic
non-gastrointestinal or gastrointestinal. The for- coincide with the most considerable alterations
mer includes altered feeding techniques, modified in intestinal microbiota colonization in newborn,
child-parent relationship, immaturity of central intestinal microbiota, gut inflammation, and
nervous system (CNS), behavioral etiology, and microbiota-gut-brain axis might represent a sig-
maternal smoking or nicotine replacement ther- nificant part in its pathophysiology. This hypoth-
apy (Gelfand 2016; Ghoshal 2011; Paradise esis has acquired more and more value in
1966). Instead, the latter involves inadequate pro- consideration of the recent research that have
duction of lactase enzyme, cow’s milk protein shown lower counts of intestinal lactobacilli,
intolerance, alteration of intestinal microbiota, bifidobacteria, and other butyrate-producing bac-
gastrointestinal immaturity, or inflammation teria and a prevalent fecal proportion of Gram-
which causes intestinal hyperperistalsis due to negative pathogens, particularly coliform bacteria
increase in serotonin secretion and motilin recep- in this population (Hatakka et al. 2001; Lehtonen
tor expression (Camilleri et al. 2017; Hyman et al. et al. 1994; Pärtty et al. 2012; Roos et al. 2013;
2006; Kaley et al. 2011). Savino et al. 2009; de Weerth et al. 2013a, b). It
Regardless of this unclear and potentially mul- may be that infant colic is related to a slower and
tifactorial etiology, modifications in gut micro- anomalous pattern of microbial colonization and
flora have been correlated with infant colic in a scanty bacterial variety of gut microbiota during
many studies by independent research groups early infancy. Those conditions may lead to
(Verduci et al. 2013; de Weerth et al. 2013a, b). alterations in the metabolome of intestinal
Through certain signaling pathway, gut-brain microbiota, the ensemble of products released
interactions influence gastrointestinal activity by resident microorganisms which has an impact
both during illness and well-being. The on human health (Konishi et al. 2013; Wikoff
microbiota may be remodeled by the brain as a et al. 2009). On the other hand, modifications in
consequence of alteration of gut barrier function, bacterial metabolome may slow down the intesti-
mucosal secretions, and gastrointestinal peristal- nal peristaltic motion, promoting a gas collection
sis. In this way, it is possible to avoid an intestinal responsible for general digestive discomfort
overgrowth of harmful microorganisms (Heijtz (Gupta 2002; Savino et al. 2004; de Weerth
et al. 2011). Cells in the gastrointestinal lamina et al. 2013a, b).
propria can release signaling molecules into the The enterobacteria typically produce gas as the
lumen under the stimulus of the CNS, and it may end product of their metabolism (Ács et al. 2015),
lead to alterations in intestinal motility, secretion, which may facilitate an accumulation of gas and a
and permeability, in this manner modifying the consequent digestive discomfort in infants with
gastrointestinal environment in which microbiota colic. Besides, Enterobacteria lipopolysac-
is located (Diamond et al. 2011). Microbiota charides (LPS), also known as endotoxins, are
interacts and modulates the gut-brain axis through particularly pro-inflammatory, in comparison
various mechanisms such as hormonal, immune, with LPS from other intestinal Gram-negative
and neural routes. On the other side, microbiota microorganisms (Lindberg et al. n.d.). Several
has a direct intercommunication with intestinal studies have suggested that the pro-inflammatory
mucosa increasing gut neuroendocrine products. effect of LPS decreases visceral sensory and pain
Microbiota can modulate expression of inflamma- thresholds, defined as allodynia, both in animal
tory mediators: in fact, when pathogen-associated and human models and this effect is mediated by
molecular patterns (PAMPs), molecule TLRs (Benson et al. 2012; Moriez et al. 2005;
characteristics of some pathogens that are not Wegner et al. 2015). For this reason, a higher
expressed by host cells, bind a family pattern of count of Enterobacteria could induce a state of
recognition receptor, called Toll-like receptor amplified visceral hypersensitivity and low-grade
52 F. Indrio et al.

inflammation in colicky babies. On the other side, expression (Akimova et al. 2011) and there are
other studies have highlighted that a high count of studies that evaluate the levels of some transcrip-
Bifidobacterium and Lactobacillus in babies’ tion factors involved in the modulation of the
microbiota has a defensive function against immune system, for instance, Fork head box
colic. A possible explanation is that intestinal P3 (FOXP3) messenger RNA, and retinoid-
lactobacilli could promote the expression of related orphan receptor-r (RORƴ) (Savino et al.
anti-inflammatory genes (van Baarlen et al. 2018). CD4+ T cells direct the immune response
2009), improving intestinal permeability and toward both Th17 cells (RORƴt) and regulatory T
motility and causing a lowering of visceral pain cells (FOXP3) in case of interaction with
(Chichlowski and Rudolph 2015; Verdu 2009). transforming growth factor beta (TGF-β). Th17/
In addition, lactobacilli and bifidobacteria may regulatory T-cell balance is considered to be a
modulate immune response playing a protective significant factor of intestinal immune-mediated
role against colic. Another important factor is pathology, contributing to infant colic (Diller
the regulation of enteric immune and microbial et al. 2016).
diversity. The gastrointestinal system represents a Moreover, as a known biomarker of gut
way of communication with the external environ- inflammation, calprotectin seems to be inversely
ment and is largely populated by potentially related with diversity in gut microbiota (Rhoads
pathogenic microorganisms; therefore a strong et al. 2009). Nevertheless, there are no studies that
presence of lymphoid system is necessary: the prove its efficacy in breastfed infants (Li et al.
gut-associated lymphoid tissue, also known as 2014). Until now, the relationship among
GALT, is structured in lymphatic nodules dysbiosis, gut inflammation, and probiotics has
occupying the lamina propria and the intestinal not been deeply researched in this condition. Fur-
submucosa, called Peyer’s patches, and immune ther studies are needed to comprehend the under-
cells residing in gut epithelium and lamina lying pathology in infant colic, for instance,
propria. GALT has a preventive role that consists performing endoscopy or colonoscopy.
in avoiding dangerous gut antigens from reaching
the circulatory system. Furthermore, through the
release of mucosal immunoglobulin A and regu- 5 Probiotics as a Preventative
latory lymphocyte activation, GALT contributes and Therapeutic Strategy
to immune tolerance induction toward non-self-
proteins in the intestinal lumen. A high number of The World Health Organization (WHO) and the
bacteria are competently detected and carried by Food and Agriculture Organization of the United
antigen-presenting cells (APC); subsequently, Nations (FAO) have internationally defined
TLRs are activated through APC. This results in probiotics as “living microorganisms that, when
a change of cell phenotype and function, trigger- administered in adequate amounts, confer health
ing signaling events causing enhanced cytokine benefits on the host”. More recently, several
secretions involved in the regulation of inflamma- new researches indicate that probiotic adminis-
tion and promotion of host immune mechanisms tration might modulate gut microbial profile
(Mason et al. 2008). increasing beneficial colonizing microbes
Several researches have shown an increase of (Boirivant and Strober 2007; Hill et al. 2014).
different cytokines in colicky infants in whom Probiotics prevent the overgrowth of coliforms
intestinal inflammatory processes and immune and inflammation-causing microorganisms and,
dysfunction coexist (Pärtty et al. 2017; Rhoads therefore, can decrease gut inflammation
et al. 2009). The role of CD4+ T cells is decisive (Gareau et al. 2010; Hochman and Simms 2005;
to comprehend the etiology of the dysfunction of Savino et al. 2011). Probiotics may play a
the immune system. We know that the expression crucial part in the manipulation of the micro-
of CD25 correlates with high helios protein biota. Probiotic administration is likely to
Preventing and Treating Colic 53

maintain intestinal homeostasis through the mod- been demonstrated breastfed infants are protected
ulation of permeability and peristalsis, against the development of colic in early infancy
influencing the gut-brain axis and inhibiting and its incidence is comparable among breast-
hypersensitivity. This is a decisive field in the and formula-fed babies.
development of preventive and therapeutic A recent randomized clinical trial showed a
strategies for infantile colic. decrease in occurrence of functional constipation,
Probiotics can favor the correct composition of gastroesophageal reflux, and inconsolable crying
normal motility patterns even if, in literature, it is administering L. reuteri DSM 17938 from birth
reported that the elements that control gut peri- until the third month of life with a preventive
stalsis have achieved the full maturity in older purpose (Indrio et al. 2014).
babies (Faussone-Pellegrini et al. 2007). Conse- In February 2017, World Gastroenterology
quently, the prime explanation of the probable Organisation global guideline (World Gastroen-
effect of probiotics on gut peristalsis is supplied terology Organisation Global Guidelines
by the intricate interactions of neuro-immune Probiotics and prebiotics 2017) recommended
components (Rhee et al. 2009). Additionally, daily administration of a dose of 108 colony-
many studies have shown the interaction between forming units (CFU) of L. reuteri DSM 17938
probiotic administration and GALT that has been during 21 days in colicky infants, as level I of
demonstrated to be essential for the specific evidence, and suggested L. reuteri DSM 17938
development of gastrointestinal functions (Wood for the prevention of infant colic too. Moreover,
2004). bis-in-die dose of Lactobacillus rhamnosus GG
The prebiotic and probiotic supplementation (LGG) 1010–1011 was recommended as a pre-
may be effective in the treatment of colic since ventive strategy of infantile colic.
their safety, tolerance (Weizman and Alsheikh The results of several studies on the L. reuteri
2006), and ability to improve gastrointestinal supplementation propose that the efficacy of
symptoms without adverse effects have been probiotics might be associated to the effect on
proved (Indrio et al. 2008). intestinal motility, immune system, nociceptive
In the subpopulation treated with probiotics, transmission, and perception of a painful stimu-
crying time reduction has been described by lus. Some other studies show that early identifica-
Savino and coworkers (Savino et al. 2007) and tion of modification in intestinal microbiota can
also reported by other authors (De Giorgio et al. be used for the prevention of infant colic. A
2004). Many clinical trials have explored the preventive strategy could be probiotic administra-
effectiveness of Lactobacillus (L.) reuteri (strain tion before the onset of colic in infants or supple-
DSM 17938) compared with placebo for treating mentation to mothers during pregnancy too.
infantile colic (Chau et al. 2015; Savino et al. However, further research would be worth doing
2010; Sung et al. 2014; Szajewska et al. 2013). in this field.
Four clinical trials showed a benefit in decreasing In the diseased population, probiotics and in
colic symptoms, and in another one, the null particular their metabolites short-chain fatty acids
hypothesis couldn’t be rejected; instead two (SCFA) can inhibit colonic movements and might
meta-analyses and one systematic review improve sphincterial activity. Indeed, SCFA
reported a significant reduction in crying rate in seems to modify the secretion of gastrointestinal
breastfed infant who received L. reuteri (5 drops/ hormones as polypeptide YY (Voortman et al.
day) (Lucassen et al. 2001; Sung et al. 2013; 2012). The research on the postinfectious intesti-
Urbańska and Szajewska 2014). More recently, nal dysmotility has found that the release of
another meta-analysis also reported that L. reuteri inflammatory mediators, including prostaglandin
effectively reduced crying and/or fussing in col- E2 (PGE2) and TGF-β, by the muscularis propria
icky infants fed with breast milk (Sung et al. of the intestinal tract is responsible for
2018). However, further research needs to be maintaining chronic neuromuscular system dys-
conducted, without considering that it has not function (De Giorgio et al. 2004). Besides,
54 F. Indrio et al.

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https://doi.org/10.1007/5584_2018_316
# Springer Nature Switzerland AG 2019
Published online: 26 January 2019

Targeting Food Allergy with Probiotics

Lorella Paparo, Rita Nocerino, Carmen Di Scala,

Giusy Della Gatta, Margherita Di Costanzo, Aniello Buono,
Cristina Bruno, and Roberto Berni Canani

Abstract microbiota composition and function, and gut

The dramatic increase in food allergy preva- microbiota dysbiosis has been associated with
lence and severity globally is demanding the development of food allergy. Selected pro-
effective strategies. Food allergy derives from biotic strains could act on immune tolerance
a defect in immune tolerance mechanisms. mechanisms. The mechanisms are multiple
Immune tolerance is modulated by gut and still not completely defined. Increasing
evidence is providing useful information on
the choice of optimal bacterial species/strains,
dosage, and timing for intervention. The
Authors Lorella Paparo, Rita Nocerino, Carmen Di Scala,
Giusy Della Gatta and Margherita Di Costanzo have been increased knowledge on the crucial role played
equally contributed to this chapter. by gut microbiota-derived metabolites, such as
L. Paparo, R. Nocerino, C. Di Scala, G. Della Gatta, butyrate, is also opening the way to a post-
and M. Di Costanzo biotic approach in the stimulation of immune
Department of Translational Medical Science, University tolerance.
of Naples “Federico II”, Naples, Italy
CEINGE-Biotecnologie Avanzate s.c.ar.l., University of Keywords
Naples “Federico II”, Naples, Italy
Butyrate · Cow’s milk allergy · Gut
A. Buono microbiota · Immune tolerance · Post-biotics ·
CEINGE-Biotecnologie Avanzate s.c.ar.l., University of
Probiotics
Naples “Federico II”, Naples, Italy
C. Bruno
Department of Translational Medical Science, University
of Naples “Federico II”, Naples, Italy
Abbreviations
R. Berni Canani (*)
Department of Translational Medical Science, University
BLG β-lactoglobulin
of Naples “Federico II”, Naples, Italy
CMA cow’s milk allergy
European Laboratory for the Investigation of Food-
EHCF extensively hydrolyzed casein formula
Induced Diseases, University of Naples “Federico II”,
Naples, Italy FA food allergy
LAB lactic acid bacteria
CEINGE-Biotecnologie Avanzate s.c.ar.l., University of
Naples “Federico II”, Naples, Italy LGG Lactobacillus rhamnosus GG
OIT oral food immunotherapy
Task Force on Microbiome Investigation, University of
Naples Federico II, Naples, Italy OVA ovalbumin
e-mail: [email protected] PBMCs peripheral blood mononuclear cells

57
58 L. Paparo et al.

SCFAs short-chain fatty acids identified as triggers of FA, there is a rather

SU sustained unresponsiveness short list of foods account for most of the more
Tregs regulatory T cells serious disease burden: namely peanuts, tree nuts,
fish, shellfish, egg, milk, wheat, soy, and seeds,
with national and geographical variations
concerning the most common FA (Boyce et al.
1 Introduction
2010; Sicherer et al. 2010; Ben-Shoshan et al.
2010; Chafen et al. 2010; Osborne et al. 2011;
Food allergy (FA) is “an adverse health effect
Gupta et al. 2011; National Academies of
arising from a specific immune response that
Sciences 2016).
occurs reproducibly” according to the 2010
Food allergy derives from a breakdown of
National Institute of Allergy and Infectious
immune tolerance. Current knowledge suggests
Diseases, National Institutes of Health (NIAID/
that the development of FA might be influenced
NIH)-supported Guidelines for the Diagnosis and
by genetics, environment, and genome-
Management of Food Allergy in the United States
environment interactions, leading to immune sys-
(Boyce et al. 2010). It is one of the most common
tem dysfunction, mediated at least in part by
allergic disorders in the pediatric age, and it has
epigenetic mechanisms (Berni Canani et al.
been recognized as a global health problem that
2015; Paparo et al. 2018). Many factors have
affects millions of persons, particularly in devel-
been postulated to contribute to the onset of
oped countries. Studies have suggested that the
FA. Among the multiple immutable risk factors
natural history of FA has changed during the last
that could influence FA onset, there are the male
two decades, with a dramatic rise in the preva-
sex, the race/ethnicity (increased risk among
lence, severity of clinical manifestations, and risk
Asian and black children compared with white
of persistence into later ages, leading to an
children), and genetics (familial associations,
increase in hospital admissions, medical visits,
HLA, and specific genes) (National Academies
treatments, and burden of care on families and
of Sciences 2016; Sicherer et al. 2017; Allen and
to an important economic impact, with significant
Koplin 2016; du Toit et al. 2016; Hong et al.
direct costs for the families and healthcare system
2015; Gupta et al. 2016). In addition, there are
(Skripak et al. 2007; McBride et al. 2012; Gupta
other risk factors that can be addressed to poten-
et al. 2013). In Europe, around seven million of
tially reduce/prevent FA, such as atopic disease
people suffer from challenge-proven FA. If this
manifestations (comorbid atopic dermatitis),
prevalence is projected onto the world’s popula-
increased hygiene, vitamin D insufficiency,
tion of seven billion, it translates into 63 million–
dietary fat (reduced consumption of omega-3-
1.16 billion of potential food-allergic people, with
polyunsaturated fatty acids), reduced
a greater incidence in children (5–8%) than in
consumption of antioxidants, increased use of
adults (1–2%) (Fiocchi et al. 2010). Furthermore,
antacids (that reduce digestion of allergens),
about 8% of people suffering from FA are
obesity, the timing and route of exposure to
exposed to the risk of potentially life-threatening
foods (increased risk for delaying oral ingestion
allergic reaction resulting in death, mainly among
of allergens with environmental exposure in the
children aged 0–14 years. According to the most
absence of oral exposure leading to sensitization
recently available epidemiological data, time
and allergy), and in particular the influence of the
trend analysis showed up to sevenfold increase
microbiome (Savage et al. 2018; Huang et al.
in hospital admissions for severe food-allergic
2017; Sicherer et al. 2010). There is mounting
reactions in children in the UK, USA, Italy, and
evidence that the alterations of microbiota com-
Australia over the last 10 years (Berni Canani
position early in life play a key role in early host
et al. 2013; Turner et al. 2015; Mullins et al.
immunological development and represent a
2015; Nocerino et al. 2015; Mullins et al. 2016).
critical factor underlying FA (Prince et al.
Although more than 170 foods have been
2015). Fig. 1 summarizes the main possible
Targeting Food Allergy with Probiotics 59

Fig. 1 Gut microbiota as a target of intervention against the risk of food allergy (red arrows), whereas on the right
allergy. Several environmental and dietary factors could side of the figure are reported the most beneficial factors
modulate the diet-microbiota-epigenetics axis influencing able to protect the baby against the occurrence of food
the occurrence of food allergy. On the left side of the figure allergy (blue arrows)
are summarized the most relevant factors able to increase

contributing factors that have been ascribed to FA in case of systemic reactions is advocated. All
development. Many subjects with FA will natu- these factors contribute to anxiety and stress and
rally outgrow it over time; however, the natural significantly affect the quality of life of patients
course highly depends on the causative allergen. and their families (Burks et al. 2018; Sicherer and
For example, cow’s milk allergy (CMA) usually Sampson 2018; Heine 2018). However, in the
resolves in more than 50% of children by age past 10 years, a significant amount of emerging
5–10 years, hen’s egg allergy approximately therapies for FA have been addressed. These
resolves in 50% of children by age 5–10 years, therapeutic strategies are focused on reducing
and 50% of children with wheat allergy outgrow levels of allergen-specific IgE, enhancing levels
by age 7 years. Other FAs (peanut, tree nut, fish) of allergen-specific IgG or IgA, suppressing Th2
have low rates of resolution or are considered effector cells, or enhancing regulatory T cells
persistent (Savage et al. 2016). There is no Food through a variety of allergen-specific and allergen
and Drug Administration (FDA)-approved treat- non-specific strategies (Berin 2014). The main
ment for FA, and the current standard of care is new therapeutic perspectives for the treatment of
the strict and careful dietary avoidance of the FA include allergen-specific (oral, sublingual,
offending food allergens with the risk of epicutaneous, subcutaneous immunotherapy and
nutritional deficiencies. In addition, the risks of heat treatment of food) and non-allergen-specific
accidental ingestion are relatively common, and therapies (humanized monoclonal antibodies,
the prompt treatment of symptoms with anti-IgE and anti-IL5, probiotics). Allergen-
antihistamines, glucocorticoids, or epinephrine specific therapies are successful to variable
60 L. Paparo et al.

degrees in achieving desensitization, defined as intestinal IgA responses (Rautava et al. 2012).
increased threshold for reaction to food allergen, The effects can vary widely, depending on the
with regular exposition to the allergen. On the probiotic strain, dose, timing, and food matrix
other hand, to date, none of these experimental used (Heine 2018). This therapeutic strategy
therapies have been shown to lead to permanent represents attempt to deliberately modify the
tolerance or cure, defined as the absence of microbiota and their metabolism, with the idea
symptoms after ingestion of the food allergen ad that manipulating microbial communities to the
libitum even after prolonged periods of avoid- host’s advantages could treat FA (Ho and
ance, and safety remains a major concern (Burks Bunyavanich 2018). This review is focused on
et al. 2018; Rachid et al. 2018). Recent data are the preclinical and clinical evidence on the role
focused also on heat treatment of food, with the of probiotics in preventing or treating FA.
modifications of allergen structure to reduce IgE
binding. The introduction of extensively heated
milk and egg protein into the diet of subjects with 2 Main Preclinical Evidence
milk and egg allergy who tolerate the baked form, on the Probiotics Efficacy
rather than strict avoidance, is becoming an alter- in Food Allergy
native approach to induce a faster acquisition of
immune tolerance (about 10 times more likely to Immune tolerance is the most important therapeu-
outgrow allergy comparing to those who strictly tic target in FA. Immune tolerance is modulated
avoid allergen), with changes in immune by the activity of a well-modulated network com-
parameters (increase in IgG4 and decrease in posed by several immune and nonimmune
allergen-specific IgE) (Wood et al. 2013; Sicherer mechanisms. Gut microbiota and its metabolites
et al. 2014). Heating to reduce allergenicity is not (mainly the short-chain fatty acid, butyrate) exert
applicable to antigens such as peanut, where high a pivotal role in immune tolerance. Main preclin-
heat increases allergenicity rather than reducing ical evidence on the probiotics activity against FA
it. In this case, allergens can be also modified are summarized in Table 1. Evidence support the
through digestion, which forms peptides that are concept that probiotics could act at different level
too short to cross-link IgE but maintain T cell on immune tolerance network in FA: modulating
epitopes that would have the capacity to generate gut microbiota composition and function
T cell-mediated immunomodulation (Ramesh (increased production of butyrate (Berni Canani
et al. 2016). However, all these strategies present et al. 2016) and interacting with enterocytes with
several pitfalls and uncertainties: optimal dose, subsequent modulation of nonimmune (gut
frequency and duration, adverse events, potential permeability and mucus thickness) (Sudo et al.
for onset of eosinophilic esophagitis, achieving of 1997; Sütas et al. 1996; Malin et al. 1997) and
desensitization but not immunological tolerance, immune tolerogenic mechanisms (stimulation of
and long-term efficacy. Thus, their precise role in sIgA and beta-defensins production) (Hardy et al.
the management of FA has yet to be determined 2013) and modulation of cytokines response by
(Rachid et al. 2018). Among the non-allergen- immune cells (Kim et al. 2008; Torii et al. 2007;
specific therapies, the more promising approach Maassen et al. 2000; Niers et al. 2005; Takahashi
for FA treatment is the use of probiotics, defined et al. 2006a, b). In vitro stimulation of human
as live microorganisms that when consumed in peripheral blood mononuclear cells (PBMCs)
adequate amounts as part of food or as oral with selected probiotic strains is a commonly
supplements confer a health benefit on the host used experimental tool for the investigation of
(Hill et al. 2014). Probiotics actions are mainly the effect of these microorganisms on immune
mediated via the innate immune system (toll-like cells. The incubation of PBMCs with lactic acid
receptors), resulting in the promotion of T helper bacteria (LAB) strains such as Lactobacillus
1 differentiation, production of regulatory plantarum (L. plantarum) and Bifidobacterium
cytokines (IL-10 and TGF-beta), and enhanced adolescentis (B. adolescentis) resulted in an
Targeting Food Allergy with Probiotics 61

Table 1 Main preclinical evidences on the protective mechanisms elicited by probiotics against food allergy
Biological effects Bacterial strains References
Intestinal barrier maturation Bifidobacterium lactis/bifidum Sudo et al. (1997)
Lactobacillus rhamnosus GG Sütas et al. (1996)
Malin et al. (1997)
Immune response modulation Th1/Th2 balance: Th1 Bifidobacterium lactis/bifidum Kim et al. (2008)
production Lactobacillus acidophilus/
reuteri
Lactobacillus rhamnosus GG Torii et al. (2007)
Maassen et al. (2000)
Immune response modulation Th1/Th2 balance: Th2 Bifidobacterium bifidum/infantis/ Niers et al. (2005)
suppression longum
Lactobacillus acidophilus/
reuteri
Lactobacillus rhamnosus GG Takahashi et al. (2006a,
b)
Kim et al. (2008)
Aitoro et al. (2017a, b)
Immune system regulation: regulatory T (Treg) cell Bifidobacterium bifidum/infantis/ Niers et al. (2005)
development lactis
Lactobacillus acidophilus/ Maassen et al. (2000)
reuteri/casei Kim et al. (2008)
Lactobacillus rhamnosus GG Smits et al. (2005)
Immune system regulation: tolerogenic dendritic cell Bifidobacterium bifidum Niers et al. (2005)
development Lactobacillus reuteri/casei
Lactobacillus rhamnosus GG Mohamadzadeh et al.
(2005)
Braat et al. (2004)
Smits et al. (2005)
Immunomodulation: suppression of IgE production Bifidobacterium bifidum/longum Kim et al. (2008)
Bifidobacterium lactis Bb-12
Lactobacillus acidophilus Takahashi et al.
(2006a, b)
Lactobacillus rhamnosus GG Borthakur et al. (2008)
Borchers et al. (2002)
Torii et al. (2007)
Epigenetic modulation of TH1/Th2 genes expression Bifidobacterium breve Ghadimi et al. (2012)
Lactobacillus rhamnosus GG

increase in the production of the regulatory cyto- in T and B cell proliferation and a reduction in
kine IL-10 by monocytes and dendritic cells and IgE production were also observed in PBMCs
to an enhance of IFN-γ production by T cells from children with FA treated for 3 months with
(Cross and Gill 2001; Karlsson et al. 2004, the same probiotic mixture (Flinterman et al.
Mohamadzadeh et al. 2005). The addition of 2007). Using a 3D co-culture model of intestinal
probiotics mixture (L. acidophilus W55, epithelial cells and PBMCs as an in vitro model
L. casei W56, L. salivarius W57, L. lactis of the intestinal mucosal immune system,
W58, B. infantis W52, B. lactis W18, and Ghadimi et al. demonstrated that a commensal
B. longum W51) to PBMCs from children with probiotics B. breve and L. rhamnosus GG (LGG)
FA resulted in increased T cell proliferation with inhibit activation of inflammatory IL-23 and
enhanced production of Th1 and regulatory IL-17 cytokines, thereby reducing histone acety-
cytokines (Flinterman et al. 2007). An increase lation and simultaneously enhancing DNA
62 L. Paparo et al.

methylation (Ghadimi et al. 2012). The limitation of VSL#3 to sensitized mice significantly reduces
of studying the effect of probiotics in vitro lies in Th2 immune responses and protects against ana-
the extrapolation of the results to in vivo benefits. phylactic reactions induced by the allergen in a
For that reason, another commonly used experi- mouse model of FA. Also, the incubation of
mental toll in this area is based on the use of animal mouse spleen cells from sensitized mice with pro-
model of FA. Differential effects in relation to biotic mixture reduced allergen-stimulated IL-5 and
molecular action of oral administration of three IL-13 production and increased of IFN-γ and IL-10
LAB strains (B. coagulans 09.712 (Bc), production (Schiavi et al. 2011). An immunoregu-
L. plantarum 08.923 (Lp), and B. infantis 11.322) latory action by LGG has been demonstrated in a
in alleviating Th2-driven intestinal inflammation murine model of CMA. LGG administration was
and other symptoms associated with food-induced able to suppress of Th2 responses such as reduced
anaphylaxis were demonstrated in a murine model hypersensitivity scores and lowered serum cow’s
induced by a major shrimp allergen, ST. In particu- milk protein (CMP)-specific IgG1 while promoting
lar, oral supplementation with Bc and Lp signifi- Th1 responses by causing elevated IFN-γ and
cantly ameliorates anaphylaxis symptoms and CMP-specific IgG2a levels (Thang et al. 2011).
increases the population of CD4 + FoxP3+ T cells Similar results have been reported by our group in
in ST-sensitized mice through mTORC inhibition, a BLG-sensitized mice model, in which we found
FoxP3 upregulation, and GATA-3 downregulation that the administration of extensively hydrolyzed
(Linglin et al. 2017). Zang et al. investigated the casein formula plus LGG elicited a significant
preventive and therapeutic effects of oral reduction of allergic reaction and of IL-4, IL-5,
C. butyricum on anaphylactic symptoms in FA IL-13, and specific IgE production (Aitoro et al.
mice model induced by a β-lactoglobulin (BLG) 2017a, b). The studies of probiotic strain-specific
as allergen, a well-established model of CMA. The modulation on gut microbiota should be considered
authors observed that the oral treatment with as a key channel that could affect its in vivo action.
C. butyricum significantly ameliorated anaphylaxis A strain-specific effect of different probiotic strains
symptoms and increased sIgA and CD4+ CD25+ could be associated with a different impact on gut
FoxP3Treg cell in the spleen from BLG-sensitized microbiota as demonstrated by Wang et al. (Wang
mice (Juan et al. 2017). Neonatal monocolonization et al. 2015). Instead, oral application of any LAB
of germ-free mice by L. casei BL23 modulated the strains seemed to have no significant effect on the
allergic sensitization to cow’s milk proteins, devel- overall gut microbiota structure, but the amount of
oped higher IgG responses against caseins, and specific species exerted notable changes (Ai et al.
elicited by L. casei that was able to hydrolyze 2016).
insoluble caseins into soluble immunogenic Additional potential mechanisms by which
peptides (Maiga et al. 2017). Using ovalbumin probiotics exert pro-tolerogenic effects in the gut
(OVA)-sensitized murine model, it was are related to the production of immunoregulatory
demonstrated that oral administration of metabolites, which interact with the host immune
B. infantis ameliorated allergic symptoms, includ- cells to promote non-responsiveness to innocuous
ing reducing OVA-specific IgE and IgG1 levels in luminal antigens (Nowak-Wegrzyn and
the serum and Th2 cytokines release in the spleen. Chatchatee 2017; Di Costanzo et al. 2016).
Moreover, gut microbiota analysis showed that the Probiotics ferment fiber to short-chain fatty acids
probiotics-mediated protection was conferred by (SCFAs): butyrate, acetate, and propionate. Evi-
upregulation of the relative abundance of dence suggests that SCFAs, particularly butyrate,
Coprococcus and Rikenella at genus level (Yang contribute to mucosal homeostasis through the
et al. 2017). Similar results were obtained by others induction of Tregs and the regulation of epithelial
that observed a decrease of concentrations of IgE, barrier integrity (Berni Canani et al. 2015). Buty-
IL-4, and IL-13 following administration of rate deficiency has been observed in allergic
B. infantis CGMCC313–2 in BLG-sensitized patients (Sandin et al. 2009; Berni Canani et al.
mice (Meng-Yun et al. 2017). Oral administration 2016). It is possible to hypothesize that different
Targeting Food Allergy with Probiotics 63

type of dysbiosis could lead to similar effects in from children affected by challenge-proven
term of SCFAs or of other microbiota-derived IgE-mediated CMA. PBMCs were stimulated
metabolites production that could facilitate the with BLG in the presence or absence of butyrate.
occurrence of allergy. Clostridia species belonging Preliminary results showed that butyrate stimulates
to cluster IV and XIVa are the prominent source of IL-10 and IFN-γ production and decreases DNA
SCFAs in the colon. Bacteria-produced SCFAs methylation rate of these two cytokines. Same
have been implicated in the regulation of both the effective butyrate dose induces FoxP3 promoter
proportions and functional capabilities of Tregs in region demethylation and HDAC6/HDAC9
the colon (Arpaia and Campbell 2013; Smith and expression downregulation. The identification of
Howitt 2013), which, in some studies, has been bacterial metabolites, that affect positively the
specifically attributed to butyrate production by immune tolerance network, may be an interesting
spore-forming Clostridiales (Furusawa and Obata strategy against FA using a post-biotic approach.
2013). SCFAs bind metabolite-sensing G-protein-
coupled receptors (GPCRs) GPR43, GPR 109A,
and GPR41 with varying affinities. These GPCRs 3 Main Clinical Evidences
are expressed on epithelial cells and immune cells. on the Probiotics Efficacy
SCFAs activate GPCRs that stimulate colonic den- in Food Allergy
dritic cells and macrophages to secrete IL-10 and
promote development of Tregs in the mesenteric The increasing evidence on the importance of the
lymph nodes. Tregs are a source of tolerogenic composition of the gut microbiota in the patho-
cytokines, such as IL-10 and TGF-β that inhibit genesis of FA supports clinical research on the
allergic and inflammatory responses. Tan et al. potential role of the probiotics against these
showed that dietary fiber/SCFAs together with conditions. In this light, several studies have
vitamin A and a healthy gut microbiota maintain revealed the efficacy of selected probiotic strains
a tolerogenic mucosal environment and protect against FA. The effect appears strain specific and
against the development of FA. This is achieved more evident in the pediatric age. Thus, in a
principally through enhancement of the randomized double-blind placebo-controlled
tolerogenic CD103+ dendritic cells function, lead- trial, it has been demonstrated that the adminis-
ing to increased Tregs differentiation. In addition, tration of L. casei CRL431 and B. lactis BB12
mice lacking GPR43 or GPR109A receptors for added to hypoallergenic formula for 12 months
SCFAs showed exacerbated FA and fewer CD103 did not affect the acquisition of immune tolerance
+ dendritic cells (Tan et al. 2016). Data from our to cow’s milk proteins in infants with CMA (Hol
laboratory showed that oral butyrate treatment et al. 2008). Whereas using a similar study
induces a dramatic inhibition of acute allergic design, we have demonstrated that the addition
skin response, anaphylactic symptom score, body of the probiotic LGG to the extensively
temperature decrease, intestinal permeability hydrolyzed casein formula (EHCF) is able to
increase, and anti-BLG-IgE, IL-4, and IL-10 pro- accelerate immune tolerance acquisition in infants
duction in a murine model of CMA, suggesting a with CMA. Infants (aged 1–12 months), consec-
protective role of butyrate against FA (Aitoro et al. utively referred for suspected CMA (IgE- or
2017a, b). The mechanisms of action of butyrate non-IgE-mediated) but still receiving cow’s milk
are multiple; many of these involve an epigenetic proteins, were invited to participate in the study.
regulation of gene expression through the inhibi- Subjects were randomly allocated to one of the
tion of histone deacetylase (HDAC). The inhibi- two groups of dietary interventions: control
tion of HDAC 9 and 6 increases FoxP3 gene group, received an EHCF, and active group,
expression, as well as the production and suppres- received an EHCF containing LGG (at least
sive function of Tregs (Tao and de Zoeten 2007). 1.4
107 CFU/100 mL). After 12 months, the
We evaluated the direct effects of butyrate on double-blind placebo-controlled food challenge
peripheral blood mononuclear cells (PBMCs) was negative in 15 of 28 control infants (53.6%)
64 L. Paparo et al.

and in 22 of 27 infants receiving EHCF with LGG prevention trial (Kukkonen et al. 2007). The
(81.5%, p ¼ 0.027) (Berni Canani et al. 2012a, b). same combination without prebiotic did not
The results were confirmed in a subsequent trial, have a significant effect (Viljanen et al. 2005).
when the effect of five different dietary strategies The addition of prebiotic may have been the
was investigated: EHCF, EHCF + LGG, partially decisive difference, although clear evidence of
hydrolyzed rice formula, soy formula, or amino its bifidogenic effect is still lacking.
acid-based formula, in children affected by IgE- Probiotics have been also proposed to rein-
or non-IgE-mediated CMA. After the treatment force the effectiveness of immunotherapy
period of 12 months, the proportion of children (Rachid and Keet 2018). Oral food immunother-
acquiring tolerance to cow’s milk proteins was apy (OIT) is currently the most investigated
significantly higher in the group receiving EHCF approach for persistent FA, and it is based on
+ LGG (78.9%) than in other groups (Berni the concept that repeated oral/intestinal exposures
Canani et al. 2013). At the 3-year follow-up of to antigens normally lead to tolerance. Tang et al.
another pediatric cohort, a further confirmation of performed a randomized double-blind placebo-
a greater rate of resolution of IgE-mediated CMA controlled trial with the probiotic L. rhamnosus
as well as a lower incidence of other atopic CGMCC 1.3724 and peanut OIT (PPOIT) in
manifestations was described after treatment 62 children with peanut allergy. Subjects received
with EHCF+LGG (Berni Canani et al. 2017). a fixed dose of probiotic (or placebo) together
These effects could derive at least in part by a with peanut OIT (or placebo) once daily for a
modulation elicited by selected LGG components total of 18 months. Sustained unresponsiveness
on immune functions through different pathways (SU), determined by DBPCFC conducted
including enterocytes, monocytes, mast cells, 2–5 weeks after discontinuation of treatment,
DCs, and Tregs (Berni Canani et al. 2013; Pan was achieved in 82.1% of patients receiving
et al. 2010; Ghadimi et al. 2008; Donato et al. PPOIT compared with 3.6% of those receiving
2010; Mileti et al. 2009) and by an expansion of placebo, the highest rate of SU reported for any
butyrate-producing gut microbiota (Berni Canani food immunotherapy treatment evaluated in a
et al. 2016). Accordingly, studies in infants with randomized controlled study to date. PPOIT also
eczema and/or CMA who received EHCF induced high rates of desensitization (90%) and
supplemented with LGG showed benefits in was associated with reduced peanut skin test reac-
decreasing gastrointestinal symptoms and inflam- tivity, decreased peanut-specific IgE, and
mation (Isolauri et al. 2000; Baldassarre et al. increased peanut-specific IgG4 levels. PPOIT
2010). Ingestion of LGG for periods ranging was well tolerated with no participants
from 4 to 12 weeks has been associated with withdrawing because of adverse reactions (six
significant decreases in atopic dermatitis score participants withdrew for reasons unrelated to
compared with placebo in some small studies, PPOIT treatment); this is in stark contrast to
but not in several more recent and larger trials OIT whereby 10–30% of participants withdraw
(Wu et al. 2017). Others were unable to detect because of adverse reactions. At approximately
significant differences in the group overall but 4 years after the study ended, 67% were still
found the LGG-associated improvements to be consuming peanut, and only 58% of the
significant in subgroups of children with 12 participants who stopped peanut ingestion for
IgE-associated disease (Viljanen et al. 2005). 8 weeks demonstrated sustained unresponsive-
The combination of four probiotics (LGG, ness. Importantly, no OIT control group was
L. rhamnosus LC705, B. breve Bb99, and evaluated to determine if the probiotic itself had
Propionibacterium freudenreichii ssp. shermanii) any effect on SU (Tang et al. 2015). Further
given in association with a prebiotic (galactooli- studies comparing peanut OIT with probiotic
gosaccharides) reduced the incidence of eczema with peanut OIT with placebo will be required
and atopic eczema and tended to reduce to evaluate this further.
IgE-associated diseases overall in a primary
Targeting Food Allergy with Probiotics 65

4 Conclusions Allen KJ, Koplin JJ (2016) Prospects for prevention of

food allergy. J Allergy Clin Immunol Pract 4:215–220
Arpaia N, Campbell C (2013) Metabolites produced by
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FA. Studies are promising, but more data are Baldassarre ME, Laforgia N, Fanelli M et al (2010) Lacto-
bacillus GG improves recovery in infants with blood in
needed to support a wide use of probiotics against the stools and presumptive allergic colitis compared
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communities and the immune system is opening Berin MC (2014) Future therapies for IgE mediated food
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Berni Canani R, Nocerino R, Terrin G et al (2012a) Effect
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therapy Allergy Clin Immunol 129:580–582
• More data on the mechanisms of action Berni Canani R, Nocerino R, Terrin G et al (2013) For-
mula selection for management of children with cow
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mal patient populations to achieve beneficial ance to dietary allergens. Curr Opin Allergy Clin
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multidisciplinary expertise in pediatrics, gas- allergic infants. ISME J 10:742–750
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https://doi.org/10.1007/5584_2018_317
# Springer Nature Switzerland AG 2018
Published online: 20 December 2018

Use of Probiotics to Prevent Celiac

Disease and IBD in Pediatrics

Gloria Serena and Alessio Fasano

Abstract In this chapter we will discuss the current

The incidence of chronic inflammatory knowledge regarding the microbiome’s contri-
diseases (CIDs) is increasing worldwide. bution to celiac disease and inflammatory
Their dramatic rise associated with limited bowel disease with a particular focus on how
effective strategies to slow down these probiotics may be used as potential preventive
epidemics calls for a better understanding of therapy for CIDs.
their pathophysiology in order to decrease the
burdens on childhood. Several cross-sectional Keywords
studies have demonstrated the association Celiac disease · Inflammatory bowel diseases ·
between intestinal dysbiosis and active Microbiome · Microbiota · Probiotics
diseases. Although informative, these studies
do not mechanistically link alterations of the
microflora with disease pathogenesis and, 1 Introduction
therefore, with potential therapeutic targets.
More prospective studies are needed to deter- 1.1 The Potential Role of the Human
mine whether intestinal dysbiosis plays a caus- Microbiome in CIDs
ative role in the onset and development of
CIDs. Furthermore, given the complexity of In the last three to four decades, the incidence of
the microflora interaction with the host, it is allergies and autoimmune diseases has been dra-
necessary to design a systems-level model of matically rising in the United States (USA) with a
interactions between the host and the develop- significant impact on the pediatric population
ment of disease by integrating microbiome, (Bach 2002). The “hygiene hypothesis” suggests
metagenomics, metatranscriptomics, and that this rise is mainly due to the reduced number
metabolomics with either clinical either of infections occurring at early age (Greenwood
environmental data. 1968, Strachan 1989); however, its accuracy is
continuously revised (Versini et al. 2015). Since
the inception of the Human Microbiome Project,
G. Serena (*) and A. Fasano our knowledge about the composition and func-
Mucosal Immunology and Biology Research Center and
tion of the human microbiome has been rapidly
Division of Pediatric Gastroenterology and Nutrition,
Massachusetts General Hospital for Children – Harvard expanding.
Medical School, Boston, MA, USA It is now well established that the microbiome
e-mail: [email protected]; afasano@mgh. can affect many key functions of the symbiotic
harvard.edu

69
70 G. Serena and A. Fasano

host, including gut mucosal barrier maturation and 1% of the worldwide population (Reilly et al.
and to the development and function of the 2012). While gluten has been recognized to be the
gut-associated lymphoid tissue (GALT). external trigger responsible for the onset of CeD,
Recent studies have shown that a specific the precise pathogenesis mechanisms responsible
microflora is present in both the human placenta for IBD are still unknown (Kaplan 2015). For
(Aagaard et al. 2014) and meconium (Vicario both conditions environmental and genetic risk
et al. 2010), therefore suggesting that the estab- factors have been described to play a role. Devel-
lishment of the microbiome may occur already opment of CeD has been strongly associated with
during the prenatal period. This is confirmed by HLA DQ2 and/or DQ8. These specific haplotypes
in vivo animal studies showing how the maternal of the human leukocyte antigen system contribute
commensal flora presented to the fetus during the to 40% of the genetic variance with 95% of CeD
last trimester of pregnancy contributes to its patients carrying an HLA DQ2 and 5% a HLA
immune system maturation and oral tolerance DQ8 (Leonard et al. 2015b). Additionally to the
establishment (Rescigno et al. 2001). Furthermore, HLA genotype, genome-wide association studies
research on the effect of delivery mode, maternal/ (GWAS) have identified more genes associated
infant diet, antibiotic exposure, and surrounding with CeD. Among these genes, interleukin-18
environment has suggested that all these factors (IL18), interleukin-21 (IL21), and chemokine
may significantly affect the early development of receptors CCR1, CCR2, and CCR5 are involved
the infant intestinal microbiome, in particular in immune function, while others, such as
within the first 3 years of life (Dominguez-Bello MYO9B, PARD3, and MAG12, are associated
et al. 2010, Azad et al. 2013). Hence, it is plausible with defects in intestinal permeability (Leonard
to infer that changes in the development of a et al. 2015b). Several studies in monozygotic and
healthy microbiome ecosystem occurring early in dizygotic twins have provided powerful evidence
life may have lasting effects (Yatsunenko et al. of the genetic susceptibility that contributes to the
2012). Several studies have demonstrated the pro- development of IBD (Thompson et al. 1996,
tective role that exposure to healthy and diverse Orholm et al. 2000). Nine IBD susceptibility
commensal species early in life have against CIDs loci have been described, some specific for
(Fig. 1). During the last decades, the need for Crohn’s disease and others related to IBD as a
knowledge about human microflora composition whole (Lees and Satsangi 2009). Furthermore
and function including the study of microbiome, GWAS studies have highlighted 50 loci specifi-
metagenome, metatranscriptome, and metabolome cally associated with IBD including genes
has been made possible by the vast array of new involved in bacterial recognition (NOD2),
technologies that have become available. With the autophagy (ATG16L1 and IRGM), immune
field growing, a multi-omic systems biology response (IL23R, IL12B, STTA3, JAK2), as
research approach in pediatrics could amplify our well as specific HLA loci (Lees and Satsangi
understanding of many common pediatric diseases 2009). Both CeD and IBD pathogeneses are
in a revolutionary way. This would help to identify characterized by increased intestinal permeability
possible targets for disease treatment, as well as for that contributes to the overall chronic intestinal
its prevention. inflammation. In CeD patients the interaction
between gliadin peptides and G protein-coupled
receptor CXCR3 on enterocytes (Lammers et al.
2 Pathogenesis of Celiac Disease 2008) triggers the MyD88-dependent release of
and Inflammatory Bowel zonulin, a potent intestinal barrier function
Diseases (Tripathi et al. 2009, Drago et al. 2006). The
consequent translocation of gliadin peptides into
Celiac disease (CeD) and inflammatory bowel the lamina propria activates the immune response
diseases (IBD) are chronic inflammatory intesti- machinery. Interleukin-15 (IL15) production
nal conditions that overall affect between 0.5% from enterocytes has an apoptotic and
Use of Probiotics to Prevent Celiac Disease and IBD in Pediatrics 71

PRENATAL POSTNATAL

2
3
1

Lymphocytes
B cells
Neutrophils
Mother derived bacteria
SIgA

Enriched Infant microbiome

Fig. 1 Stages of immune system maturation and enhance- (via delivery and breast milk) and environment-derived
ment of barrier function during the first years of life. The bacterial antigens. This interaction induces epithelial
initial seeding of the microbiota starts during the prenatal cells differentiation, tight junction enhancement, and
stage in utero where the commensal flora present in the immune cells proliferation (2). Secretory IgA (SIgA)
maternal placenta initiates signaling with the fetal mucosa favor diversity in the bacteria community (3)
(1). At birth the newborn is directly exposed to the mother

pro-inflammatory effect on the epithelium disease (Chassaing and Darfeuille-Michaud

(Escudero-Hernandez et al. 2017, Maiuri et al. 2011). IBD patients are characterized by
2000, Malamut et al. 2010); release of increased paracellular permeability and
intereleukin-8 (IL8) leads to neutrophils recruit- abnormalities in tight junctions (TJs) proteins
ment (Lammers et al. 2011, Lammers et al. 2015), expression and distribution. Furthermore the
followed by macrophages activation and prolifer- abundant production of pro-inflammatory TNFα
ation of intraepithelial cytotoxic lymphocytes. has been associated with TJs transcription regula-
After transglutaminase-induced deamidation, gli- tion, and patients with ulcerative colitis have been
adin peptides are recognized in the lamina propria shown to have defective mucosal barrier structure
by Th1 and Th17 cells (Garrote et al. 2008). This (Rosenstiel et al. 2007). The increased intestinal
leads to production of pro-inflammatory permeability characterizing IBD patients
cytokines such as interferon gamma (IFNγ), promotes bacterial translocation through the
interleukin-17 (IL17) and tumor necrosis factor- intestinal mucosa (Chassaing and Darfeuille-
alpha (TNFα), and activation of humoral immune Michaud 2011). The interaction between com-
response with IgA and IgG anti-Ttg antibodies mensal microbes and specific epithelial receptors
production (Fasano 2011). Finally the inflamma- triggers in susceptible individuals a chronically
tory response is exacerbated by the defective active inflammation that causes the perpetuation
function of T regulatory cells (Serena et al. of the disease (Fiorucci et al. 2002). Dendritic
2017, Granzotto et al. 2009). cells and macrophages recognize microbes
Contrary to CeD, the external trigger for IBD through pattern recognition receptors. Their acti-
is not known, and its pathogenesis is still unclear vation stimulates pro-inflammatory signaling
(Kaplan 2015). Increasing evidence suggests the cascades that lead to production of a wide range
contribution of impaired barrier function to the of cytokines (IL6, IL12, TGFβ) and initiation of
72 G. Serena and A. Fasano

adaptive immune response with proliferation of specimen collection, analysis pipelines, and
Th1/Th17 cells and production of IFNγ, TNFα, patients’ information make it difficult to compare
and interleukin-2 (IL2) (Lees and Satsangi 2009). these studies.
Patients with IBD have been shown to exhibit a Given the high HLA DQ2/DQ8 penetrance
reduced number of peripheral T regulatory cells that characterizes CeD patients, evaluating the
with decreased suppressive function in the association between differences in the coloniza-
mucosa (Eastaff-Leung et al. 2010). tion of the gastrointestinal tract and the genetic
Overall both CeD and IBD are multifactorial makeup may contribute to determine the infant’s
and complex conditions in which environment future risk of developing CeD (Tjellstrom et al.
and genetic susceptibility equally contribute to 2013, Olivares et al. 2015). Previous studies have
their onset and development. shown that genetically predisposed infants carry-
ing an HLA DQ2 are characterized by an
increased abundance of Firmicutes and
3 Role of Microbiome in CeD Proteobacteria compared to control infants
and IBD (Sellitto et al. 2012). Similarly, at-risk infants
(first-degree relatives of CeD patients) genetically
3.1 Celiac Disease compatible with CeD showed a reduction in
Bacteroidetes, a higher abundance of Firmicutes,
CeD represents a unique model of autoimmunity in and slower microbiota during the first 2 years
that the genetic predisposition with HLA DQ2 after birth (Sellitto et al. 2012).
and/or DQ8 (Schuppan 2000) and the environmen- Lionetti et al. have shown that a later introduc-
tal trigger (gluten) are known. Furthermore disease- tion of gluten in the infant’s diet only transiently
specific autoantibodies have been identified and delays the development of CeD but do not prevent
can be routinely measured. The precise moment its onset (Lionetti et al. 2014). Another study has
in which the loss of tolerance to gluten occurs can demonstrated that introducing small amounts of
be precisely determined by knowing when the gluten between 16 and 24 weeks does not influence
environmental trigger (gluten) has been introduced the overall incidence of CeD (Vriezinga et al.
in the diet and, most importantly, by frequently 2014). While the timing of gluten introduction
screening genetically predisposed individuals for does not seem to have pathological and preventive
the appearance of tissue transglutaminase (tTG) relevance, the effect that gluten has on altering the
autoantibodies (Schuppan 2000). host microbiome in genetically predisposed
Dysbiosis has been associated with the develop- individuals is well established. Firmicutes and
ment of CeD by several groups. In vitro studies Proteobacteria phyla have been shown to change
have shown that commensal bacteria can affect the in abundance upon introduction of gluten in the diet
digestion of gliadin, as well as cytokines produc- (Sellitto et al. 2012). Furthermore, this same study
tion and intestinal barrier function upon stimulation showed how kids that developed some kind of
with gliadin (Laparra and Sanz 2010, Lindfors et al. autoimmunity were characterized by high levels
2008). Numerous studies have described the micro- of lactate and high abundance of Lactobacilli spe-
bial composition and phylogenetic diversity of cies in the stools preceding the first detection of
fecal and small intestinal samples in CeD patients positive CD antibodies, therefore suggesting the
compared to healthy controls and how these can be possibility of identifying biomarker predictors in
related to age, disease status, and symptoms the microbiota (Sellitto et al. 2012).
(Wacklin et al. 2013). Additionally to alterations
in the microbiome compositions, also changes in
production of specific metabolites such as short 3.2 Inflammatory Bowel Disease
chain fatty acids (SCFA) in the stools have been
described in active CeD patients and associated Although the involvement of microorganisms in
with the described dysbiosis (Di Cagno et al. the pathogenesis of IBD has been previously
2011). However, the lack of standardization in suggested, data confirming the causative role of
Use of Probiotics to Prevent Celiac Disease and IBD in Pediatrics 73

specific pathogens in triggering the chronic inflam- Data on intestinal dysbiosis in pediatric IBD
matory cascade that characterizes IBD are still patients are scarcer. Two unreported strains of
missing. Although specific mutations in pattern adhesive-invasive E. coli (AIEC) were in pediat-
recognition receptors (PRR) have been linked to ric IBD patients and were associated with
IBD (Corridoni et al. 2014), these genetic upregulation of CEACAM6 (carcinoembryonic
mutations appeared to be present only in a sub- antigen-related cell adhesion molecule 6), tumor
group of IBD patients. This finding suggests that necrosis factor-α, and IL8 gene/protein expres-
genetic makeup can only partially explain IBD. sion (Negroni et al. 2012). Furthermore children
Hence the hypothesis that additionally to the with ulcerative colitis that were not responsive to
genetic predisposition, other factors may contribute steroids appeared to have decreased fecal
to the immune dysregulation that characterizes IBD microbiome diversity with abundant Firmicutes,
(Barclay et al. 2008). Recent studies have shown Verrucomicrobia, and Lentisphaerae (Michail
that an altered immune response to commensal et al. 2012).
microflora is able to trigger IBD pathogenesis and While the above studies are mainly descrip-
that this exacerbated immune response that follows tive, Morgan et al. described the association
is driven, in turn, by specific genetic makeup and between microbial dysfunction metabolism and
dysbiosis (Khor et al. 2011, Manichanh et al. 2012, bacterial signaling pathways suggesting a mecha-
Gevers et al. 2014, Morgan et al. 2012). Although nistic link between intestinal dysbiosis and IBD
the lack of consistency in studies design and data onset (Morgan et al. 2012).
analysis makes the comparison among these stud-
ies very difficult, reduction in biodiversity and
altered abundance of different taxa (Morgan et al. 4 Possible Probiotics
2012, Manichanh et al. 2006) are findings that have Interventions to Manipulate
been confirmed by the majority of research groups. Microbiome and Challenges
A study from Manichanh et al. performed in to Their Implementation
Crohn’s (CH) patients reported an increased abun-
dance in Bacteroidetes and Firmicutes as compared For long time, probiotics have been considered
to control individuals. Interestingly the number of promising candidates for treatment and preven-
Firmicutes’ rybotypes is reduced in CH patients tion of CIDs such as CeD and IBD. If the causa-
(Manichanh et al. 2006). A study looking at the tive role of dysbiosis on CIDs was confirmed, the
microbiome composition in CH patients and how manipulation of microbiome composition via
this relates to disease activity shows that the acute probiotics in order to resupply/rebalance the gut
phase of the disease is characterized by decreased ecosystem should improve the overall health of
diversity of Bacteroidetes as compared to the the microflora and therefore contribute to the
remission patients (Seksik et al. 2003). In addition disease prevention. While the theory of probiotics
to the disease activity, also the site of the disease use is exciting, its efficacy is still under debate,
inflammation appears to contribute to the mainly due by the still uncertain idea of what a
microbiota composition with higher Firmicutes healthy microbiota consists of. Currently, diver-
diversity in subjects with colonic disease and sity is considered the main characteristic for a
reduced diversity in patients with affected ileum healthy microbiome. Several studies have shown
(Willing et al. 2010). As expected, the site of sam- the association between reduced microbial diver-
pling has been shown to influence microbiome sity and many diseases (Falony et al. 2016). The
analysis data in IBD studies. Mucosal samples use of probiotics can be challenging also for tech-
from CH patients show increased abundance of nical reasons: while most studies describe
Enterobacteriaceae and reduced diversity of dysbiosis at phylum level, the most commonly
Feacalibacteria, while analysis from fecal samples available probiotics contain specific species,
gave opposite results (Swidsinski et al. 2002, therefore creating a gap between our knowledge
Chassaing and Darfeuille-Michaud 2011). on microbiome and potential therapies.
74 G. Serena and A. Fasano

Mechanistic studies looking at how specific et al. 2001). Specific Lactobacilli species have
probiotics may positively impact intestinal health been shown to lyse the proline /glutamine-rich
failed in taking in considerations the complex gluten peptides reducing their immunotoxicity
biological networks that play a part in disease and to decrease gluten concentration to less than
development. Therefore, it is desirable for the 10 ppm (Rizzello et al. 2007). The efficacy of
future research to focus on how specific Lactobacilli in digesting gluten has been proved
microbiota signatures may contribute to the dis- in vivo by challenging CD patients in remission
ease development before exploring therapeutic with probiotic predigested sweet gluten
treatments. An example relative to what stated containing baked goods. The lack of worsening
above is presented by a recent phase 3 clinical in hematological, serological parameters, and
trial about NEC prevention in which data showed intestinal permeability during 60 days of chal-
the lack of efficacy in administering the probiotic lenge in the participants suggested that
Bifidobacterium breve to premature infants Lactobacilli-derived endopeptidases were able to
(Costeloe et al. 2016). While this probiotic was completely degrade gluten and annul its toxicity
chosen among others because it is commercially for CeD patients (Francavilla et al. 2017). VSL#3,
available and because of its reported positive a probiotic mixture of eight lactic acid and
nutritional outcomes, no data confirmed its Bifidobacteria, has been shown to hydrolyze
benefits in preventing NEC. A prospective completely several α2-gliadin-derived epitopes
cross-sectional observational birth cohort study and regulate the epithelial barrier function by
describing the microbiome profile of premature stabilizing tight junctions (De Angelis et al.
infants later affected by NEC showed no evidence 2006, Madsen et al. 2001). Orlando et al. have
that reduction of Bifidobacteria could be shown that Lactobacillus rhamnosus is able to
associated with NEC development, therefore reduce the gliadin-mediated production of
predicting the failure of the preventive interven- polyamines, therefore restoring the intestinal bar-
tion with Bifidobacterium breve. rier function and reducing zonulin release
This work highlights the importance of (Orlando et al. 2014). In a similar study in
identifying the microbiome signature associated which CaCo2 cells were stimulated with gliadin
to a specific condition before designing an inter- peptides, the authors have demonstrated that fer-
ventional study. Knowledge about the mentation of gliadin synthetic peptides with Lac-
microbiome composition paired with mechanistic tobacillus paracasei was interfering with their
understanding of how specific bacteria contribute entrance in the epithelial compartment (Sarno
to the disease development would greatly help in et al. 2014).
using probiotics in a more effective manner. Additionally to the endopeptidases activities,
probiotics have been also demonstrated to reduce
the gluten-induced inflammation by modulating
5 Use of Probiotics for CeD the immune response. Bifidobacterium breve has
and IBD Prevention and/or been shown to prevent intestinal inflammation in
Treatment CeD pediatric patients by inducing production of
anti-inflammatory cytokine interleukin-10 (IL10)
5.1 Celiac Disease by Tr1 cells and reducing TNFα serum level
(Zheng et al. 2014, Jeon et al. 2012). Other two
Probiotics have been used in several clinical trials studies conducted in children with CeD have
as an additional treatment to the gluten free diet demonstrated that consumption of different
(Table 1). Probiotics, in fact, are not only potent strains of B. breve for 3 months reconstituted the
modulator of immune response and barrier func- microbiome composition by reducing the abun-
tion, but they also represent an important source dance of Verrucomicrobia and production of
of endopeptidases to detoxify nondigestible glu- short chain fatty acids (Primec et al. 2017),
ten epitopes (Francavilla et al. 2017, Madsen re-establishing the physiological Firmicutes/
Use of Probiotics to Prevent Celiac Disease and IBD in Pediatrics 75

Table 1 Summary of main human studies on use of probiotics for treatment of chronic intestinal diseases
Study Type of probiotic Condition Number of participants Outcome
Rizzello et al. Lactobacilli CD 12 +
(2007)
Francavilla et al. Lactobacilli CD 10 +
(2017)
Olivares et al. Bifidobacterium longum CD 127 +
(2018)
Bibiloni et al. VSL#3 and immunosuppressants UC 34 +
(2005), Tursi et al. and/or amynosalicylates
2010
Miele et al. (2009) VSL#3 UC 29 +
Kato et al. (2004) Bifidobacteria-fermented milk UC 20 +
(BFM)
Ishikawa et al. BFM UC 21 (11 subjects, BFM group, +
(2003) and 10 subjects, control group)
Oliva et al. 2012 Lactobacillus reuteri UC 40 +
Furrie et al. (2005) Bifidobacterium longum and UC 18 +
Bifidobacterium breve
Butterworth et al. Lactobacillus rhamnosus GG CH 11 +
(2008) pediatric
patients
Schultz et al. Lactobacillus rhamnosus GG CH 11 +
(2004) pediatric
patients
Gupta et al. (2010) Lactobacillus rhamnosus GG CH 4
pediatric
patients
Fujimori et al. Bifidobacterium breve, CH 10 +
(2007) Lactobacillus casei, and
Bifidobacterium longum
Steed et al. (2010) Bifidobacteria CH 35 +

Bacteroides ratio and increasing the enrolled subjects for extensive time. Further stud-
Actinobacteria abundance (Primec et al. 2017). ies are deemed to better understand the long-term
Similarly, Olivares et al. have shown that admin- effect of the use of probiotics in the contest of
istration of Bifidobacterium longum in children CeD to better understand how to use them in a
with CeD triggered a reduction of peripheral safe and proper manner.
CD3+ T cells as well as decreased levels of
TNFα and fecal IgA (Olivares et al. 2018).
While the number of studies involving probi-
5.2 Inflammatory Bowel Disease
otic preparations as a treatment for CeD is sub-
stantial, the literature about the use of probiotic as
Probiotics have been considered possible thera-
preventive measure in genetically predisposed
peutic tools for IBD for long time (Table 1). They
individuals is rather modest. Given the important
can influence the microbiome composition and
role that the microbiome plays in the develop-
function, produce antibacterial substances,
ment of the immune system, a deeper understand-
enhance barrier function, reduce intestinal perme-
ing on how shaping the microbiome composition
ability, and modulate innate and adaptive immune
may influence the final outcome of the disease is
response (Orel and Kamhi Trop 2014).
fundamental. Additionally the clinical trials
Several groups have explored the potential of
developed until these days do not follow the
VSL#3 as possible treatment for IBD. The
76 G. Serena and A. Fasano

probiotic mixture has been shown to significantly While probiotics have great potential for future
enhance the remission stage of patients with therapeutic approaches in IBD, further studies are
ulcerative colitis (UC) when administered in necessary to ameliorate the specific conditions of
combination with immunosuppressants and/or the treatment.
amynosalicylates (Bibiloni et al. 2005, Tursi
et al. 2010). Furthermore VSL#3 has been also
shown to be useful in maintaining the remission 6 Prospective Longitudinal
stage in UC patients both in adult and pediatric Studies as Ideal Approach
populations (Miele et al. 2009). Kato et al. tested to Identify Targets for Disease
Bifidobacteria-fermented milk (BFM) supple- Prevention Through Probiotics:
mentation as a dietary adjunct to treat UC and The CDGEMM Proof of Concept
concluded that the supplementation was more Project
effective than conventional treatment (Kato
et al. 2004). Similarly, Ishikawa et al. found Different groups have reported that genetic risk
that BFM supplementation triggered a reduction for developing autoimmune diseases is associated
in the relative proportion of Bacteroidaceae and with alterations in the microbiota composition
butyrate production in UC patients (Ishikawa (Sellitto et al. 2012, Olivares et al. 2015). For
et al. 2003). A prospective randomized example, reduced abundance of Bacteroidetes
placebo-controlled study showed that Lactoba- and increased Firmicutes were described in
cillus reuteri improved the inflammation in UC infants at risk for CeD (Sellitto et al. 2012). The
patients by increasing IL10 production and microbiota of these infants also showed a delay in
reducing the levels of pro-inflammatory interleu- maturation at 2 years of age (Sellitto et al. 2012)
kin-1β (IL1β), TNFα, and IL8 (Oliva et al. as compared to not at-risk control group, while
2012). Similarly, also Bifidobacterium longum the maturation was complete in not at-risk infants
and Bifidobacterium breve have been shown to at 1 year (Palmer et al. 2007). Additionally, this
reduce inflammation in UC patients as well as same study showed that preceding the detection
increase regeneration of epithelial cells (Furrie of positive antibodies, the microbiome of infants
et al. 2005). Treatment with Lactobacillus that developed an autoimmunity was
rhamnosus GG significantly improved the clini- characterized by reduction in Lactobacillus spe-
cal activity of CH pediatric patients by enhanc- cies (Sellitto et al. 2012). Similar data were con-
ing barrier function (Butterworth et al. 2008) as firmed in a later study in which differences
well as maintaining the disease remission between the microbiota compositions at 1 month
(Schultz et al. 2004). Other studies however did of age were observed, with infants genetically
not find the same promising results in their study predisposed showing increased Firmicutes and
(Gupta et al. 2000). Fujimori et al. conducted an Proteobacteria compared to infants without a
open-label study in which patients with CH were genetic predisposition (Olivares et al. 2015).
treated with two probiotic preparations Although extremely informative, these studies
containing Bifidobacterium breve, Lactobacillus have the limitation of having a cross-sectional
casei, and Bifidobacterium longum. The study design, therefore assuming that alterations in the
showed an amelioration of clinical symptoms microbiome are causative of the disease. How-
in the cohort and concluded that high doses of ever, changes in the microbiome may also repre-
probiotics could be safely used as co-therapy for sent a consequence of the disease rather than the
the treatment of CH (Fujimori et al. 2007). cause. To correct this limitation, longitudinal pro-
Finally different studies utilizing Bifidobacteria spective studies are needed to link the
strains have shown promising results in CH microbiome composition and function with the
patients with improvement of the mean histolog- pathogenesis of CIDs.
ical score and reduction of pro-inflammatory Two longitudinal studies prospectively
TNFα (Steed et al. 2010). screened infants, with a first-degree family
Use of Probiotics to Prevent Celiac Disease and IBD in Pediatrics 77

member with CeD, from birth and found that CeD on the bacterial component of the microbiome
can develop very early in life. The first study excluding analysis on virome, fungi, and
found that 16% of infants with a first-degree parasites. Additional studies looking at the
relative with CeD and carrying HLA DQ2 mechanisms through which the microflora
and/or DQ8 will develop CeD by age 5 (Lionetti interacts with the host are needed. This is the
et al. 2014). The second study demonstrated that only way we will be able to understand how to
38% of infants with first-degree relatives of CeD design interventions for treatment and prevention.
patients and carrying two copies of DQ2 will The transition from descriptive to mechanistic
develop CeD by age 5 (Vriezinga et al. 2014). microbiome studies is fundamental to succeed in
These findings support the hypothesis that early translational medicine. Researchers working with
environmental factors may strongly contribute to pediatric populations are the only ones that can
the development of CeD. start this transition given the hypothesis that the
Unfortunately, the abovementioned studies did development of the microbiome during the first
not look at microbiome composition or function. 1000 days of life has a lasting effect on an
To date no large-scale, longitudinal studies have individual’s future health and risk of disease.
defined the mechanistic contribution of the gut Large-scale, multicenter, prospective, longitu-
microbiota in the pathogenesis of CeD. The dinal, multi-omic studies are required to design
Celiac Disease Genomic, Environmental, therapeutic strategies and targeted prevention
Microbiome, and Metabolomic (CDGEMM) has therapies using probiotics as tool to modulate
been designed to answer this question. Its aim is the microbiome composition and function. Ide-
to understand the role that the gut microbiome ally, data collection should start before the devel-
plays in early steps involved in the pathogenesis opment of the disease in order to understand the
of autoimmunity (Leonard et al. 2015a). This role that the microbiome plays in the develop-
study is conceptualized based on the hypothesis ment of disease.
that the combination of gluten and specific
microbiota in infants genetically at risk for CeD
triggers specific metabolic pathways that can epi-
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 Part III
The Child
Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2019) 1125: 85–100
https://doi.org/10.1007/5584_2018_318
# Springer Nature Switzerland AG 2018
Published online: 22 December 2018

Fighting Fatty Liver Diseases

with Nutritional Interventions,
Probiotics, Symbiotics, and Fecal
Microbiota Transplantation (FMT)

Valerio Nobili, Antonella Mosca, Tommaso Alterio,

Sabrina Cardile, and Lorenza Putignani

Abstract conditions, including type 2 diabetes (T2D),

Pediatric obesity is rising worldwide leading obesity, and nonalcoholic steatohepatitis
the worrying phenomenon of nonalcoholic (NASH). One of the emerging areas of the
fatty liver disease (NAFLD) to shift into one study is the link between liver diseases and
of the most frequent causes of chronic liver gut microbiome, which has added new infor-
illness in childhood. Occurrence of NAFLD mation to the understanding of the so-called
depends on several factors such as the geo- gut-liver axis.
graphical area and the diagnostic modalities In order to address the role of gut
used; overall it ranges between 3% and 10% microbiome in NAFLD onset and progression,
of pediatric population, increasing up to 70% it is necessary to “decipher” operational codes
in patients with metabolic comorbidities for microbiome investigation within the con-
(Manco M, Bottazzo G, DeVito R et al, J Am text of advanced laboratory medicine to cap-
Coll Nutr 27:667–676, 2008). ture microbiome features and, hence, to
Recent findings have related the intestinal address the function of the intestinal
microbiota to a plethora of pathological microbiome within the gut microbiota-
liver axis.
Results of these investigations have allowed
V. Nobili (*) the beginning of implementing the usage of
Hepatology, Gastroenterology and Nutrition Unit, IRCCS probiotics and symbiotics in the medical
“Bambino Gesù” Children’s Hospital, Rome, Italy approach of obesity and NAFLD in adults and
Department of Pediatric – University “La Sapienza,” children. Several randomized clinical trials
Rome, Rome, Italy (RCTs) have been already published on fecal
e-mail: [email protected]
microbiota transplantation (FMT), T2D,
A. Mosca, T. Alterio, and S. Cardile NASH, and inflammatory bowel disease (IBD).
Hepatology, Gastroenterology and Nutrition Unit, IRCCS
“Bambino Gesù” Children’s Hospital, Rome, Italy
This review proposes to describe the cur-
rent state of knowledge on the ways fatty liver
L. Putignani
Unit of Parasitology, Children’s Hospital and Research
diseases can be addressed with nutritional
Institute Bambino Gesú, Rome, Italy interventions, probiotics, symbiotics,
Unit of Human Microbiome, Children’s Hospital and
and FMT.
Research Institute Bambino Gesú, Rome, Italy

85
86 V. Nobili et al.

Keywords causes such as sedentary lifestyle, excessive

NAFLD · Probiotics · Fecal microbiota intake of calories, genetic susceptibility, epige-
transplantation netic regulation, and altered gut microbiome
homeostasis (Clemente et al. 2016).
Currently, there are no licensed treatments for
NAFLD and lifestyle interventions remain the main
1 Introduction approach, with lack of compliance representing,
however, a major limiting factor. A variety of
Pediatric obesity is rising worldwide leading the diets have been proposed such as the “low-fat,”
worrying phenomenon of nonalcoholic fatty liver “low-carbohydrate,” and “low-fructose” ones.
disease (NAFLD) to shift into one of the most Pharmacological interventions include vitamin
frequent causes of chronic liver illness in child- D and E, polyunsaturated fatty acids (PUFAs),
hood. Occurrence of NAFLD depends on several metformin, and probiotics (Manco et al. 2008).
factors such as the geographical area and the Both lifestyle and pharmacological
diagnostic modalities used; overall it ranges interventions have been tested in several RCTs
between 3% and 10% of pediatric population, (either in association or as single intervention)
increasing up to 70% in patients with metabolic using as end points the improvement of LTFs,
comorbidities (Manco et al. 2008). histology, and liver ultrasound features. Cur-
NAFLD consists of a range of conditions, rently, weight reduction is considered the primary
rather than a single entity, from hepatic steatosis intervention in fighting fatty liver, while vitamin
to nonalcoholic steatohepatitis (NASH) with or E seems to be the only medical option which may
without fibrosis to final-stage hepatic disease have a direct effect on the liver; other options,
(Manco et al. 2008). instead, cannot be recommended as a treatment to
Children with NAFLD are diagnosed either date (Manco et al. 2008).
through an assessment for obesity or fortuitously Bariatric surgery techniques such as intragastric
by liver ultrasound or deranged function liver tests balloons, gastric bypass or adjustable banding, and
(FLTs); however a formal diagnosis of NAFLD laparoscopy sleeve gastrectomy have also been
has to include a liver biopsy (Mann et al. 2018). suggested for obese adolescents with comorbidities
Based on histological criteria, pediatric such as diabetes, obstruction sleep apnea syn-
NAFLD has been divided into two entities: drome, and pseudotumor cerebri (Clemente et al.
“type 1 NAFLD” and “type 2 NAFLD.” Type 2016). It must be noted however that weight loss
1 NAFLD describes histological aspects seen in surgical procedures, in children, account for less
obese adult with NASH characterized by steatosis than 1% of the total (Del Chierico et al. 2018).
mostly severe in the centrilobular area associated As pharmacological treatments are not a first-
with hepatocyte damage known as ballooning, choice option for pediatric NAFLD, there is a
lobular inflammation, and perisinusoidal fibrosis. need to investigate and identify potentially
Type 2 NAFLD, instead, consists of steatosis and modifiable risk factors.
portal field inflammation or periportal fibrosis. In this regard, one of the most exciting areas is
This aspect is mostly seen in childhood although the link that has been found to exist between the
association between type 1 and type 2 NAFLD is gut microbiota and liver diseases which has
commonly described in pediatric cohorts (Manco opened new avenues to our understanding of the
et al. 2008). so-called gut-liver axis.
Etiology and pathogenesis of primary NAFLD
in children are not completely understood, and to
date the disease may be explained by a progres- 2 The Intestinal Microbiome
sive “multiple-hit” hypothesis characterized, at
disease onset, by adipocyte overload (steatosis) The microbiota is the community of commensal,
and insulin resistance subjected to a compound of coactive, and pathogenic microorganisms that
Pediatric Fatty Liver and Probiotics Approach 87

inhabit the whole human body, and it is damaging the membrane of the enterocytes and/or
represented by about 100 trillion cells with a DNA, and this produces secondary particles which
proportion of human cell of 100 to 1 (Nobili may activate host receptor such as the nuclear
et al. 2018). farnesoid X receptor (FXR) (Poeta et al. 2017).
The microbiota composition dramatically TLRs are specific pattern recognition receptors
differs between geographical communities, normally inactive in the healthy liver that repre-
individuals, and races, and it varies with gender, sent a first line of protection against microorgan-
age, diets, and many other factors. ism and bacteria-derived molecules by producing
Microbial colonization starts already in utero; TNFα, interleukin-1β (IL-1β), and interferons.
it is influenced by delivery localization (vaginal Identified TLR receptors are TLR-2, TLR-4,
or abdominal) and feeding modalities (maternal TLR-5, and TLR-9 which identify pathogen-
or formula feeding) and then dynamically associated molecular patterns (PAMPs) such as
changes over time (Leung and Yimlamai 2017). endotoxins and lipopolysaccharide (LPS). TLRs
Approximately more than 100 microbial spe- hyperactivity, however, lead to liver damage and
cies inhabit the intestine, and, at the phylum level, it is known to be involved in NAFLD/NASH
pediatric gut of healthy children is dominated by pathogenesis (Miura and Ohnishi 2014) (Fig. 1).
Bacteroidetes and Firmicutes (Hollister et al. Bile acids derive from cholesterol in the liver,
2015) which absolve important physiological and they are firstly bound with amino acids tau-
functions such as mucosal permeability homeo- rine and glycine and then transferred to the bile
stasis and vitamin, carbohydrate, and bile acid through the bile salt export pump (BSEP) before
metabolism. Moreover, gut microbiota produces being released into the small intestine. Gut
pathogen-associated molecules which, in particu- microbiome has a crucial role as it takes part in
lar conditions, are known to be responsible for the bile acid metabolism by controlling choles-
inflammatory process within the liver (Clemente terol 7α-hydroxylase or oxysterol 7α-hydroxylase
et al. 2016). Therefore, as 70% of the blood flown (bile acid synthesis) and upregulating the ileal
goes to liver through the portal system, which apical sodium-dependent bile acid transporter
drains gut-derived products, it can be easily seen (ASBT) (bile acid uptake). Bile acid synthesis is
that intestinal dysbiosis may directly affect the regulated by a negative feedback inhibition
first line of defense represented by the liver through the FXR mainly expressed in the liver
(Abdou et al. 2016). and the ileum (Adolph et al. 2018). FXR has
normally a key role in lipid and glucide metabo-
lism, and dysbiosis leads to abnormal bile acid
3 Gut-Liver Axis modifications which change FXR activation and
signaling resulting in retained bile salt and leaky
The term “gut-liver axis” describes the close link gut that secondarily involve the liver (Leung and
among the gut and the liver that begins during early Yimlamai 2017).
prenatal life (Del Chierico et al. 2017). The liver
derives from a sprout of the ventral foregut endo-
derm, and it mainly gathers blood from the portal 4 Intestinal Microbiota in NAFLD
system that is rich in metabolites. As such it is the
first solid organ which faces the gut micro and Overweight and obesity are red flags for fatty liver
macro products. On the one hand, intestinal and NAFLD. In humans it has been shown that
dysbiosis and impaired intestinal barrier increase microbiome can alter body weight through many
bacterial/metabolite translocation breaking the ways. One is the variance of fermentation of com-
immunological equilibrium and leading to the plex carbohydrates in short-chain fatty acid
inflammation of the liver via the stimulation of (SCFA) (acetate, propionate, and butyrate). Buty-
hepatic Toll-like receptors (TLRs); on the other rate and propionate are considered anti-
hand, bile acids modify gut microbiome by obesogenic, while acetate is obesogenic (Schwiertz
88 V. Nobili et al.

Gut microbiota

Disrupted TJs

Portal vein
bacteria products

TLR-4
LPS
FFA
Tryglicerides

HSCs Kuppfer cells

NAFLD NASH

Oxidative Stress & ER stress

Fig. 1 Gut-liver axis: pathophysiological mechanisms of NAFLD development and progression of liver injury to NASH

et al. 2010; Morales et al. 2010). Interestingly, Furthermore, it has been shown that a choline-
while propionate and acetate mainly derive from deficient diet (CDD) may induce continual
the Bacteroidetes phylum, Firmicutes (Clostrid- modifications in the intestinal metabolome and
ium IV and XIVa: Eubacterium rectale, microbiota (Ye et al. 2018) and that a high intake
Faecalibacterium prausnitzii, and Roseburia of fructose may lead to alterations in bile acids
intestinalis) primarily produce butyrate. Butyrate composition and impaired epithelial stability
represents an important fount of energy for the (Zöhrer et al. 2017).
cells of the colon, and it is known to increase Choline is present in eggs and red meat, but it
intestinal eubiosis and potentially decreases intes- can also be biosynthesized (Shu et al. 2016).
tinal penetrability preventing the release of meta- Colonic bacteria can split choline into the
bolic toxins. Butyrate also appears to ameliorate precursors of dimethyl nitrosamine called
insulin sensitivity by stimulating the secretion of dimethylamine and trimethylamine, all factors
glucagon-like peptide-1 (GLP-1) and gastric inhib- participating in the formation and secretion of
itory polypeptide (GIP) incretins. SCFA have very low density lipoproteins (VLDLs) from the
many other beneficial effects: propionate and buty- liver. Both deficient and excess nutrition could
rate enhance the expression of the anorexigenic negatively influence the liver. In fact, a shortage
adipokine leptin, propionate alone inhibits resistin of choline participates in the development of
expression in the adipose tissue, and it is responsi- steatosis. Another nutrient that has been exten-
ble for the blockage of acetyl-CoA synthetase and sively studied is fructose. In cavies, a high fruc-
therefore gluconeogenesis at the expense of cho- tose intake contributes to NAFLD, alters the
lesterol synthesis. In contrast, acetate represents microbiota structure, and promotes endoplasmic
the most absorbed SCFA, and it is the basis for reticulum stress and apoptosis (Mouzaki et al.
lipogenesis of cholesterol both in hepatocytes and 2013; Gao et al. 2014). In humans, the link
adipocytes (Remely et al. 2014). The result is a between high fructose intake, microbiota, and
greater absorption of fatty acids leading to the NAFLD has not been demonstrated yet. A study
accumulation of adipose tissue and thus the evolu- of 427 adults with NAFLD however showed that
tion into fatty liver (Camp et al. 2012). those with increased fructose intake had less
Pediatric Fatty Liver and Probiotics Approach 89

steatosis and more fibrosis. A pediatric NAFLD following: enhancement in the bloodstream of
population study showed that uric acid, one of the intestinal mucosa, improving the absorption of
final products of fructose metabolism, was con- nutrients, and suppression of an activated protein
siderably higher in children with NASH com- kinase, the adenosine monophosphate-AMPK, in
pared to the NAFLD ones (Mosca et al. 2017). hepatocytes and myocytes, leading to inhibition
Hence, high fructose intake could be related with of oxidation of fatty acids and the appearance of
NASH, but how fructose is implicated in intesti- insulin resistance (Weber and Polanco 2012). To
nal dysbiosis, with relative alteration of the date, several studies have attempted to character-
gut-liver axis, has not been demonstrated in ize the microbiota of obese subjects. Khan et al.
human models so far. showed that the microbiota of obese subjects who
Moreover, it is hypothesized that, outside the followed a hypocaloric dietary program examined
enterohepatic system, bile acids may develop a before and after the program was predominantly
signaling function involving lipids, glucose, and composed of Firmicutes at the beginning, while
energy homeostasis by activating the FXR and G Bacteroidetes increased from 10% to 15% at the
protein-coupled receptor (TGR5). FXR is end of the study (Khan et al. 2016).
recognized to act as an important player in the
control of hepatic conversion of carbohydrate into
fat (de novo lipogenesis), in the formation of 5 Microbiota and NASH
VLDLs with the release of triglycerides
(TG) into plasma (Boursier and Diehl 2015). All Numerous studies, held on humans, highlighted a
these data demonstrated that bile acids can mod- convincing correlation between intestinal
ify the intestinal microbiota and vice versa, which microbiome and NASH (Zhu et al. 2015).
could have a function in NAFLD genesis. Changes in the microbiome alter the gut mucosa,
A study comparing the gut microbiome of permit intestinal access to the liver, and favor the
Western European children with that of African progression of fatty liver and its evolution to
children showed that the latter had much more NASH. Contrasting the results obtained in adult-
Bacteroidetes than Firmicutes with a significant hood, the only study held on children showed that
presence of bacteria able to hydrolyze cellulose gut mucosal permeability was greater in children
and xylan. African children also had a high con- with steatohepatitis than in children with steatosis
tent of SCFA. Based on these data, the modula- only (Abdou et al. 2016). This finding indicates a
tion of intestinal microflora may be, in the future, role for bacterial translocation that exposes the
one of the cornerstones for preventing and liver to multiple microbial metabolites and
treating of metabolic disorders related to food influences the spreading and then the progression
(Grześkowiak et al. 2012; Suzuki and Worobey of NAFLD. A study demonstrating a high bacte-
2014). rial proliferation in the small intestine and high-
Microbiota can be modified by obesity, level TNF-α in NASH patients suggests that chil-
regardless of food intake. Obese individuals dren with NASH are prone to systemic dysbiosis
have an increased level of an adipokine called and inflammation. Comparable results were suc-
leptin which has an explicit role that regulates cessively described in another study where higher
the constitution of the intestinal microbiota, via degrees of gut bacterial proliferation associated
the modulation of the secretion of antimicrobial with enhanced TLR4 expression and increased
molecules by the Paneth cells in the intestine, release of inflammatory mediators were found in
namely, a vicious circle between the microbiota NASH patients (Zhu et al. 2013). The character-
and the adipose tissue, that promotes further istic of NASH inflammation is probably the result
worsening of the degree of obesity (Musso of the interaction of several causes such as vis-
et al. 2011). ceral alcohol production, releasing of toxins, and
Extra mechanisms through the intestinal inflammatory intermediators. Alcohol consump-
microbiota which promote obesity are the tion causes fatty liver and is correlated with
90 V. Nobili et al.

disease evolvement. Endogenous alcohol produc- this family are often dispensed as probiotics.
tion may have a function in the pathogenesis of Members of the Lactobacillus spp. are known to
NASH and, interestingly, Escherichia coli, from regulate the immune response and enhance the
the Enterobacteriaceae family, is most expressed function of the epithelial barrier, all processes
in ethanol fermentation (Gao et al. 2014). that seem to prevent the progression into
At the same time, increased products of etha- NAFLD/NASH (Pace et al. 2015; Nobili et al.
nol metabolism are also seen in the feces of chil- 2018). However, Lactobacillus might lead to the
dren diagnosed with fatty liver. It has been shown generation of volatile organic substances such as
that alcohol dehydrogenase (ADH) and aldehyde acetate and ethanol, which are involved in the
dehydrogenase genes are highly upregulated in development of obesity and NAFLD (Zhu et al.
the liver of children with nonalcoholic fatty liver 2013). In fact, Lactobacillus family includes
(Liu et al. 2015). A novel study showed that there more than 180 species and a broad diversity of
is a positive correlation between blood ethanol organisms; some of them only implied in the
levels and insulin resistance in children with fermentation of sugars leading the production of
NAFLD. Moreover, ob/ob mice were found to lactic acid (e.g., L. salivarius and L. acidophilus),
have a considerably lower hepatic ADH activity and others, instead, are also involved in the syn-
compared to controls (Engstler et al. 2015). thesis of ethanol (e.g., L. casei, L. plantarum, and
According to these results, the authors suggest L. brevis). Moreover, in this case, the diminution
that the increase in ethanol in NAFLD could be of Ruminococcaceae may lead to a lower genera-
the result of the insulin-dependent variations in tion of SCFA and primarily butyrate. A reduction
liver ADH activity. Ethanol induces TG accumu- in the genus Faecalibacterium which produce
lation in hepatocytes and is a common source for butyrate has been also seen in NASH patients.
the generation of reactive oxygen species (ROS), On the contrary, in the study by Del Chierico
causing steatohepatitis and compromising the et al. (2017), it was found that in the NASH
intestinal entireness. These findings indicate that patients, the Ruminococcaceae and Dorea species
dysbiosis in NASH is responsible for elevated are more often present while the Oscillospira
production of autogenous alcohol, augmented species are less represented. Therefore the associ-
intestinal permeability, and exposes the liver to ation of a small concentration of Oscillospira
high concentrations of bacterial agents. In view of with increased levels of 2-butanone could charac-
these data, it can be assumed that, despite the terize the metagenomic profile of the intestinal
numerous differences in phylum, family, and gen- flora in children with hepatic steatosis. The ele-
der among healthy people and patients with obe- vated presence of Lachnospiraceae, Dorea, and
sity (with or without NASH), patients with Ruminococcus, seen in children with NASH,
obesity and patients with NASH tend to have an indicates that alterations in the intestinal flora
overlap of the gut microbiome composition. are linked with a more severe behavior of the
Results from a limited prospective study held by disease (Del Chierico et al. 2018). Increase in
Mouzaki and colleagues (Mouzaki et al. 2016) pathogenic bacteria such as those from
highlighted the dissimilarity of the gut microbiota Escherichia and Streptococcus genus, known to
among healthy individuals and NAFLD and promote gut inflammation as seen in inflamma-
NASH patients. NASH patients were found to tory bowel diseases, has been shown in patients
have a lesser proportion of Bacteroidetes than with NAFLD with a higher percentage compared
patients with steatosis only and healthy ones. to healthy individuals. Malaguarnera et al.
The two studies comparing patient with NAFLD (Malaguarnera et al. 2012) showed a prevalent
versus healthy controls to date have found an distribution of three species of Bifidobacterium
expansion in the genus Lactobacillus and a reduc- (Bifidobacterium longum, Bifidobacterium
tion in the Ruminococcaceae species in NAFLD. adolescentis, Bifidobacterium bifidum) and five
The association of NAFLD with Lactobacillus species of Lactobacillus (Lactobacillus vaginalis,
comes as a surprise, since numerous species of Lactobacillus zeae, Lactobacillus brevis,
Pediatric Fatty Liver and Probiotics Approach 91

Lactobacillus ruminis, Lactobacillus mucosa). In Lactobacilli has been highlighted both in children
particular, L. ruminis was only described in the with normal weight and in obese ones. To explain
NASH and NAFLD groups. L. mucosa and this apparent discrepancy, a hypothesis could be
L. vaginalis were more present in NAFLD than that Lactobacilli improve the ability of the gut to
obese and NASH patients. In contrast, an oppo- absorb and metabolize the nutrients, thus increas-
site correspondence between the presence of ing the transformation of food and also leading to
bifidobacteria and NAFLD/NASH was described. an increase in body weight (Vajro et al. 2011).
Especially, the concentrations of B. longum,
B. bifidum, and B. adolescentis were markedly
reduced in all patients, while they were much 6 Probiotics and Symbiotics
higher in healthy controls (CTRLs). In previous in NAFLD Therapy
studies, the presence of Bifidobacteria has been
related with a decreasing progression of liver Using probiotics to overthrow the intestinal
injury, reducing the exposure of hepatocytes to microbiota may result in a successful therapeutic
intestinal metabolites, especially LPS, which option to cease the onset and progression of
stimulate the production of inflammatory NAFLD. Several studies assessed the usage of
cytokines and oxidative stress, with increased probiotics in patients with NAFLD. Three
hepatic damage (Solga and Diehl 2003). RCTs, with approximately 128 participants,
B. longum has been reported as a probiotic that evaluated the effects and differences between
reduces endotoxin levels and hence inflammation probiotics and placebo. Three different probiotic
and progression of NASH, reducing hepatocellu- preparations have been used for 2–4 months. In a
lar, portal, and parenchymal damage (Ren et al. RCT, an 8-week probiotic treatment involving
2014). Ren et al. showed the role that 20 children with increased LFTs and ultrasound-
Bifidobacteria has in enhancing the liver expres- documented steatosis showed that 12 billion CFU
sion of sirtuin 1 (SIRT1-α), which increases the per day of Lactobacillus rhamnosus GG strain
expression of peroxisomal proliferation receptors caused a substantial improvement in ALT com-
(PPARa) and downregulates cholesterol level pared to placebo independently from BMI
through the modulation of transcription factor of variations (Alisi et al. 2014). In another pediatric
sterols 1c (SREBP-1c), two markers involved in RCT, the authors demonstrated an important ame-
lipid metabolism, thus preventing the progression lioration in ultrasound findings in patients with
of NAFLD (Yin et al. 2010). This study has also NAFLD who underwent biopsy after a 4-months
shown that obese patients had a substantial reduc- regimen of VSL#3 compared to placebo (Alisi
tion in Bifidobacteria in comparison with healthy et al. 2014). More recently, another RCT with
controls, corroborating the much-discussed probiotics – a formulation comprising Lactoba-
hypothesis of the function of Bifidobacteria in cillus acidophilus ATCC B3280, Bifidobacterium
the development of obesity (An et al. 2011). bifidum ATCC SD66576, Bifidobacterium lactis
Previous evidence in animals had in fact showed DMSZ 32269, and Lactobacillus Rhamnosus
that B. longum significantly reduced weight gain DSMZ 21690 – for 12 weeks showed reduction
in a cohort of cavies nurtured for 5 weeks with a of ALT and lipid profile in children with obesity
diet rich in fat (Glenny et al. 1997). It is important and ultrasound evidence of steatosis (Famouri
to underline that the obesity reduction activity, et al. 2017). These promising outcomes along
seen in some probiotics, is frequently correlated with low costs and no side effects make probiotics
with a cholesterol-lowering result, obtained in a great and futurist treatment option for NAFLD
consequence of the inhibition of cholesterol syn- in children. In earlier studies, Bifidobacteria
thesis, the increased leak of cholesterol in the reduced the exposure to LPS and thus the produc-
feces, and the inhibition of the reabsorption of tion of hepatic inflammatory mediators and oxi-
the bile salts (Casas and Dobrogosz 2000). At dative stress which resulted in a reduction of the
the same time, however, the presence of NAFLD activity (Feldstein et al. 2009; Tilg
92 V. Nobili et al.

2010). Bifidobacterium longum was shown as a group had enhanced insulin sensitivity in compar-
probiotic to decrease endotoxin release, hence ison to no modification in amoxicillin cohort.
inducing a reduced hepatocellular damage, portal Amoxicillin group had a reduction in gram-
and parenchymal inflammation, and NASH activ- positive bacteria (primarily Firmicutes) and a
ity (Tilg 2010). One study stressed the potential- countervailing proliferation in gram-negative
ity of Bifidobacteria in increasing hepatic bacteria (primarily Proteobacteria) and dimin-
expression of sirtuin 1a (SIRT1-α), which ished microbial variety (Albenberg and Wu
modulates the expression of peroxisome 2015). Unfortunately, there was no evaluation of
proliferator-activated receptor a (PPAR-a) and the microbiota after antibiotic suspension. The
rectifies the low level of the sterol-binding regu- result of oral rifaximin given with a dosage of
lator element in 1c (SREBP-1c), hence avoiding 1200 mg/day for 28 days in NAFLD (n ¼ 15) and
the development of NAFLD (Giorgio et al. 2014). NASH (n ¼ 27) adults was examined in a study
Furthermore, it has been found in some studies published in 2014 (Vrieze et al. 2014). The
that the use of probiotics with a high content of NASH cohort showed an important fall in the
Lactobacillus spp. may have a hypocholes- average ALT, AST, LDL, IL-10, LPS, and BMI
terolemic effect, through the inhibition of choles- when compared to baseline. Nonetheless, it must
terol synthesis, increasing the loss of fecal be noted that this study was not placebo-
cholesterol, reducing the absorption of choles- controlled. In essence, to date, antibiotics cannot
terol in the body, and causing the inhibition of be considered a treatment option for NAFLD as
the recycling of the bile (Scorletti et al. 2018) they do not seem to show auspicious efficiency in
(Table 1). targeting microbial manipulation (Table 2).
Additional approaches that could appear more
promising in the near future include an alteration
of the intestinal microbiota through the usage of
7 Diet and Modulation
prebiotics, substances effective for the flowering
of Microbiota
of GMOs, and symbiotics, products that combine
probiotics and prebiotics in one synergistic form,
Impact of diets on microbiome modulation
which seem to decrease intestinal inflammation
represents a flourishing field of study. Targeting
and enhance the epithelium barrier activity
the synergism between food, gut microbiome, and
(Mullish et al. 2018; Principi and Esposito 2016).
the health of the host is nowadays at the center of
In recent years, antibiotic therapies have also
attention for the development of new therapeutic
been used for manipulation of the microbiota.
interventions.
Numerous studies conducted with antibiotics in
Since early life, nutrition has important role for
patients with NAFLD/NASH have shown
growth and development of human species. A
conflicting results (Fernandes et al. 2014). The
study (Hevia et al. 2015) analyzed two different
macrolide solithromycin decreases inflammation
types of early diets such ad breast-feeding and
and ballooning of hepatocytes in animals, without
formula feeding and their link with gut
altering the fat load of the liver, independently
microbiome. It has been shown that breast milk
from LPS activity (Cobbold et al. 2017). Studies
is rich in healthy bacteria of Bifidobacterium
conducted on rifaximin, an antibiotic with a
species which are known to modulate the intesti-
gram-negative activity spectrum, show diverging
nal immune system and increase the production
results in NASH varying from a substantial
of Immunoglobulin A (IgA) (Lees et al. 2016).
decrease of ALT, IL-10 levels, and endotoxins
The same study highlighted that aerobic
to efficacy on fatty liver (Gangarapu et al. 2015)
organisms seem to be higher in breast-fed
(Vrieze et al. 2014). In a double-blind RCT
children, whereas anaerobic species such as
conducted in 20 adults with metabolic syndrome,
Clostridium difficile prevails in formula-fed
10 took vancomycin and 10 amoxicillin with a
children (Ferrocino et al. 2015).
dosage of 1,5 g/day for 1 week; vancomycin
Pediatric Fatty Liver and Probiotics Approach 93

Table 1 Trials on NAFLD and microbiota

Title Status Conditions Interventions Locations
1 Intestinal microbiota and Recruiting Morbid obesity University health
NAFLD pre and post bariatric Nonalcoholic network, Toronto,
surgery fatty liver Ontario, Canada
disease
2 Probiotics and microbiota in Active, not NAFLD Dietary Tel Aviv Sourasky
bariatric surgery recruiting supplement: medical center, Tel Aviv,
Bio-25 Israel
(Supherb)
Dietary
supplement:
Placebo (for
Bio-25,
Supherb)
3 Dietary intervention and Completed Obesity Other: Geneva university
intestinal microbiota in Nonalcoholic Hypocaloric hospital, Geneva,
non-alcoholic fatty liver fatty liver diet Switzerland
disease
(NAFLD)
4 Prebiotic fibre supplementation Recruiting Nonalcoholic Dietary University of Calgary,
and gut microbiota in non- fatty liver supplement: Calgary, Alberta, Canada
alcoholic fatty liver disease disease Prebiotic fiber
Dietary
supplement:
Placebo
Behavioral:
Weight loss
5 Gut microbiota and modulation Unknown Simple Procedure: Stefano Ginanni
of liver damage in NAFLD status steatosis (SS) Liver and Corradini, Rome, Italy
Nonalcoholic white adipose
steatohepatitis tissue biopsies
(NASH)
Obesity
6 Study on metabolic flexibility Recruiting Nonalcoholic Dietary Stefano Ginanni
and gut microbiota composition fatty liver supplement: Corradini, Rome, Italy
in obese subjects with NAFLD, disease Mediterranean Department of clinical
taking into account diet medicine, Sapienza
PNPLA3 and TM6SF2 Obesity Dietary University of Rome,
polymorphisms. Effects of supplement: Umberto I hospital,
nutritional intervention Low-fat diet Rome, Italy
combined to exercise Other:
Aerobic
exercise
7 Effects of probiotic on Enrolling Nonalcoholic Biological: Northwell health,
inflammation and microbiota in by fatty liver Probiotic Manhasset, New York,
patients with NASH invitation disease formulation United States
Probiotics VSL#3
MeSH
Descriptor
Data 2018
Interleukin-17
MeSH
Descriptor
Data 2018
(continued)
94 V. Nobili et al.

Table 1 (continued)
Title Status Conditions Interventions Locations
8 Fecal microbiota transplantation Recruiting Nonalcoholic Drug: Fecal Rhode Island hospital,
(FMT) in nonalcoholic fatty liver microbiota Providence, Rhode Island,
steatohepatitis (NASH). A pilot disease transplantation United States
study
9 Investigation of Synbiotic Recruiting Nonalcoholic Dietary Southamption general
treatment in NAFLD fatty liver supplement: hospital, Southampton,
disease Synbiotic Hants, United Kingdom
Dietary
supplement:
Maltodextrin
10 Effects of dietary fructose on gut Recruiting Nonalcoholic Other: The Rockefeller
microbiota and fecal metabolites fatty liver Fructose University, New York,
in obese men and disease solution (75 g) New York, United States
postmenopausal women: A pilot Obesity Other:
study Glucose
solution
(75 g)
11 Rifaximin in fatty liver disease Terminated Nonalcoholic Drug: Liver unit, St Mary’s
fatty liver Rifaximin hospital, Imperial College
disease London, London, United
NAFLD Kingdom
Nonalcoholic
steatohepatitis
12 Fatty acids, genes and Completed Nonalcoholic Toronto general hospital,
microbiota in fatty liver fatty liver Toronto, Ontario, Canada
disease
Steatosis
Nonalcoholic
steatohepatitis
13 Fecal microbiota therapy versus Recruiting Nonalcoholic Biological: Institute of Liver & biliary
standard therapy in NASH steatohepatitis Fecal sciences, New Delhi,
related cirrhosis. microbiota Delhi, India
transplant
Other:
Standard
medical
treatment
14 Histological improvement of Completed Nonalcoholic Dietary . No reported
NASH with prebiotic fatty liver supplement:
disease Prebiotic
oligofructose
Dietary
supplement:
Placebo
maltodextrin

Micro- and macronutrients such as proteins, concluded that the “Western diet,” which is rich
fats, carbohydrates, polyphenol, and probiotics in animal and protein fat, correlates with a reduc-
are known to modify gut microbiome with conse- tion of the total number of bacteria as well as
quential metabolic and immunological effects on beneficial bacterial species such as
the host. A recent review analyzed the impact of Bifidobacterium and Eubacterium; healthy bacte-
several popular diets on human microbiome and ria reduction has been seen in the “gluten-free
Pediatric Fatty Liver and Probiotics Approach 95

Table 2 Trial in obesity and NAFLD pediatric

Title Status Conditions Interventions Locations
1 Effect of prebiotic intake on Completed Overweight Dietary supplement: University of Calgary,
adiposity, satiety and gut Prebiotic fiber Calgary, Alberta, Canada
microbiota in overweight Obesity Dietary
and obese children supplement:
Placebo
2 Composition and collection Recruiting Childhood University of Illinois
feasibility of gut microbiota obesity College of medicine at
in children with and without Peoria, Peoria, Illinois,
obesity United States
3 Effects of HMOs on the Active, not Obesity Dietary supplement: Department of Paediatrics,
Faecal microbiota and on recruiting HMO Holbaek hospital,
host metabolism in obese Dietary Holbaek, Denmark
children supplement:
Dextropur
4 The effect of probiotics on Completed Metabolic Dietary supplement: Copenhagen university,
low-grade inflammation, syndrome Ls-33 Frederiksberg, Denmark
microbiota and risk factors Inflammation
for metabolic syndrome in Obesity
obese children
5 Effects of VSL#3 on Completed Obesity Dietary supplement: Bambino Gesù children
metabolic, endocrine, VSL#3 active hospital, Rome, Italy
Lipidomic and probiotic
inflammatory parameters of NAFLD Other: VSL#3
pediatric patients with placebo
BMI > 90
6 Effects of probiotics in Completed Obesity Dietary supplement:
obese children Probiotics mixture
7 Gut microbiome, adiposity, Completed Obesity, Dietary supplement:
and probiotics (GMAP) childhood Probiotic – VSL#3
Dietary
supplement:
Placebo pill – Soy
protein powder
8 Study on dietary nutrition Unknown Obesity Dietary supplement: Shenzhen Meilin high
intervention techniques for status Intestinal Dietary nutrition school, Shenzhen,
children obesity bacteria flora based on plant Guangdong, China
disturbance fermentation extract
Body weight
9 Effects of VSL#3 on Completed Obesity Dietary supplement: Bambino Gesù children
metabolic, endocrine, VSL#3 active hospital, Rome, Italy
Lipidomic and probiotic
inflammatory parameters of Other: VSL#3
pediatric patients with placebo
BMI > 90
10 BIFI-OBESE: Clinical trial Completed Obesity, Drug: AOU Maggiore della
in Paediatric obesity childhood Bifidobacterium Carità – Clinica Pediatrica
breve BR03 and – Ambulatorio di
Bifidobacterium Auxologia
breve B632 ed. Endocrinologia
Drug: Placebo SE Pediatrica, Novara, Italy
11 Pilot study of probiotics in Not yet Obesity, Dietary supplement:
pre-diabetic adolescents recruiting childhood Vivomixx
Dietary
supplement:
Placebo
(continued)
96 V. Nobili et al.

Table 2 (continued)
Title Status Conditions Interventions Locations
12 The effect of probiotics on Completed Metabolic Dietary supplement: Copenhagen university,
low-grade inflammation, syndrome Ls-33 Frederiksberg, Denmark
microbiota and risk factors Inflammation
for metabolic syndrome in Obesity
obese children
13 Nutrition and pregnancy Active, not Pregnancy Dietary supplement: University of Turku,
intervention study recruiting Comparison of Turku, Finland
Obesity probiotics, fish oil, Turku university hospital,
Gestational and their Turku, Finland
diabetes combination to
mellitus placebo
14 Probiotics and the gut Terminated Obesity, Dietary supplement: University of Southern
microbiome in obese abdominal VSL#3 California, Los Angeles,
Hispanic youth Adiposity Dietary California, United States
supplement:
Placebo
ClinicalTrials.gov Search Results 29/07/2018

diet” with a decrease in Bifidobacterium and Lac- and adults with obesity to normal weight ones,
tobacillus and a proliferation of unhealthy bacte- finding substantial differences between the four
ria such as E. coli, Enterobacteriaceae. With groups; the principal dissimilarities in microbiota
regard to vegan and vegetarian diets, Putignani content were between adolescents with obesity,
and Dallapiccola (Putignani and Dallapiccola rich in Faecalibacterium prausnitzii and Actino-
2016) found discrepant results as one study myces, and normal weight adolescents, whose
showed a significantly lower count of microbiota was characterized by
Bifidobacterium and Bacteroides species, while Parabacteroides, Oscillospira, Bacteroides
another indicated no differences when compared caccae, Rikenellaceae, and Barnesiellaceae. All
to an unrestricted control diet. Of interest, the detections suggesting that microbiota modulation
Mediterranean, rich in monounsaturated and may exemplify a target for obesity treatment.
polyunsaturated fat, polyphenols, fiber, low
glycemic carbohydrates, and vegetables, was
accompanied with an augmented count of the
8 Future Perspectives in NAFLD
total amount of bacteria and specifically healthy
Treatment: From Microbiome
bacteria such as Bifidobacterium, Prevotella, and
Profiling by “Fused Data”
Lactobacillus and a reduction in Clostridium spe-
Models to the Interventional
cies (Putignani and Dallapiccola 2016).
Procedure “Fecal Microbiota
Strictly related to diet is the phenomenon of
Transplantation” (FMT)
obesity which has been, in the past few years,
linked with the gut microbiome composition. A
Based on the relationship between gut
2018 Italian study (Del Chierico et al. 2018)
microbiome and NAFLD onset and progression
found that microbiome gut in both human and
(Del Chierico et al. 2018), it is necessary to “deci-
animal obese models is decreased in
pher” operational codes for microbiome investi-
Bacteroidetes quantity with a corresponding
gation within the context of advanced laboratory
increase in Firmicutes phylum (Nobili et al.
medicine to capture microbiome features and,
2018). In addition, the authors compared intesti-
hence, to decode the gut microbiome activities
nal microbiota ecological profiling of adolescents
in the complex gut microbiota-liver axis.
Pediatric Fatty Liver and Probiotics Approach 97

To perform microbiome profiling, an integrated clinicians with meta-omics microbiome profiles and
workflow with different levels of complexity must samples. The eventual aim is to assist the scientific
be conceived, going through the following steps: community, but especially the clinicians, with a
(i) to unscramble gut microbiome ecology by new tool of laboratory medicine investigation.
using either 16S rRNA targeted-metagenomics Therefore, a big effort is now focused on
(relative microbiome profiling, RMP) or investigating microbiota-mediated or
genomics-based (quantitative microbiome microbiota-linked disease mechanisms, with the
profiling, QMP) strategies (descriptive level, hope that a deep understanding of NAFLD
enterotypes or enterogradients’ profiling); (ii) to phenotype-enterophenotype interactions will fuel
predict gut microbial metabolic networks based the design and usage of novel therapeutic and
on metabolite measurements and assessment of preventive strategies.
their interaction with host metabolism by In the meantime, microbiome exploration has
exploiting metabolomics-based approaches (func- already become, for its high rate of complexity, a
tional level, metabotypes’ profiling); and (iii) to discipline, with two main branches, the
evaluate diet contribution to the microbial and host microbiome analytics and the microbiome clinics.
co-metabolic networks by metabonomics analyses The latter is now representing one of the biggest
(nutritional level, nutrients’ profiling). This revolution in the patient management and for sure
microbial community complexity can only be tack- a future perspective in clinical medicine.
led by an approach of systems biology, which can Fecal microbiota transplantation (FMT) is
integrate multifactorial NAFLD disease features nowadays proposed as a new therapeutic oppor-
and big data from gut microbiota profiling into tunity in a wide spectrum of diseases, such as
integrated disease-microbiome frameworks (Del Clostridium difficile (CD) and multidrug-resistant
Chierico et al. 2014). The identification of physi- (MDR) Enterobacteriaceae infections, inflamma-
cochemical modifications by unsupervised tory bowel diseases (IBDs), behavior alterations
profiling of the gut considered as “bioreactor” of (i.e., autism), obesity, type 2 diabetes (T2D), and
the gut-liver axis can provide novel information for NAFLD.
cutting-edge strategies to prevent and control For CD infections in adults, FMT has been
NAFLD, directed toward modulation and targeted shown to be even superior to antibiotic treatment.
modification of gut microbiota function, through For IBD, T2D, and NASH, some placebo-
diet-linked and personalized probiotic-driven controlled randomized controlled trials are cur-
solutions until FMT intended as bacterial therapy. rently open for the adult patients (De Groot
Advances in omics- and meta-omics-based et al. 2017). Techniques for FMT standardization
procedures (Bakker and Nieuwdorp 2017) have are fully developing for adults, and dedicated
pushed forward culture-independent technologies, protocols for pediatric cases are currently under
radically changing the current microbiology which considerations; in all cases they are still consid-
is now able to describe microbial communities’ ered experimental and research practices. For the
diversity and functional patterns, going beyond the metabolic syndrome, the gut microbiota could be
pathogen characterization. The current challenge related to the progression of insulin resistance and
now is to produce datasets of microbiome profiling diabetes and really open new avenues to new
data of disease-associated and reference control promising therapies by revealing in which patient
population (epidemiological screening) to address insulin resistance may be driven by gut
disease trajectories and age-associated dynamics of microbiome.
the human microbiome. For this reason, our institu- In a preclinical study, Zhou et al. (2017) have
tion (“Bambino Gesù” Children’s Hospital) has investigated the hypothesis regarding whether
created an infrastructure for a biobank of more FMT is effective in attenuating high-fat diet
than 6500 microbiota, prevalently associated to (HFD)-induced NAFLD in mice (Zhou et al.
gut, and is establishing a related dataset to provide 2017). With this purpose, 36 mice were
98 V. Nobili et al.

Table 3 The randomized controlled trials published of probiotics treatment for pediatric NAFLD
Number of
Authors Country participants Response Tolerability Comments
Vajro et al. Italy 10 placebo Improved ALT (2 placebo, No drop out. BMI z-score loss: 0.1
(2011) 30.2 probiotics, p ¼ 0.03) PLA vs 0.1 probiotic
10 No adverse Improved ALT only
Lactobacillus effects with treatment
GG
Alisi et al. Italy 22 placebo Improved US (81% 8% dropout BMI SD loss: 0 PLA vs
(2014) plus lifestyle 0.4
22 VSL#3 plus VSL#3 vs 23% PLA, No adverse VSL#3
lifestyle p < 0.001) effects Improved US with
reported VSL#3 treatment
Famouri Iran 32 placebo Improved ALT (9.7probiotics Not reported WC reduction: 1.5 cm
et al. vs 2.7 PLA, p < 0.05) PLA vs 2 cm probiotic
(2017) 32 probiotics Improved US (53% probiotics Improved C0 + HDL for
(mixture of 4) vs 16% PLA, p < 0.05) probiotic

randomized into control, HFD and HFD + FMT

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Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2019) 1125: 101–107
https://doi.org/10.1007/5584_2018_319
# Springer Nature Switzerland AG 2019
Published online: 11 January 2019

Probiotics in the Treatment

of Inflammatory Bowel Disease

Stefano Guandalini and Naire Sansotta

Abstract hand, there is clinical evidence of efficacy for

While considerable progress has been made in some specific strains and especially for multi-
the treatment of inflammatory bowel diseases strain preparations.
(IBD), alternative options are constantly In summary, more data are needed very
sought by adult patients as well as frustrated likely to yield a better understanding on what
parents of young patients. These include die- strains and in what doses should be used in
tary modifications, food supplements, and, different specific clinical settings.
more recently, probiotics.
Their potential use is based on the Keywords
demonstrated role of the altered mucosal Crohn’s disease · IBD · Inflammatory bowel
immune response to bacterial agents that even- disease · Lactobacillus · Probiotics · Ulcerative
tually leads to the chronic intestinal inflamma- colitis · De Simone Formulation
tion that characterized IBD. In fact, probiotics
might conceivably be beneficial due to multi-
ple mechanisms: stimulation of anti- 1 Introduction
inflammatory cytokines, inhibition of inflam-
matory cytokines, strengthening of intestinal Crohn’s disease (CD), ulcerative colitis (UC), and
barrier, and antagonistic action on pathogens. undetermined colitis, grouped under the name of
Such mechanisms have been largely exten- inflammatory bowel diseases (IBD), are chronic,
sively investigated in animal models both currently incurable inflammatory diseases of the
in vitro and in vivo. gastrointestinal tract of largely unknown causes.
Despite such premise, a relatively scarce While IBD can arise at any age, approximately
number of clinical trials are available, and of 10% of cases occur in children and teenagers,
them only a handful in pediatric age. Overall, with Crohn’s disease more commonly affecting
available evidence is very disappointing in the girls and UC boys (Loftus 2004). IBD appears to
treatment of Crohn’s disease (CD), where no be increasing in prevalence worldwide (Duricova
recommendation for probiotic use can be et al. 2014).
made. In ulcerative colitis (UC), on the other The balance between beneficial and harmful
intestinal microorganisms leads to either
S. Guandalini (*) and N. Sansotta
Section of Gastroenterology, Hepatology and Nutrition mucosal homeostasis or chronic inflammation,
Department of Pediatrics, University of Chicago, Chicago, and it is known that endogenous intestinal
IL, USA microbiota is involved in the onset of IBD
e-mail: [email protected]

101
102 S. Guandalini and N. Sansotta

(Scharl and Rogler 2012), as extensively 2016; Nishida et al. 2018), in spite of
recently reviewed in Basson et al. (2017) and uncertainties about the fact that the dysbiosis
Zuo and Ng (2018). Additionally, it has been could be the consequence, and not the cause, of
shown that microbiota in IBD patients differs the intestinal inflammation per se (Mack 2011) as
from healthy individuals, as it is characterized well described in a thorough recent review by Ni
by a reduced diversity, reduced abundance of et al. (Ni et al. 2017).
Firmicutes and Bacteroidetes, and increased In addition, an altered intestinal permeability
abundance of Enterobacteriaceae (Gosiewski has been found in patients with IBD, especially
et al. 2012). CD, and this may involve alterations in the muco-
The possible mechanisms of probiotic efficacy sal barrier eventually promoting bacterial translo-
have been explored in a variety of experimental cation (Chassaing and Darfeuille-Michaud 2011).
conditions both in vitro and in vivo (reviewed in Whether the environmental trigger for IBD
(Abraham and Quigley 2017)). The effects of any responsible for these changes in a condition of
probiotic depend on a number of factors, includ- dysbiosis is a single bacterium or, more likely, a
ing its metabolism, the molecules expressed on its number of microorganisms is still unclear. Fur-
surface, as well as the metabolites they produce thermore, also unclear is if the role played by an
(Oelschlaeger 2010). abnormal immune reaction of the commensal
In fact, a widely accepted theory on the microorganisms is important (Orel and Kamhi
pathogenesis of IBD calls for a complex interac- Trop 2014).
tion between genes (NOD2 and ATG16L1 In fact, it appears that individuals who are
variants being the best known) and a number of genetically predisposed at some point lose the
abnormalities like an imbalance between tolerance to commensal bacteria, thereby devel-
pro-inflammatory and anti-inflammatory T cell oping an inflammatory process chronically trig-
responses (including TNF-alfa and nitric oxide), gered and maintained by the microbiota (Fiorucci
an impaired epithelial barrier function, and more et al. 2002).
in general, as mentioned above, a condition of To further stress the role of the endogenous
dysbiosis (Ganji-Arjenaki and Rafieian-Kopaei microbiota, it was found that the vast majority of
2018). IBD patients’ intestinal mucosal biopsies showed
However, despite the valid conceptual basis evidence of bacterial invasion, while control
for the use of probiotics in IBD, well supported samples were devoid of bacteria (Seksik et al.
by encouraging experimental data in animal 2003; Swidsinski et al. 2002).
models, there is a paucity of published clinical An imbalance between beneficial and patho-
trials in humans. We will examine randomized genic bacteria as a contributory element in the
clinical trials (RCTs) that have used a comparator, development of IBD is further suggested by the
either a placebo or an accepted standard therapy. reduction in mucosa-associated Bifidobacteria
The possible utilization of probiotics for the pre- and a concomitant increase in E. coli and
vention of IBD is discussed elsewhere in this Clostridia (Ott et al. 2004; Ott and Schreiber
book (see Chapter 6). 2006; Manichanh et al. 2006).
Further changes in the IBD microbiome were
found using a metagenomic approach that
2 Gut Microbiota in IBD revealed fewer Bacteroidetes and Firmicutes,
bacteria that are known to possess an important
As mentioned, the different composition of butyrate-generating and anti-inflammatory activ-
microbiota in IBD patients in respect to healthy ity (Sokol et al. 2006; Frank et al. 2011; Martinez
individuals has been well documented in the past et al. 2008).
several years (Sokol et al. 2006) to the point that a To support the possible utilization of
dysbiotic microbiome is considered to be a criti- probiotics in IBD, Kim et al. (Kim et al. 2018)
cal factor for the onset of IBD (Prosberg et al. recently showed that Lactobacillus acidophilus
Probiotics in the Treatment of Inflammatory Bowel Disease 103

(LA1) significantly interferes with endoplasmic RCT with this probiotic failed to show significant
reticulum stress, suppresses NF-κB activation differences in the frequency of relapses that
(a major step in the pathogenesis of IBD), and occurred in 47.5% of the S. boulardii arm com-
thus possesses a potential to be utilized as an pared to 53.2% in the placebo arm (Bourreille
immunomodulator in the treatment of IBD. et al. 2013). A recent systematic review on the
Focusing now on data available in children, use of S. boulardii in IBD (Sivananthan and
the first large investigation on pediatric Petersen 2018) confirms the current lack of evi-
microbiota (Conte et al. 2006) showed a higher dence of its efficacy in these disorders.
number of mucosa-associated bacteria in the A multicenter RCT on 75 children with CD
small and large intestine of patients with IBD testing the efficacy to maintain remission of the
than in healthy controls. Of note, the highest probiotic Lactobacillus rhamnosus GG (LGG) in
concentrations of mucosa-associated bacteria addition to standard therapy (Bousvaros et al.
were found in children with CD and with 2005) also failed to demonstrate any additional
undeterminate colitis (Conte et al. 2006). benefit of the probiotic. Indeed, the children
Kellermayer et al. (Kellermayer et al. 2012) receiving LGG even relapsed sooner, though not
reported in an investigation on still untreated significantly so, than those who were on placebo.
pediatric patients with CD that those patients Similarly disappointing data have been
who had granulomatous CD had a higher number recently reported for adults in a systematic review
of genera and species, significantly differentiating and meta-analysis on the efficacy of probiotics in
them from controls and from patients with CD CD (Derwa et al. 2017). The meta-analysis
with no granulomas (Kellermayer et al. 2012). included 22 RCTs on IBD, and of these 8 tested
the efficacy of probiotics either in inducing remis-
sion, maintaining remission, or preventing
3 Probiotic Treatment relapses after surgery in patients with CD: none
of Pediatric IBD of these studies showed any significant benefit of
probiotics.
As properly stated in a recent position paper by an Thus, at present it seems clear that there is no
ESPGHAN task force (Miele et al. 2018), high- evidence of any beneficial effects of probiotics in
quality studies on the effect of probiotics in pedi- the treatment of CD either in adults or in children,
atric IBD are extremely limited: only two RCTs and this conclusion has been recently reiterated
in UC (Miele et al. 2009; Oliva et al. 2012) and by a consensus paper of the European Society for
one in CD (Bousvaros et al. 2005). Thus, while Paediatric Gastroenterology, Hepatology and
certainly one can extrapolate conclusions from Nutrition (ESPGHAN) that does not recommend
data obtained in adults, briefly summarized probiotics in the induction or maintenance of
below, caution must be exerted. remission of pediatric CD (Miele et al. 2018).
However, one needs to remain mindful of the
very limited number of RCTs performed, espe-
3.1 Crohn’s Disease cially in children, and of the various genotypes
and phenotypes of this condition, that are likely to
Most studies on testing the efficacy of probiotic require a more personalized therapeutic approach.
interventions in CD (either in children or in
adults) have assessed their role in maintaining
remission achieved with standard medical therapy 3.2 Ulcerative Colitis
or surgical resection.
The probiotic yeast S. boulardii in addition to While probiotics in CD have so far failed to show
mesalamine proved effective in an initial study in any clinical usefulness, the same is not true for
adults with CD in reducing the rates of relapses ulcerative colitis (UC), where some of the many
(Guslandi et al. 2000); subsequently, however, a RCTs have indeed shown promising results, both
104 S. Guandalini and N. Sansotta

in adults and in children, as reviewed recently in patients. The probiotic mixture was well tolerated
Derwa et al. (2017). without any clinical adverse effects.
Among the most studied probiotics in animal L. reuteri (ATCC 55730) administered in the
models of colitis (Rodriguez-Nogales et al. 2018), amount of 1010 CFU by enema was tried against
E. coli Nissle 1917, widely used in Europe, placebo in a small study assessing in children,
showed encouraging results in an open-label also treated with oral mesalazine, remission of
trial in children in 2008 (Henker et al. 2008); their mild to moderate ulcerative proctitis (Oliva
however, this initial unblinded study was not et al. 2012). Clinical, endoscopic, and histologic
followed by any RCT in children. In adults, five scores improved more in the children treated with
studies conducted between 1997 and 2014, probiotic.
reviewed by Scaldaferri et al. (Scaldaferri et al. In their 2017 meta-analysis on probiotics and
2016), showed essentially a “non-inferiority” as IBD including 22 RCTs all performed in adults,
compared to treatment with mesalazine in Derwa et al. (Derwa et al. 2017) concluded that
maintaining clinical remission. In the most probiotics – when considered overall – did not
recently published RFCT on 100 patients, no show benefits over placebo in inducing remission
additional benefit of Nissle 1917 when of patients with active UC. However, clumping
administered with mesalazine was shown com- different strains and different doses of probiotics
pared to mesalazine alone (Petersen et al. 2014). may end up by masking efficacy of some strains;
One of the most studied probiotic preparations and indeed, when they restricted the analysis to
for UC both in adults and in children is however RCTs using De Simone Formulation, a significant
De Simone Formulation, a proprietary mixture probiotic beneficial effect over placebo was
that combines eight strains of lactic acid- demonstrated with a RR of 0.74. In addition,
producing bacteria (L. plantarum, L. delbrueckii probiotics also proved to be just as efficacious
subsp. bulgaricus, L. casei, L. acidophilus, as aminosalicylic acid in preventing relapses of
Bifidobacterium breve, B. longum, B. infantis, UC, once in remission. Had the meta-analysis
and Streptococcus salivarius subsp. included also the pediatric study by Miele et al.
Thermophilus). In children, Miele et al. (2009), clearly the case for the usefulness of De
performed a 1-year prospective, placebo- Simone Formulation as a potent coadjuvant in the
controlled, RCT investigation to assess the effi- treatment of UC, already supported by a previous
cacy on this preparation in the treatment of chil- meta-analysis (Mardini and Grigorian 2014),
dren with active UC. More than 90% of the would have been even stronger.
children achieved remission when treated with
De Simone Formulation and standard therapy,
while only 36% of those treated with mesalazine 4 Conclusions
and placebo did (Miele et al. 2009). Of note, De
Simone Formulation was also an effective adju- Modifications of the microbiota in children with
vant in maintaining remission. In fact, children IBD are a conceptually logical and exciting
who underwent remission on De Simone Formu- means to control in a more “natural” way these
lation had lower endoscopic and histological aggressive conditions, especially in consideration
scores (p < 0.05) than those on placebo, and of the high burden of the medical treatment. Such
only 21% of them experienced a relapse within modifications could theoretically be achieved by
1 year, compared to 73% of the children on pla- several approaches: intervention on nutrition
cebo (p ¼ 0.014). (Miele et al. 2018; Lane et al. 2017), administra-
In the same year, De Simone Formulation was tion of probiotics, and – more recently – also fecal
also tested by Huynh et al. (Huynh et al. 2009) in microbial transplant (Jeon et al. 2018). While the
an open-label study in children with UC, mild to use of probiotics may appear to be the “lowest
moderately active. Remission was achieved in hanging fruit,” unfortunately the evidence so far
56%, with no change or worsening in 39% of accumulated is not hard enough, especially in
Probiotics in the Treatment of Inflammatory Bowel Disease 105

children, to be translated in clinical Bourreille A, Cadiot G, Le Dreau G, Laharie D,

recommendations. More specifically, in the case Beaugerie L, Dupas JL, Marteau P, Rampal P,
Moyse D, Saleh A, Le Guern ME, Galmiche JP,
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of any role for probiotics: however, it must be prevent relapse of Crohn’s disease. Clin Gastroenterol
stressed again that CD is a complex condition, Hepatol 11:982–987
extremely heterogeneous in its genetic asset, phe- Bousvaros A, Guandalini S, Baldassano RN, Botelho C,
Evans J, Ferry GD, Goldin B, Hartigan L,
notype presentations, and outcomes. It is there- Kugathasan S, Levy J, Murray KF, Oliva-Hemker M,
fore conceivable that the trials so far conducted Rosh JR, Tolia V, Zholudev A, Vanderhoof JA,
have not yet addressed with the proper probiotic Hibberd PL (2005) A randomized, double-blind trial
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https://doi.org/10.1007/5584_2018_320
# Springer Nature Switzerland AG 2019
Published online: 17 January 2019

Acute Infectious Diarrhea

Andrea Lo Vecchio, Vittoria Buccigrossi, Maria Cristina Fedele,

and Alfredo Guarino

Abstract research should investigate probiotic efficacy

Acute infectious diarrhea (AID) is one of the in at-risk populations and settings where the
most common diseases in pediatric age with evidence is missing.
relevant burden both in high- and in Their role in prevention of AID is however
low-income countries. questionable in healthy population, whereas it
Thanks to their direct action on enterocyte should be considered in at-risk population.
functions and indirect actions on mucosal and Evidence for prevention of diarrhea in
systemic immune system and intestinal micro- day-care centers and communities is lacking,
environment, probiotics are an ideal interven- but consistent evidence supports efficacy in
tion to manage AID in childhood. However, prevention of hospital acquired diarrhea.
their efficacy is strictly related to strains and Overall, AID is the most convincing area
indications, and practitioners should take this for probiotic use in children, and effective
information into account in clinical practice. strains should be used early after onset of
This chapter summarizes the main symptoms.
mechanisms of action of probiotics in AID,
with a focus on proof of efficacy supporting Keywords
their use in prevention and treatment of Diarrhea · Gastroenteritis · Gastrointestinal
infant AID. infections · Probiotics
The use of selected strains in appropriate
doses is strongly recommended by guidelines
of AID, based on large and consistent proofs 1 Introduction
of efficacy and safety. At present, therapy with
probiotics of AID is arguably the strongest Acute infectious diarrhea (AID) is one of the most
indication for probiotics in medicine. Future common diseases in pediatric age with relevant
burden both in high- and in low-income
countries.
A. Lo Vecchio, V. Buccigrossi, and A. Guarino (*)
In most cases, acute diarrhea, usually defined
Department of Translational Medical Sciences – Section of
Pediatrics, University of Naples Federico II, Naples, Italy by a decrease in the consistency of stools (loose
e-mail: [email protected] or liquid) and/or an increase in the frequency of
M. C. Fedele evacuations (typically >3 in 24 h), is
Department of Woman, Child and of General and accompanied by the presence of fever and/or
Specialized Surgery, University of Campania “Luigi vomiting, defining the clinical features of acute
Vanvitelli”, Naples, Italy

109
110 A. Lo Vecchio et al.

gastroenteritis (Guarino et al. 2015a). Every pre- inhibiting viral replication, enhancing gut epithe-
school child experiences at least one episode of lial barrier integrity, and modulating innate
AID per year. Those episodes usually last not immune response (Lemberg et al. 2007; Yan
more than 7 days (Guerrant et al. 2001) and in et al. 2007). In addition, there is evidence that
high-income settings represent a relatively mild selected probiotics may have a direct antidiarrheal
and self-limiting condition with rare effect counteracting ion secretion and promoting
complications. However, due to the high fre- their absorption through a direct effect on
quency of the diseases, AID is a major cause of enterocytes. However, not all probiotics
medical consultations both in general practice or demonstrated the same efficacy, and, as it
in emergency department and a leading cause of happens for drugs, the clinical efficacy is related
hospitalization with major impact on the quality with strains, specific mechanisms, dosages, and
of life of children and their families, as well as indications.
major impact on health-associated costs. Clinical In this chapter we review the main
features and outcomes significantly change in mechanisms of action of probiotics in AID and
low-income areas, where AID still is the second summarize the clinical evidence supporting their
leading cause of mortality in under-5-year chil- use in prevention and treatment of infant AID.
dren. Although patients’ underlying conditions,
presence of malnutrition, and limited access to
health-care facilities may affect the clinical out- 2 Models of Infectious Diarrhea
come of AID, the severity of infection ultimately and Mechanisms of Actions
depends on the etiological agent. In most cases of Probiotics
AID is caused by viral agents (Rotavirus,
Norovirus, adenovirus), but in some cases bacte- In physiological conditions, intestinal epithelium
rial pathogens (Salmonella, Shigella, entero- balances the secretory and absorptive processes
pathogenic E. coli, Campylobacter jejuni, or managing the movements of several ions (Na+,
Clostridium difficile) or parasites (i.e., Giardia Cl , HCO3 , and K+) and nutrients, mainly glu-
lamblia, Cryptosporidium parvum, Entamoeba cose. Water secretion is a passive process driven
histolytica) may be involved. by Cl secretion. The cyclic AMP-dependent
Despite large-scale immunization programs, chloride channel (namely, cystic fibrosis trans-
Rotavirus still represents the leading cause of membrane conductance regulator or CFTR) is
severe diarrhea and hospitalization (Lo Vecchio responsible for water secretion in basal conditions
et al. 2017) (Tate et al. 2016). and under the effects of many enterotoxins.
Oral, and in some circumstances parenteral, Several bacterial enterotoxins (e.g., cholera
rehydration is the key treatment of AID. How- toxin from Vibrio cholerae and heat-stable
ever, the replacement of fluids does not affect enterotoxin from enterotoxigenic Escherichia
duration of symptoms neither does it restore intes- coli) induce chloride secretion by stimulating
tinal barrier function (Blum et al. 2011; Desjeux intracellular cyclic nucleotides (Guandalini et al.
et al. 1997). In the last 20 years, many different 1982), inducing the activation of CFTR. This
interventions have been proposed to limit the effect is mediated by a specific interaction of
disease progression and to heal intestinal damage, TMEM16A and CFTR in a functional signaling
and probiotics gained a leading role thanks to a compartment at the plasma membrane level
large amount of consistent evidence of efficacy (Benedetto et al. 2017).
(Hill et al. 2014; Guarino et al. 2015b). On the contrary, Rotavirus (RV) infection
The efficacy of selected strains is related to causes chloride secretion as well as structural
their capacity of restoring damaged mucosal damages in the intestinal epithelium (Greenberg
intestinal barrier, competing with intestinal and Estes 2009) by elevation of cytoplasmic Ca2+
pathogens for nutrients and for adhesion concentration induced by NSP4 enterotoxin
receptors, producing antimicrobial substances, activity (Buccigrossi et al. 2014).
Acute Infectious Diarrhea 111

The effects of bacterial and viral toxins on indirectly on intestinal mucosa (Surendran Nair
intestinal mucosa have been studied using both et al. 2017). For example, Galvao demonstrated
in vitro and in vivo animal models (Lucas 2008; that the administration of a mix of three probiotics
Jafari et al. 2016) especially in RV infection reduced the colonization of Salmonella enterica
models (Kawahara et al. 2017; Gagnon et al. ser. Typhimurium in germ-free mice (Galvao
2016). et al. 2017) suggesting that probiotic efficacy is
Since secretory diarrhea results from an alter- related to their ability to produce mucus-binding
ation of electrogenic ion transport across the polar proteins that compete for host cell surface
intestinal epithelium, electrophysiology studies receptors (Surendran Nair et al. 2017). Other
provide an effective experimental approach direct properties of probiotics include the neutral-
(Herrmann and Turner 2016). ization of toxins and the competition for nutrients
The Ussing chamber technique allows (Surendran Nair et al. 2017). The production of
maintaining tissues alive several hours in con- bacteriocins and metabolites with specific antimi-
trolled conditions positioning the tissue between crobial effects is a further protection mechanism
two half chambers defined as apical compartment (Iraporda et al. 2017).
(i.e., intestinal lumen of the intestine) and Probiotic metabolites secreted in the intestinal
basolateral compartment (i.e., serosa). The elec- environment counteract damage induced by
trical parameters measured at different time points enteropathogens such as the enterotoxic effect
include short-circuit current (Isc) expressed in (Fig. 2) (responsible for secretory diarrhea) and
μA/cm2, transepithelial potential difference the cytotoxic effect (responsible for osmotic diar-
(PD) in mV, and tissue ionic conductance (G) in rhea as a consequence of reduction of functioning
mS/cm2. Ussing chamber system can also be used digestive surface) acting on oxidative stress
for permeability studies (Wuyts et al. 2015). (Buccigrossi et al. 2014; Anand et al. 2016) and
The Ussing chamber system can be used both anti-inflammatory pathways (Compare et al.
for epithelial monolayers from cell culture and for 2017). The release of soluble factors also triggers
intestinal mucosa from biopsies or surgical a signaling cascade leading to host immune mod-
specimens (humans or animal models). This ulation. This type of interaction, defined
approach is very useful to study the efficacy of “postbiotic” effects, has been the target of several
probiotics in counteracting diarrhea focusing on studies, in the attempt to find stable moieties to
biological and molecular mechanisms involved. eventually produce drugs rather than rely on
On the other hand, clinical trials are needed to active microorganisms that are difficult to quanti-
study the efficacy of functional nutrients, drugs, tate and to store (Tsilingiri and Rescigno 2013;
and probiotics on clinical outcomes (duration, Simeoli et al. 2017; Haileselassie et al. 2016;
intensity of diarrhea, or side effects of testing Levy et al. 2015).
moieties). Probiotics also provide a second line of
Probiotics exert different direct and indirect defense through indirect mechanisms. The
actions on the intestinal mucosal layer and micro- upregulation of mucins and the expression of
environment that can be expected to possess tight junctions proteins lead to reinforce or restore
antidiarrheal effects (Fig. 1). They target the a damaged mucosa, thereby enhancing the intes-
small intestine inducing a substantial host muco- tinal barrier function. Pathogens find a physical
sal transcription response affecting several genes barrier that prevents their direct interaction with
(Thomas et al. 2015). intestinal epithelium, and the concept of epithelial
Several diseases may be effectively treated barrier has been updated with epithelial
with probiotics as shown by randomized con- microbiological barrier. Such a change explains
trolled trials (Rondanelli et al. 2017) in pediatric well the harmful use of antibiotics in disrupting a
setting (Szajewska et al. 2016, 2014). defense against invasion of intestinal pathogens
Beneficial effects exerted by probiotics depend as well as the increased risk of allergy related to
on several mechanisms acting directly or multiple antibiotic courses (Mitre et al. 2018). In
112 A. Lo Vecchio et al.

Fig. 1 Schematic effects exerted by probiotics on intesti- enterotoxins are direct and early mechanisms. Gut
nal mucosa microbiota modulation, promotion of barrier functions,
Probiotics exert protective effects against intestinal and immune system modulation are indirect, late, but
pathogens by direct and indirect mechanisms. Competition lasting effects
for nutrients, antiadhesive effects, and inhibition of

addition, probiotics also enhance beta-defensin enteropathogenic bacteria by modulation of

production in intestinal epithelial cells preventing antiviral response, namely, the reduction of A20
the proliferation of pathogens (Surendran Nair and improvements of IRF-3, IFN-beta, MxA, and
et al. 2017). RNaseL, and by acetate produced by themselves.
A recent review focuses on immune modula- All these mechanisms are effective both in the
tion by probiotics. In particular, lactobacilli are prevention and during pathogen’s infection
able to induce IgA production enhancing IL-6 and (Anand et al. 2016). Rotavirus infection is the
TGF-beta production by dendritic cells, natural most common gastroenteritis in children, and sev-
killer cell activity by IL-12 production; inhibit eral studies were conducted on probiotic effects in
Th2 response enhancing IL-12 production and animal models.
apoptosis of activated T cells; and finally induce Bifidobacterium bifidum G9–1 therapeutic
an anti-inflammation effect attenuating administration significantly restored epithelial
pro-inflammatory cytokines activity barrier function, normalizing intestinal functions
downregulating TLR-signaling and INF-beta in an RV-infected suckling mice model
(Hachimura et al. 2018). (Kawahara et al. 2017). Jiang et al. demonstrated
Bifidobacterium genus also induces IgA pro- that the protective effects of the well-studied pro-
duction by upregulation of pIgR expression. In biotic Lactobacillus rhamnosus GG (LGG) were
addition, bifidobacteria induce protection from due to the modulation of the differentiation and
Acute Infectious Diarrhea 113

Fig. 2 Direct mechanisms

of S. boulardii in
RV-induced intestinal
epithelial damage
S. boulardii, one of the
probiotic strain
recommended by
ESPGHAN in RV-induced
diarrhea, inhibits the
alteration of chloride
secretion induced by RV in
the early stage of infection
(a) and later protects from
the cytotoxic damage
measured by transepithelial
resistance (TEER) (b). This
inhibition is exerted by
soluble moieties released in
the milieu by S. boulardii
(Buccigrossi et al. 2014)

maturation of dendritic cells, the increased pro- induces changes in the gut microbiota, promoting
duction of TLR-3 and NF-κB, and the modulation the development of butyrate producers with cor-
of inflammatory cytokines (Jiang et al. 2017). In relative associations to immune biomarkers
addition, LGG is protected from RV infection by (Berni Canani et al. 2017).
upregulating TLR-3 gene expression (Aoki-
Yoshida et al. 2016).
A new multiple host and probiotic metabolic
3 Prevention of Infectious
network, involving lipid and amino acid/peptide
Diarrhea
metabolism, was described. These findings impli-
cate intestinal and serum metabolomes (Nealon
In the last two decades, there have been an
et al. 2017) and miR-148a modulation (Taibi et al.
increasing number of trials investigating the role
2017) in the effects elicited by microorganisms
of probiotics in prevention of common infections
which may be exploited for clinical purposes.
in children. Although the first trials published
Last but not least, probiotics are able to remodel
more than 15 years ago showed promising results
gut microbiota inducing positive regulation of the
(Saavedra and Tschernia 2002; Saran et al. 2002),
mutual interaction between the immune system
more recent data show conflicting results in the
and gut microbiota. Cow’s milk fermented with
efficacy of probiotic-enriched formulas for pre-
the probiotic Lactobacillus paracasei CBA L74
vention of AID episodes in healthy children.
114 A. Lo Vecchio et al.

Intervention tested to prevent AID, as well as incidence of health-care-associated diarrhea,

respiratory infections, included various probiotic including Rotavirus-related diarrhea (RR 0.37,
strains, dosages, and formulations (enriched for- 95% CI 0.23–0.59)(Szajewska et al. 2011).
mula or probiotic as such) and showed a very More recently, a controlled trial comparing a
modest statistically significant effect but of ques- combination of LGG and micronutrients
tionable clinical importance (Agustina et al. 2012; (vitamin B, vitamin C, zinc) with placebo
Guandalini 2011; Ranucci et al. 2018; Szajewska demonstrated a significant reduction in the inci-
et al. 2001, 2006). dence, duration, and severity of gastrointestinal
The European Society of Pediatric Gastroen- infections and in the length of hospitalization. Inter-
terology, Hepatology and Nutrition (ESPGHAN) estingly, the protective effect was extended well
defined as “insufficiently convincing” the evi- beyond hospital discharge (Bruzzese et al. 2016).
dence supporting the use of probiotic-enriched Conversely, several studies did not show efficacy of
formulas for prevention of infections, including other probiotic strains in preventing AID. Two trials
AID (Braegger et al. 2011). A recent study in evaluating, respectively, the probiotic
children allocated to LGG-enriched hydrolyzed Bifidobacterium lactis and L. reuteri DSM 17938
formulas did not demonstrate any difference in as preventative of nosocomial-acquired diarrhea
the incidence of AID at 3 and 5 years follow-up failed to show their efficacy (Hojsak et al. 2015;
(Scalabrin et al. 2017). It should be noted that the Urbanska et al. 2016). Similarly, Bifidobacterium
bacterial load in formula is considerable lower lactis failed to prevent common infections in
than that available in lyophilized preparations hospitalized children (Wanke and Szajewska 2012).
used for treatment. Based on this moderate evidence, the
However, based on present results, the admin- ESPGHAN concludes that if probiotics for
istration of probiotics for prevention of diarrhea preventing nosocomial diarrhea in children are
on a large scale appears neither reliable nor real- considered, LGG (at least 109 CFU/day, for the
istic. As declared in 2010 by the American Acad- duration of hospital stay) should be used (Hojsak
emy of Pediatrics, probiotics may find a et al. 2018).
preventive role in “special circumstances, (such
as) in children in long-term health-care facilities
or in child care centers” (Thomas et al. 2010). 3.2 Children in Day-Care Centers

Children attending day-care centers have 2–3

3.1 Children in Hospital Settings times more infections than children at home and
receive more frequent medical visits and antibi-
Nosocomial infections strike about 15–20% of otic prescriptions. There is evidence that
children during hospital stay and may prolong probiotics (mainly LGG) in these children may
the length of hospitalization, worsen the treatment significantly reduce the incidence of intestinal
outcome, and increase hospital costs (Hojsak and upper respiratory infections.
et al. 2010). However, different strains were used, and
The use of probiotics has been proposed in conflicting results were obtained even with the
association to standard preventive measures to same strain. Two studies using LGG and other
reduce the incidence and severity of nosocomial two using Bifidobacterium lactis did not show
diarrhea (Posfay-Barbe et al. 2008). any efficacy. More recently Gutierrez-Castrellon
LGG has been tested in large populations of and colleagues demonstrated that the daily
hospitalized children with reduction of the inci- administration of Lactobacillus reuteri DSM
dence of diarrheal episodes, vomiting, and other 17938 to healthy children attending day-care
gastrointestinal symptoms (Hojsak et al. 2010). In may reduce the episodes of AID with cost-saving
2011, a meta-analysis including two RCTs con- benefit to the families and health-care system
firmed the potential of LGG to reduce the overall (Gutierrez-Castrellon et al. 2014).
Acute Infectious Diarrhea 115

4 Treatment of Infectious other probiotic preparations in reducing the dura-

Diarrhea tion and intensity of symptoms by about 25–30%
(Szajewska et al. 2014).
Treatment of AID is currently the main indication The effect of probiotics is maximal with early
for probiotic use in childhood. administration during the disease course and an
Since its publication in the 1990s (Guandalini appropriate dosage being doses of LGG  1010
et al. 2000; Guarino et al. 1997), the initial result CFUs/day more effective than lower doses in
of a probiotic-induced reduction in the duration of reducing the duration of diarrhea (Szajewska
diarrhea and rotaviral shedding in children living et al. 2013). In addition, the efficacy seems to be
in high-income countries, a 20-year-long way greatest in viral (mainly rotaviral) and watery
built on consistent evidence of efficacy, led to diarrhea, and it should be considered that the
conclusive recommendations for the use of spreading of Rotavirus immunization might
selected probiotic strains in children living change in part the current scenario.
worldwide. Based on consistent evidence available in vir-
A recent study comparing the guidelines for tually all parts of the world and in all settings,
the management of AID produced in different probiotics have been included as active treatment
countries reported that probiotics are currently of AID, in adjunct to rehydration, in the universal
recommended by the World Gastroenterology recommendations for management of gastroenter-
Organization and by scientific societies in itis in children (Guarino et al. 2018).
Europe, Latin America, China, Australia, and
Malaysia and are considered in specific situation
in South Africa and the USA according to paren- 4.1 Evidence in Low-Income Settings
tal perspective (Lo Vecchio et al. 2016).
Compelling evidence show a statistically sig- Although the use of probiotics is at least formally
nificant effect in reducing the duration of diarrhea defined as recommended in children with AID
of about 24 h (17–30 h) and the risk of living in high-income countries, the evidence in
complications independently of settings and low-income countries is not as consistent
conditions. although supporting evidence is growing. In
According to the Consensus Group on Out- settings where the incidence on bacterial and par-
come Measures Made in Paediatric Enteral Nutri- asitic intestinal infections is higher than in Europe
tion Clinical Trials (COMMENT), the duration of or the USA, the pediatric population is more
diarrhea and the need and length of hospitaliza- frequently affected by nutritional deficiency, and
tion are the most relevant outcomes to be consid- the access to medications may be limited. There-
ered in trials focusing on the management of AID fore, the use of probiotics needs to be considered
(Karas et al. 2015). However only selected probi- according to local proofs of efficacy and also to
otic strains demonstrated a relevant impact on all cost-benefit analysis.
these outcomes. In the last few years, at least four clinical trials
Lactobacillus rhamnosus GG (LGG) and Sac- tested the efficacy of different probiotics in India,
charomyces boulardii are the most studied strains Indonesia, and Vietnam, reporting conflicting
and are strongly recommended by several results.
guidelines as primary choice for the active treat- In 2015, Hegar and colleagues tested the effi-
ment of children with AID. These two strains cacy of a probiotic food supplement containing
demonstrated a rapid (within 48 h) efficacy in L. rhamnosus R0011 and L. acidophilus R0052 in
reducing the duration of diarrhea, the risk of Indonesian children with AID and moderate
prolonged diarrhea, and the length of hospital dehydration. The duration of diarrhea and daily
stay. Other approximately 20 RCTs of poor frequency of evacuation were similar in the inter-
quality provided results showing the efficacy of vention and control groups (Hegar et al. 2015).
116 A. Lo Vecchio et al.

Similarly, an RCT in a quite large population of A new open-label RCT is currently enrolling
Vietnamese hospitalized children with acute children in a similar setting involving three areas
watery diarrhea was not able to demonstrate the of Uganda, with the aim of reducing diarrhea,
efficacy of Lactobacillus acidophilus in reducing incidence of other infectious diseases, and impact
any of the outcomes (time to stop diarrhea, dura- on overall infant mortality. The product tested
tion of diarrhea and hospitalization, daily stool will be a blend of Bifidobacterium breve BR03
frequency, treatment failure, daily fecal and B632 and Lactobacillus delbrueckii (Del
concentrations of rotavirus and norovirus) Piano et al. 2016). However, based on available
(Hong Chau et al. 2018). data, the current recommendations for use of
In our opinion, to define the usefulness of probiotics in children from Asian-Pacific area
probiotics in low-income settings, strains that are for the use of LGG or Saccharomyces
demonstrated strong efficacy in developed areas boulardii as active therapy in adjunct to rehydra-
should be evaluated as a priority. tion of AID (Cameron et al. 2017).
In support to this approach, a large RCT carried
out in 2000 Indian children with acute watery
diarrhea accessing outpatient department and 5 Conclusions and Future
emergency room reported a shorter duration of Perspectives
diarrhea, lower number of daily stools, and a faster
improvement in stool consistency in children Thanks to their direct action on enterocyte
receiving LGG than in control group. Interestingly, functions and indirect actions on mucosal and
the clinical benefits were seen irrespective of rota- systemic immune system and intestinal microen-
virus positivity (Aggarwal et al. 2014). vironment, probiotics are an ideal intervention to
In 2016, Das and colleagues demonstrated a manage AID in childhood. However, their effi-
significant reduction in duration of diarrhea cacy is strictly related to strains and indications,
(between 17 and 40 h) and hospitalization and practitioners should strictly adhere to
(between 0.5 and 33 h) in Indian children with recommendations in clinical practice (Table 1).
rotaviral diarrhea receiving Saccharomyces The use of selected strains (such as LGG and
boulardii, although the intervention was not able S. boulardii) in appropriate dose is strongly
to reduce the occurrence of fever, vomiting, and recommended by current guidelines for the treat-
the rate of severe dehydration or prolonged diar- ment of AID, based on large and consistent proofs
rhea (Das et al. 2016). of efficacy and safety.
The underlying conditions and, particularly, the Recently, two large RCTs have appeared
degree of malnutrition of the enrolled population addressing the potential efficacy of probiotics in
may impact of the efficacy of such interventions. children with acute gastroenteritis (Freedman
In fact, most studies usually exclude children with et al. 2018; Schnadower et al. 2018). The
moderate to severe malnutrition that represent in investigations had equal design and primary end
some cases a relevant portion of the population in point (a truly unusual “moderate to severe gastro-
low-income countries. There are few trials that enteritis within 14 days”).
specifically assess the use of probiotics in malnour- The first study (Freedman et al. 2018),
ished children. Recently, an RCT assessing the conducted in Canada, utilized a unique mix of
efficacy of a mix of Bifidobacterium lactis and strains (of note: including neither LGG nor
L. rhamnosus in children with diarrhea and severe S. Boulardii) and failed to show efficacy of this
acute malnutrition demonstrated a reduction of the intervention. The second RCT (Schnadower et al.
duration of diarrhea by 2 days in outpatients’ chil- 2018) was made in the USA and utilized LGG.
dren, but was not able to demonstrate any effect on This study too, unexpectedly, yielded negative
weight gain, incidence of other infections, and results: in fact, no significant differences were
duration of diarrhea and recovery in hospitalized found between the LGG and the placebo group
children (Grenov et al. 2017). in the primary end point as well as in the
Acute Infectious Diarrhea 117

Table 1 Level of evidence supporting the use of specific probiotic strains for each indication
Strains
showing Highest
higher level of Guidelines’
Potential indication Most commonly tested strains efficacy evidence Recommendation
Prevention of AID in Probiotic-enriched formulas – RCT No
healthy children containing LGG, B. lactis, alone or in
combination with Str. thermophilus,
and L. reuteri, L. rhamnosus – not GG
– and L. acidophilus
Prevention of AID in LGG, B. lactis alone or in combination LGG, RCT No
children attending day-care and L. reuteri L. reuteri
centers
Prevention of AID in LGG, B. lactis, L. reuteri LGG MA Weak
hospitalized children
Treatment of AID in LGG, S. boulardii, L. reuteri LGG, MA Strong
otherwise-healthy children S. boulardii,
L. reuteri
Treatment of AID in LGG, S. boulardii, L. acidophilus LGG, RCT No
malnourished children and alone or in combination with S. boulardii
those living in low-income L. rhamnosus (not LGG), B. lactis
countries alone or in combination with
L. rhamnosus (not LGG)

secondary ones (duration of diarrhea, vomiting). the reductions of gastrointestinal infections in

However, one needs to consider that LGG is at-risk settings should be considered. Evidence
particularly effective against diarrhea by Rotavi- for prevention of diarrhea in day-care centers
rus, the most common single agent of diarrhea and communities are preliminary, but those in
worldwide, and notably the majority of US chil- nosocomial diarrhea are rapidly growing leading
dren are immunized against it. Furthermore it’s to more common use of active preventive
not clear when in the course of the diarrheal strategies.
illness was the probiotic started, and it is known
that a delayed onset of probiotic treatment may
decrease efficacy.
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https://doi.org/10.1007/5584_2018_321
# Springer Nature Switzerland AG 2018
Published online: 22 December 2018

Probiotics in Functional Gastrointestinal

Disorders

Iva Hojsak

Abstract Keywords
Infantile colic, constipation, functional Functional abdominal pain · Infantile colic ·
abdominal pain (FAP), and irritable bowel Irritable bowel syndrome · Lactobacillus ·
syndrome (IBS) are the most common Probiotics
functional gastrointestinal disorders (FGID).
This chapter will review current evidence on
the role of probiotics in the treatment of these 1 Introduction
FGID. The etiology of FGID is considered
multifactorial, but the importance of intestinal The definition of functional gastrointestinal
microbiota in their development has been disorders (FGID) varied over the last decades,
repeatedly emphasized. As a consequence, mainly due to evolving consensus between experts
the potential role of probiotics in their treat- but also due to constantly emerging new scientific
ment is being increasingly scrutinized. Pres- evidence. Furthermore, FGID encompass various
ently, the strongest evidence of efficacy is for disorders in all age groups and are responsible for a
the use of Lactobacillus reuteri (L reuteri) large proportion of pediatric gastroenterologists’
DSM 17938 at the dose of 108 CFU/day for consultations. FGID definition became more strin-
the treatment of infantile colic in breastfed gent after the introduction of Rome criteria around
infants. Limited, yet encouraging, evidence 20 years ago, criteria aiming at providing diagnos-
exists for Lactobacillus rhamnosus GG tic guidance for every specific FGID. The last
(LGG) at the dose of 3  109 CFU and for a revision of these criteria, Rome IV criteria, was
multi-strain preparation for the treatment of published in 2016 in two separate documents,
IBS. In the treatment of FAP, there is some FGID in neonates, infants, and toddlers (Benninga
evidence for the use of L reuteri DSM et al. 2016) and FGID in children and adolescents
17938 at the dose of at least 108 CFU/day. (Hyams et al. 2016), and they are summarized in
Tables 1 and 2.
This chapter will focus on the most common
I. Hojsak (*) FGID: infantile colic, constipation, functional
Referral Center for Pediatric Gastroenterology and abdominal pain not otherwise specified, and irri-
Nutrition, Children’s Hospital Zagreb, Zagreb, Croatia table bowel syndrome.
School of Medicine, University of Zagreb, Zagreb, Croatia
School of Medicine Osijek, University J.J. Strossmayer,
Osijek, Croatia
e-mail: [email protected]

121
122 I. Hojsak

Table 1 Rome IV criteria for functional disorders in neonates, infants, and toddlers (Benninga et al. 2016)
Disorder Criteria
Infant regurgitation Must include both of the following in otherwise healthy infants 3 weeks to 12 months of age:
1. Regurgitation 2 or more times per day for 3 or more weeks
2. No retching, hematemesis, aspiration, apnea, failure to thrive, feeding or swallowing
difficulties, or abnormal posturing
Infant rumination Must include all of the following for at least 2 months:
syndrome 1. Repetitive contractions of the abdominal muscles, diaphragm, and tongue
2. Effortless regurgitation of gastric contents, which are either expelled from the mouth or
rechewed and re-swallowed
3. Three or more of the following:
(a) Onset between 3 and 8 months
(b) Does not respond to management for gastroesophageal reflux disease and
regurgitation
(c) Unaccompanied by signs of distress
(d) Does not occur during sleep and when the infant is interacting with individuals in the
environment
Cyclic vomiting Must include all of the following:
syndrome 1. Two or more periods of unremitting paroxysmal vomiting with or without retching,
lasting hours to days within a 6-month period
2. Episodes are stereotypical in each patient
3. Episodes are separated by weeks to months with return to baseline health between
episodes of vomiting
Infant colic For clinical purposes, must include all of the following:
1. An infant who is <5 months of age when the symptoms start and stop
2. Recurrent and prolonged periods of infant crying, fussing, or irritability reported by
caregivers that occur without obvious cause and cannot be prevented or resolved by caregivers
3. No evidence of infant failure to thrive, fever, or illness
Functional diarrhea Must include all of the following:
1. Daily painless, recurrent passage of 4 or more large, unformed stools
2. Symptoms last more than 4 weeks
3. Onset between 6 and 60 months of age
4. No failure to thrive if caloric intake is adequate
Infant dyschezia Must include in an infant <9 months of age:
1. At least 10 min of straining and crying before successful or unsuccessful passage of soft
stools
2. No other health problems
Functional Must include 1 month of at least 2 of the following in infants up to 4 years of age:
constipation 1. 2 or fewer defecations per week
2. History of excessive stool retention
3. History of painful or hard bowel movements
4. History of large-diameter stools
5. Presence of a large fecal mass in the rectum
In toilet-trained children, the following additional criteria may be used:
6. At least 1 episode/week of incontinence after the acquisition of toileting skills
7. History of large-diameter stools that may obstruct the toilet
Probiotics in Functional Gastrointestinal Disorders 123

Table 2 Rome IV criteria for functional disorders in children and adolescents (Hyams et al. 2016)
Disorder Criteria
Functional nausea and vomiting disorders
Cyclic vomiting syndrome Must include all of the following:
1. The occurrence of 2 or more periods of intense, unremitting nausea and
paroxysmal vomiting, lasting hours to days within a 6-month period
2. Episodes are stereotypical in each patient
3. Episodes are separated by weeks to months with return to baseline health
between episodes
4. After appropriate medical evaluation, the symptoms cannot be attributed to
another condition
Functional nausea Must include all of the following fulfilled for the last 2 months:
1. Bothersome nausea as the predominant symptom, occurring at least twice
per week, and generally not related to meals
2. Not consistently associated with vomiting
3. After appropriate evaluation, the nausea cannot be fully explained by
another medical condition
Functional vomiting Must include all of the following:
1. On average, one or more episodes of vomiting per week
2. Absence of self-induced vomiting or criteria for an eating disorder or
rumination
3. After appropriate evaluation, the vomiting cannot be fully explained by
another medical condition
Rumination syndrome Must include all of the following:
1. Repeated regurgitation and rechewing or expulsion of food that:
(a) Begins soon after ingestion of a meal.
(b) Does not occur during sleep.
2. Not preceded by retching
3. After appropriate evaluation, the symptoms cannot be fully explained by
another medical condition. An eating disorder must be ruled out
Aerophagia Must include all of the following:
1. Excessive air swallowing
2. Abdominal distention due to intraluminal air which increases during the
day
3. Repetitive belching and/or increased flatus
4. After appropriate evaluation, the symptoms cannot be fully explained by
another medical condition
Functional abdominal pain disorders
Functional dyspepsia Must include 1 or more of the following bothersome symptoms at least 4 days
per month:
1. Postprandial fullness
2. Early satiation
3. Epigastric pain or burning not associated with defecation
4. After appropriate evaluation, the symptoms cannot be fully explained by
another medical condition
Irritable bowel syndrome Must include all of the following:
1. Abdominal pain at least 4 days per month associated with one or more of
the following:
(a) Related to defecation
(b) A change in frequency of stool
(c) A change in form (appearance) of stool
2. In children with constipation, the pain does not resolve with resolution of
the constipation (children in whom the pain resolves have functional
constipation, not irritable bowel syndrome)
(continued)
124 I. Hojsak

Table 2 (continued)
Disorder Criteria
3. After appropriate evaluation, the symptoms cannot be fully explained by
another medical condition
Abdominal migraine Must include all of the following occurring at least twice:
1. Paroxysmal episodes of intense, acute periumbilical, midline or diffuse
abdominal pain lasting 1 h or more (should be the most severe and distressing
symptom)
2. Episodes are separated by weeks to months
3. The pain is incapacitating and interferes with normal activities
4. Stereotypical pattern and symptoms in the individual patient
5. The pain is associated with 2 or more of the following:
(a) Anorexia
(b) Nausea
(c) Vomiting
(d) Headache
(e) Photophobia
(f) Pallor
6. After appropriate evaluation, the symptoms cannot be fully explained by
another medical condition
Functional abdominal pain – not Must be fulfilled at least 4 times per month and include all of the following:
otherwise specified 1. Episodic or continuous abdominal pain that does not occur solely during
physiologic events (e.g., eating, menses)
2. Insufficient criteria for irritable bowel syndrome, functional dyspepsia, or
abdominal migraine
3. After appropriate evaluation, the abdominal pain cannot be fully explained
by another medical condition
Functional defecation disorders
Functional constipation Must include 2 or more of the following occurring at least once per week for a
minimum of 1 month with insufficient criteria for a diagnosis of irritable bowel
syndrome:
1. Two or fewer defecations in the toilet per week in a child of a
developmental age of at least 4 years
2. At least 1 episode of fecal incontinence per week
3. History of retentive posturing or excessive volitional stool retention
4. History of painful or hard bowel movements Presence of a large fecal mass
in the rectum
5. History of large-diameter stools that can obstruct the toilet
Non-retentive fecal incontinence At least a 1-month history of the following symptoms in a child with a
developmental age older than 4 years:
1. Defecation into places inappropriate to the sociocultural context
2. No evidence of fecal retention
3. After appropriate medical evaluation, the fecal incontinence cannot be
explained by another medical condition

otherwise healthy infant (Biagioli et al. 2016). The

2 Infantile Colic
symptoms tend to resolve by 3–4 months of age or,
in the case of premature babies, 3–4 months after
Infantile colic is a common problem in healthy
term; crying peak usually occurs at 4–6 weeks and
growing infants and is characterized by
then steadily diminishes by 12 weeks (Benninga
inconsolable crying, irritability, and fussing in an
et al. 2016).
Probiotics in Functional Gastrointestinal Disorders 125

Although it affects a high number of young fecal bacterial diversity and a higher fecal counts
infants worldwide, it remains poorly understood of gram-negative bacteria, especially coliform
and is an often frustrating problem for parents and bacteria (Partty and Kalliomaki 2017; Dubois
caregivers, mainly due to the lack of effective and Gregory 2016). More specifically, infants
treatment options (Anabrees et al. 2013). The with colic were shown to have more
reported incidence varies from 4% to 28% Proteobacteria and less Bifidobacteria and Lac-
(Lucassen 2015): the wide range can be at least tobacillus than healthy controls (Dubois and
partially explained by the influence of caregivers’ Gregory 2016; de Weerth et al. 2013). Further-
perception of the intensity and duration of crying more, it appears that microbes from the
bouts, the method by which data are collected, as Bacteroidetes phylum are inversely associated
well as culturally influenced infant care practices with the expression of colic symptoms (Dubois
(Benninga et al. 2016). and Gregory 2016; de Weerth et al. 2013; Roos
The inconsolable crying often causes psycho- et al. 2013). Additionally, bacteria from the phyla
social distress and depression for caregivers and Actinobacteria and Firmicutes appear to have a
could delay the maternal-infant bonding process protective effect against the development of
(Jadresin et al. 2017; Maragkoudaki et al. 2017). infantile colic (Partty et al. 2012). The finding
Therefore, parents frequently visit health care that Actinobacteria and Bifidobacterium quickly
facilities and often turn to alternative, unproven, dominate the intestine of newborn who did not
and potentially unsafe methods to soothe crying have the colic phenotype (Partty et al. 2012) as
infants (Sung et al. 2012; Landgren and Hallstrom well as the presence of Bifidobacterium longum in
2011). human breast milk (Ruiz-Moyano et al. 2013)
As previously mentioned, the etiology of the suggests that these bacteria may play a central
syndrome remains elusive, with a number of pro- role in reducing inflammation and infant fussiness
posed etiologies having been described. Some of and crying. The importance of interplay between
the theories like immaturity of the nervous/diges- microbiota and inflammation was further
tive system, altered gut flora, and mild gastroin- strengthen by a study showing a negative associ-
testinal inflammation focus on the changes in the ation between pro-inflammatory biomarkers and
digestive system (Schreck Bird et al. 2017; Clostridium leptum and Clostridium coccoides,
Savino et al. 2014). Still other theories, primarily the major constituents of the normal colonic
psychosocial, have also been proposed, including microflora (Sghir et al. 2000). Additionally,
maternal anxiety, inadequate maternal-infant increased levels of cytokines MCP-1, MIP-1b,
bonding, or challenging infant temperament and IL-8 were found in infants with colic
(Schreck Bird et al. 2017). suggesting the role of low-grade systemic inflam-
Recently, more attention has been given to the mation in the pathogenesis of infantile colic
role of gut microbiota. This is mainly related to (Partty et al. 2017).
the fact that occurrence of infant colic coincides Although a progress in our knowledge has
with the most remarkable changes in the micro- been done in the last several years, no definitive
bial intestinal colonization during the first weeks conclusions can be drawn, and there is a clear
and months of life (Partty and Kalliomaki 2017). need for more species-specific research, since
The role of microbiota was further emphasized by not all microbes interact with a host in the same
the fact that microbes involved in intestinal colo- way (Dubois and Gregory 2016). Moreover, as is
nization communicate with host cells and signifi- the case in other dysbiosis, it is still not clear
cantly impact and shape the immune system and whether the observed changes are the cause or
future inflammatory response (Dubois and the consequence of the clinical condition.
Gregory 2016). Thus, this relatively new evidence, together with
There is a growing evidence that infants with lack of effective treatment, has prompted a research
colic have an intestinal microbiome that differs for new therapeutic options aiming at influencing
from that of healthy infants; they have decreased intestinal microbiome with the use of probiotics.
126 I. Hojsak

2.1 Treatment of Infantile Colic breastfed infants, the study which found no dif-
with Probiotics ference (Sung et al. 2014) included both breast-
and formula-fed infants. These studies were com-
Different treatment strategies have been proposed pared in an individual participant data meta-
for the treatment of infantile colic, although most analysis which showed that the probiotic group
of them were not analyzed in well-designed had averaged less crying and/or fussing time than
randomized control trials. A recently published the placebo group at all time points (Sung et al.
network meta-analysis (Gutierrez-Castrellon 2018). The probiotic group was almost twice as
et al. 2017) summarized the available evidence likely as the placebo group to experience treat-
in the treatment of infantile colic, concluding that ment success at all time points (Sung et al. 2018).
Lactobacillus (L.) reuteri DSM 17938 at a dose of As previously mentioned, intervention effects
108CFU/day significantly reduced the duration of were more dramatic in breastfed infants and
crying episodes. The superiority was were not significant in formula-fed infants (Sung
demonstrated not only when L. reuteri DSM et al. 2018).
17938 was compared to placebo but also when No data are available for other probiotic
indirectly compared to other types of intervention strains.
including dietary or manipulative interventions, Proposed algorithm for the treatment of infan-
reassurance, massage, herbal treatment, acupunc- tile colic based on current evidence (Vandenplas
ture, and drugs (Gutierrez-Castrellon et al. 2017). et al. 2013; Vulin and Hojsak 2015) is presented
Overall, probiotics were used in several in Fig. 1.
randomized controlled trials (RCT) (Szajewska There are several proposed mechanisms
et al. 2013; Savino et al. 2010; Chau et al. 2015; whereby probiotics could ameliorate infantile
Mi et al. 2015; Sung et al. 2014; Partty et al. colic. Probiotics are capable to antagonize enteric
2015). Five RCTs used same dose of L. reuteri pathogens, to modulate the gut microbiota
DSM 17938, and four studies showed positive (Savino et al. 2018; Roos et al. 2013), and to elicit
effect (Table 3). The difference between the innate or adaptive immune responses (Walsham
results of those studies can be partially explained et al. 2016). It has been shown that the adminis-
by the difference in the infants’ feeding; while tration of L. reuteri induces a significant increase
studies with positive effect mainly included in FOXP3 mRNA levels which is a Treg marker

Table 3 Randomized controlled trials using probiotics in the treatment of infantile colic
Number of
included Crying
Author Predominant diet children Intervention duration
Savino 2010 (Savino Breastfeeding 50 L. reuteri DSM 17938, 108 CFU Decreased
et al. 2010)
Szajewska 2013 Breastfeeding 80 L. reuteri DSM 17938, 108 CFU Decreased
(Szajewska et al.
2013)
Chau 2014 (Chau Breastfeeding 52 L. reuteri DSM 17938, 108 CFU Decreased
et al. 2015)
Mi 2015 (Mi et al. Breastfeeding 42 L. reuteri DSM 17938, 108 CFU Decreased
2015)
Sung 2014 (Sung et al. Breastfeeding and 167 L. reuteri DSM 17938, 108 CFU Not
2014) formula feeding significant
Partty 2015 (Partty Breastfeeding and 30 LGG 4.5  109 CFU Not
et al. 2015) formula feeding Supplemented together with significant
behavioral and nutritional
intervention
CFU colony-forming units, LGG Lactobacillus rhamnosus GG
Probiotics in Functional Gastrointestinal Disorders 127

Fig. 1 Proposed algorithm for the management of infantile colic (based on (Vandenplas et al. 2013; Vulin and Hojsak 2015))
*L. reuteri is currently the only probiotic strain with proven efficacy
128 I. Hojsak

(Savino et al. 2018). The effects on Treg neurological responses to stress and bowel sensi-
lymphocytes could result from increased IL-10 tivity (Korterink et al. 2015; Smith et al. 2007).
secretion which is reduced not only in colitis Other forms of stress, like psychological stress, is
models but also in children with colic (Partty also recognized as a risk factor together with
et al. 2017). The effect could also be the result genetic predisposition and recent acute intestinal
of reduced response of capsaicin- and distension- infections (Korterink et al. 2015).
evoked firing of spinal nerve action potentials, Another important factor in the pathogenesis is
meaning reduction of the sensation of pain the alteration in the gut microbiota. In fact, chil-
shown by L. reuteri DSM 17938 (Perez-Burgos dren with IBS have a greater proportion of the
et al. 2015). Finally, the administration of phylum Proteobacteria, and genera such as
L. reuteri was associated with a significant Dorea, Haemophilus, Ruminococcus, and Clos-
decrease in the fecal calprotectin level, which is tridium spp., and increased ratio of the Firmicutes
a clinical marker of intestinal inflammation in to Bacteroidetes (Rajilic-Stojanovic et al. 2011;
both adults and children (de Weerth et al. 2013). Saulnier et al. 2011). A recently published meta-
analysis identified downregulation of bacterial
colonization with Lactobacillus, Bifidobacterium,
3 Functional Abdominal Pain and Faecalibacterium prausnitzii in patients with
and Irritable Bowel Syndrome IBS, particularly in diarrhea-predominant IBS
(Liu et al. 2017). Pediatric patients with
FAP in children has a similar prevalence all over diarrhea-predominant IBS seem to have increased
the world, ranging between 10% and 12% levels of Veillonella, Prevotella, Lactobacillus,
(Korterink et al. 2015), while the prevalence of and Parasporbacterium and reduction in levels
IBS ranges between 6% and 14% in children and of Bifidobacterium and Verrucomicrobium
it is even higher in adolescents (Giannetti and (Rigsbee et al. 2012). It has been proposed that
Staiano 2016). Although usually considered as these microbes might alter or influence visceral
very benign, FAP and IBS can significantly perception, gut motility, gut permeability, and
decrease quality of life and increase school absen- intestinal gas production that can lead to pain-
teeism comparable to some very severe organic predominant FGIDs (Korterink et al. 2015;
gastrointestinal diseases, like inflammatory bowel Ohman and Simren 2013).
disease (Assa et al. 2015; Warschburger et al.
2014).
Like for infantile colic, the pathogenesis of 3.1 Treatment of FAP/IBS
FAP and IBS is not completely understood, but with Probiotics
it is supposed to involve visceral hypersensitivity
and altered gastrointestinal motility (Korterink Unfortunately, treatment options for children with
et al. 2015). It has been demonstrated that chil- IBS and FAP are limited (Abbott et al. 2018).
dren with FAP or IBS have a lower sensory Very limited data are available for pharmacologi-
threshold for gastric or rectal balloon distension cal treatments, and currently no firm recommen-
than healthy controls, suggesting the role of vis- dation for their use can be made (Abbott et al.
ceral hypersensitivity in the pathogenesis of these 2018). There is also limited data on the effect of
disorders (Faure and Wieckowska 2007). dietary changes on symptoms of FAP and IBS.
Interestingly, there are early life events, Although a diet low in fermentable oligo-, di-,
including cow’s milk protein allergy, pyloric ste- and monosaccharide and polyol (FODMAP) has
nosis, and vasculitis, associated with the develop- been found effective in several adult studies, it
ment of visceral hyperalgesia and consequently was evaluated only by one small RCT showing
abdominal pain (Bonilla and Saps 2013; improvement of symptoms in children with IBS
Korterink et al. 2015). It has been proposed that (Chumpitazi et al. 2015). Other therapies avail-
these early life events might cause altered able are cognitive behavioral therapy (CBT) and
Probiotics in Functional Gastrointestinal Disorders 129

Table 4 Randomized controlled trials using probiotics in the treatment of functional abdominal pain (FAP) and irritable
bowel syndrome (IBS)
Author N Intervention Main results
Bauserman 2005 (Bauserman 50 LGG, 1010 CFU Not significant for IBS
and Michail 2005)
Francavilla 2010 (Francavilla 60 LGG, 3  109 CFU Increases treatment success in
et al. 2010) IBS
Gawronska 2007 (Gawronska 104 LGG, 3  109 CFU Moderately increases
et al. 2007) treatment success in IBS
Giannetti 2017 (Giannetti et al. 48 Bifidobacterium longum Increases treatment success in
2017) BB536, 3  109 CFU, Bifidobacterium IBS
infantis M-63, 109 CFU;
Bifidobacterium breve M-16 V, 109 CFU
Guandalini 2010 (Guandalini 59 VSL#3 Increases treatment success in
et al. 2010) IBS
Eftekhari 2015 (Eftekhari et al. 40 L reuteri DSM 17938, 108 CFU Not significant for FAP
2015)
Jadresin 2017 (Jadresin et al. 55 L reuteri DSM 17938, 108 CFU Decreased pain intensity
2017) mainly in FAP
Maragkoudaki 2017 54 L reuteri DSM 17938, 2  108 CFU Not significant for FAP
(Maragkoudaki et al. 2017)
Romano 2010 (Romano et al. 141 L reuteri DSM 17938, 2  108 CFU Decreased pain intensity in
2014) FAP
Weizman 2016 (Weizman et al. 101 L reuteri DSM 17938, 108 CFU Decreased pain intensity in
2016) FAP
CFU colony-forming units, LGG Lactobacillus rhamnosus GG

hypnotherapy. Indeed, these approaches were (MD -0.44, 95% CI -0.82 to 0.05) (Horvath
reported to significantly reduce pain in the short- et al. 2011). Importantly, this effect was more
and medium-term (Abbott et al. 2018), but unfor- pronounced when only children with IBS were
tunately they are not widely available. It has to be taken into account (MD -0.60, 95% CI -0.97 to
emphasized that the positive diagnosis of FGIDs 0.23) (Horvath et al. 2011). Two randomized,
from the start increases a chance of symptoms placebo-controlled clinical trials investigating
relief (Trivic and Hojsak 2018). Therefore, it is multi-strain preparations are available: one with
important to acknowledge the existence of pain a proprietary specific mixture of Lactobacillus,
and to explain the benign nature of the disease to Bifidobacterium, and a Streptococcus (VSL#3)
patient and parents from the beginning in order to (Guandalini et al. 2010) and the other with a
achieve a better control of symptoms. combination of different Bifidobacterium strains
Similar to the treatment of infantile colic, sev- (Giannetti et al. 2017). Both trials showed pain
eral RTCs investigated the effect of probiotics in reduction in children with IBS significantly better
FAP and IBS (Table 4). Two probiotic strains than placebo.
have been studied the most: Lactobacillus On the contrary, L. reuteri DSM 17938 was
rhamnosus GG (LGG) (Bauserman and Michail mainly used in children with FAP. As it can be
2005; Francavilla et al. 2010; Gawronska et al. appreciated from Table 5, most of these studies
2007) and L. reuteri DSM 17938 (Romano et al. (Jadresin et al. 2017; Romano et al. 2014;
2014; Weizman et al. 2016; Jadresin et al. 2017; Weizman et al. 2016) found pain intensity reduc-
Eftekhari et al. 2015; Maragkoudaki et al. 2017) tion when compared to placebo. A recent meta-
(Table 5). A meta-analysis of published RCTs analysis of these trials reported borderline signifi-
showed that the use of LGG significantly cance (Abbott et al. 2018), but it should be
decreases the post-intervention pain intensity stressed that the included investigation with
130 I. Hojsak

Table 5 Randomized controlled trials using probiotics in the treatment of functional constipation
Number of
Author Intervention patients (age) Results
Banaszkiewicz LGG, 2  109 CFU 84 (2–16 years) Not significant difference
2005 + lactulose
(Banaszkiewicz
and Szajewska
2005)
Bu 2007 (Bu et al. L. casei rhamnosus 27 (<10 years) In probiotic group higher defecation
2007) Lcr35, 8x108 CFU frequency, higher percentage of
treatment success, less use of
glycerin enema
Coccorullo 2010 L. reuteri DSM 17938, 1x108 CFU 44 (>6 months) Positive effect on bowel frequency
(Coccorullo et al.
2010)
Guerra 2011 A goat yogurt (1 ml) containing 59 (5–15 years) Improved stool consistency
(Guerra et al. B. longum 109CFU, in addition to
2011) yogurt starters
Sadeghzadeh L. casei PXN 37, L. rhamnosus PXN 48 (4–12 years) At the end of the first week, fecal
2014 54, S. thermophilus PXN 66, B. breve incontinence and abdominal pain
(Sadeghzadeh PXN 25, L. acidophilus PXN improved significantly in
et al. 2014) 35, B. infantis PXN 27, L. bulgaricus intervention group; at the end of the
PXN 39, 1x109 CFU fourth week, this difference was not
+ lactulose significant
Tabbers 2011 A fermented dairy product containing 159 (3–16 years) Not significant difference
(Tabbers et al. B. lactis DN- 173010 and yogurt
2011) starter cultures
Wojtyniak 2017 L. casei rhamnosus Lcr35, 8x108 94 (<5 years) Not significant difference
(Wojtyniak et al. CFU, twice daily
2017)
Wegner 2018 L. reuteri DSM 17938, 129 (3–7 years) Not significant difference
(Wegner et al. 108 CFU + macrogol
2018)
CFU colony-forming units, LGG Lactobacillus rhamnosus GG

negative results (Eftekhari et al. 2015) had several effective in IBS, and L. reuteri DSM 17938
major biases. Of note, in none of these studies, the showing limited effectiveness in FAP.
issue of the duration of the relief of pain has been An algorithm for the treatment of FAP and IBS
adequately investigated (Abbott et al. 2018). based on current evidence (Korterink et al. 2015)
A finding repeatedly reported in RCTs is a is presented in Fig. 2.
positive effect of placebo (Guandalini et al.
2010; Jadresin et al. 2017; Maragkoudaki et al.
2017); as the extent of the placebo effect can be
4 Constipation
substantial, this needs to be critically taken into
account when examining the potential treatment
Constipation is considered a widespread disorder in
of functional disorders.
children, with a recent meta-analysis reporting a
Taking into account all of the above, it seems
prevalence of 9.5% in children worldwide (Koppen
fair to state that probiotics may well have a role in
et al. 2018). In more than 90% of the affected
the pain amelioration in children with IBS and
children, no underlying organic cause can be
FAP. Specifically, based on currently available
found (de Meij et al. 2016). The etiology of func-
evidence, only two strains were investigated in
tional constipation is considered multifactorial, with
more than two RCTs: LGG, proven to be
no specific cause(s) identified. There is however
Probiotics in Functional Gastrointestinal Disorders 131

Fig. 2 Proposed algorithm for the management of functional abdominal pain or irritable bowel syndrome (based on
(Korterink et al. 2015))
CBT cognitive behavioral therapy

agreement that an important factor is withholding The importance of intestinal microbiota in the
behavior, often seen in children with functional development of the constipation was emphasized
constipation (Benninga et al. 2004). Other causes by the finding that intestinal microbiota can influ-
include psychological, social factors, and alterations ence gastrointestinal motility (de Meij et al. 2016).
in intestinal microbiota (de Meij et al. 2016). This notion originated by studies in germ-free mice
132 I. Hojsak

in whom, in the absence of a gastrointestinal whether that dysbiosis causes constipation or is

microbiota, gastric emptying and gut transit time only its consequence remains unclear (Dimidi
are delayed compared with wild-type mice (Dimidi et al. 2017).
et al. 2017). Furthermore, colonization with a spe-
cific microbiota tends to normalize small-bowel
migrating motor complexes (Dimidi et al. 2017). 4.1 Treatment of Functional
Colonization with Lactobacillus acidophilus A10 Constipation with Probiotics
and Bifidobacterium bifidum B11 in germ-free rats
normalizes gut transit time, whereas colonization According to the 2014 guidelines developed by
with E. coli inhibits intestinal myoelectric activity the European and North American Societies for
(Husebye et al. 2001; Dimidi et al. 2017). While the Pediatric Gastroenterology, Hepatology, and
precise mechanism of that association is unknown, Nutrition (ESPGHAN/NASPGHAN), polyethyl-
several possible explanations exist, including ene glycol (PEG) is the first-line treatment for
(de Meij et al. 2016) microbiota-induced modula- children presenting with functional constipation
tion of the gene expression involved in motor (Tabbers et al. 2014). If PEG is not sufficient or
responses (Hooper et al. 2001), pH-dependent available, other laxatives, such as lactulose, may
motility stimulation by fermentation products, also be considered as a second-choice treatment
osmotic effects of microbiota metabolites, and (Tabbers et al. 2014). For many patients, current
intestinal distension caused by increased treatment options do not provide sustained relief
intraluminal gas production (Bar et al. 2009). of the symptoms, and new strategies are pro-
Studies in humans have partially confirmed posed, including probiotics.
results observed in animal studies. Slow transit Based on 14 randomized controlled trials
constipation in children has indirectly been linked (including 1182 participants), adult data showed
to microbial changes by detection of increased that overall, probiotics reduced whole-gut transit
methane levels in exhaled breath (Soares et al. time by 12 h and increased stool frequency by 1.3
2005) which can be contributed to increased bowel movements per week (Dimidi et al. 2014).
amount of methanogenic microorganisms (Attaluri Importantly, the effect of probiotics was species
et al. 2010). In adults, studies consistently show and strain specific, and various B. lactis strains
decreased Bifidobacterium and Lactobacillus and improved gut transit time, stool frequency, con-
increased Bacteroidetes compared with controls, sistency, and flatulence, whereas the strain
while in children data are not so conclusive (Dimidi L. casei Shirota conferred no benefit (Dimidi
et al. 2017). Conventional culturing techniques et al. 2014, 2017).
showed significant increase in colonic Clostridium There are several RCTs using different probi-
and Bifidobacterium species in constipated children otic strains in children (Banaszkiewicz and
(Zoppi et al. 1998). Other study used 16S rRNA Szajewska 2005; Bu et al. 2007; Coccorullo et al.
gene pyrosequencing with aim to compare stool 2010; Guerra et al. 2011; Sadeghzadeh et al. 2014;
microbial composition of constipated obese chil- Tabbers et al. 2011; Wojtyniak et al. 2017; Wegner
dren comparing to obese controls (Zhu et al. 2014). et al. 2018) (Table 5). These RCTs were compared
Significant decrease of Prevotella and increase rep- in a recent meta-analysis including 7 RCTs that
resentation in several genera of Firmicutes was showed overall no effect of the probiotics in the
observed in constipated patients, but probiotic treatment of constipation in children (Wojtyniak
genera Lactobacillus and Bifidobacterium were and Szajewska 2017). From those strains only
not decreased (Zhu et al. 2014). Lactobacillus rhamnosus casei Lcr35 was
Although there could be a difference in micro- investigated in two RCTs and found no positive
bial composition between constipated children effect in the meta-analysis (Wojtyniak and
comparing to adults, studies clearly show Szajewska 2017). The other strain investigated in
different microbial composition when compared two RCTs is L. reuteri DSM 17938. The first study
to healthy age-matched controls. However, included 44 infants who received no laxatives and
Probiotics in Functional Gastrointestinal Disorders 133

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# Springer Nature Switzerland AG 2019
Published online: 29 January 2019

Clostridium difficile Colitis Prevention

and Treatment

Meltem Dinleyici and Yvan Vandenplas

Abstract 1 Introduction
Clostridium difficile (C. diff) is the most
common causative agent of antibiotic- Antibiotic-associated diarrhea is estimated to
associated diarrhea and colitis. This spore- occur with an incidence of about 15 to 20% in
forming, obligate anaerobic, gram-positive children treated with antibiotics (Turck et al.
bacillus is becoming responsible for an 2003; Goldenberg et al. 2015; Johnston et al.
increasing number of infections worldwide, 2016). Clostridium difficile (C. diff) is the most
both in community and in hospital settings, common causative agent of antibiotic-associated
whose severity can vary widely from an diarrhea and colitis, designated as C. diff infection
asymptomatic infection to a lethal disease. or disease. C. diff is a spore-forming, obligate
While discontinuation of antimicrobial agents anaerobic, gram-positive bacillus and is acquired
and antibiotic treatment of the infection remain from the environment or by the fecal-oral route.
the cornerstone of therapy, the use of Although the nomenclature is being modified to
probiotics, especially Saccharomyces Clostridioides difficile, we keep in this
boulardii, and more recently of fecal manuscript Clostridium difficile (C. diff). C. diff
microbiota transplantation have become valid is a common healthcare-associated pathogen. The
forms of prevention and/or therapy and are toxins (A, B, and binary) are the agents that cause
here critically examined. injury and disease, although binary toxin is prob-
ably not causing disease but may aggravate the
Keywords damage caused by toxins A and B (McKonnie
Antibiotic associated diarrhea · Clostridium and Kasti 2017). Clinical symptoms vary widely,
difficile · Colitis from asymptomatic colonization to
pseudomembranous colitis with bloody diarrhea,
fever, and severe abdominal pain. C. diff is world-
wide, a frequent and still increasing etiology of
healthcare-related diarrhea causing significant
morbidity (Pant et al. 2013). In some parts of
M. Dinleyici
Eskisehir Osmangazi University Faculty of Medicine, the world, mainly in North America, a hyperviru-
Department of Social Pediatrics, Eskisehir, Turkey lent strain is of major health concern and has
Y. Vandenplas (*) caused C. diff infection among low-risk
KidZ Health Castle, UZ Brussel, Vrije Universiteit populations, including in ambulatory care
Brussel, Brussels, Belgium (Sammons et al. 2013). Several studies have
e-mail: [email protected]

139
140 M. Dinleyici and Y. Vandenplas

shown also a rise in C. diff infection in children, emergence and circulation of strains with peculiar
both in community and in hospital settings toxins’ profiles and/or multidrug resistance
(Sammons et al. 2013). Severe C. diff infection, strongly highlight the necessity of a rapid C. diff
including with the North American strain, is on disease diagnosis, a careful monitoring of C. diff
the rise as well. disease in pediatric patients, and a stricter control
Nucleic acid amplification testing appears to of antibiotics usage (Spigaglia et al. 2017).
be the most efficient test to detect the gene that For patients with moderate or severe disease,
encodes for toxins A and B and thus to diagnose proper empirical antibiotic treatment should be
C. diff-induced disease (McKonnie and Kasti started as soon as the diagnosis is suspected.
2017). Infants were believed to not develop Oral metronidazole is recommended by the
C. diff disease, even when toxins are detected American Academy of Pediatrics as the first-line
(McKonnie and Kasti 2017), although some therapy for a first episode, due to concerns about
reports suggest different (Buts et al. 1999; Ooi the increased risk of resistant bacteria and excess
et al. 2009). Obviously, colonoscopy with costs associated with vancomycin (Sammons
biopsies is the best way to detect colitis et al. 2013, Schutze et al. 2013). When C. diff
(McKonnie and Kasti 2017). Recent exposure to disease is mild to moderate, discontinuation of the
fluoroquinolones, clindamycin, third-generation administered antibiotics in patients receiving
cephalosporins, proton pump inhibitors, and mul- these drugs when C. diff infection develops or
tiple classes of antibiotics were each strongly administration of metronidazole is sufficient to
associated with the diagnosis of C. diff disease solve this problem (Esposito et al. 2015). Evi-
(Adams et al. 2017). Recent exposure to outpa- dence for oral vancomycin use in adults with
tient healthcare clinics or to a family member with severe C. diff disease is growing (Zar et al.
C. diff infection was associated with C. diff dis- 2007). Vancomycin is recommended by the
ease (Adams et al. 2017; Wei et al. 2017). Also American Academy of Pediatrics for a second
H2-receptor antagonists induce an increased risk recurrence of mild or moderate CDI (Schutze
(Faden and Ma 2016). Thus, since acid suppres- et al. 2013). In severe or frequently relapsing
sion medications are associated with an increased cases, vancomycin is the drug of choice.
risk of C. diff infection and recurrent C. diff infec- Rifaximin has been shown to have cure rates
tion, a judicious use of acid suppression medica- comparable to vancomycin (Gerding and Johnson
tion should be considered, especially among 2010). Fidaxomicin has been approved in adults
those at highest risk to develop C. diff disease but needs further studies in children (Tannock
(Nylund et al. 2014). Recurrence of C. diff diar- et al. 2010; D’Ostroph and So 2017).
rhea and pseudomembranous colitis occur in up Non-antimicrobial treatments for C. diff dis-
to 20% of patients after standard therapy of a first ease exist as well. The use of probiotics has a
C. diff infection (Surawicz et al. 1989a, b). theoretic benefit in restoring the normally protec-
tive function of intact lower intestinal microbiota
after disruption of antimicrobial therapy.
2 Management of Clostridium Probiotics are not (yet) part of the treatment rec-
difficile Disease ommendation of the American Academy of Pedi-
atrics (Koon et al. 2016; Schutze et al. 2013).
The optimal management of pediatric C. diff dis- Buts et al. reported more than 25 years ago that
ease remains unclear (Sammons et al. 2013). Dis- the yeast Saccharomyces boulardii (S. boulardii)
continuation of antimicrobial agents is the first was effective in 18/19 infants (median age
step in treating C. diff disease and may suffice in 8 months) with persistent intestinal symptoms
many instances. Resistance to antibiotics (persistent or protracted diarrhea, malabsorption
characterizes almost 50% of the toxigenic and failure to thrive, colic, emesis, flatulence)
isolates, and, in particular, 20% of them are related to a C. diff-secreting toxin B (Buts et al.
multidrug resistant (Spigaglia et al. 2017). The 1993). Toxin B cleared in 16 cases, and
Clostridium difficile Colitis Prevention and Treatment 141

eradication of C. diff occurred in 14 (Buts et al. disease. Prevention is possible in different ways:
1993). The efficacy of S. boulardii could be (i) by minimizing the duration and frequency of
related to yeast concentration in the stools antimicrobial treatments, (ii) by minimizing other
(Elmer et al. 1999). In adults with either an initial medications such as proton pump inhibitors
episode or recurrent C. diff disease, S. boulardii associated with C. diff disease, and (iii) by
was shown to be effective and safe (McFarland decreasing the number of antibiotics. Exposure
et al. 1994). Other treatment options must be to antibiotics is recognized as the single most
reserved for severe cases and be considered as a important risk factor. Nearly all antimicrobials
salvage treatment, although potential advantages are associated with the risk to develop C. diff
in pediatric patients remain unclear (Esposito disease (Sammons et al. 2013). Antibiotic stew-
et al. 2015). ardship programs will enhance this approach.
Fecal microbiota transplantation (FMT) may Adequate hand hygiene and prevention of contact
have a substantial effect with few short-term are recommended. Cleaning with chlorine
adverse events for recurrent C. diff disease. Evi- containing products or other agents destroying
dence is insufficient to recommend a FMT for an spores has also been shown to be useful. Repeti-
initial C. diff infection. Efficacy varies according tive hospitalizations are considered another risk
to the donor, preparation, or delivery method factor (Sammons et al. 2013). Both risk factors
(Crow et al. 2015; Drekonja et al. 2015), but are of course interrelated.
FMT is becoming part of the treatment algorithms It has not been well documented that a system-
against recurrent C. diff disease both in adults and atic co-treatment of probiotics with each adminis-
children (Davidovics et al. 2018). Cure rates in tration of antibiotics would result in a decrease of
adults approach 90% (Kassam et al. 2013). FMT, C. diff disease. A large retrospective data analysis
arguably the most effective treatment in case of in a tertiary care hospital in the USA concluded
recurrent C. diff disease, has become a standard that the incidence of C. diff infection related to
part of therapeutic algorithms (Davidovics et al. antibiotic administration remained unchanged
2018). At this stage, FMT should only be with or without co-administration of S. boulardii
performed in established centers (Davidovics (Flatley et al. 2015).
et al. 2018). Current studies have demonstrated Some of the human milk oligosaccharides
minimal adverse effects, with no reports of trans- (HMOs) bind to the toxin A carbohydrate-binding
mission of infectious diseases (Kassam et al. site of C. diff (Nguyen et al. 2016). However,
2013). Hospitals should encourage the develop- while HMOs are expected to bind extensively to
ment of FMT programs to improve therapy of these bacterial toxins, it is unlikely that HMO
local patients (Cammarota et al. 2014). Severe binding will effectively inhibit their interactions
C. diff disease and inadequate bowel preparation with their cellular receptors (El-Hawiet et al.
appear to be independent predictors of failure 2015). HMOs do not significantly inhibit the
after single fecal infusion in patients treated with cytotoxic effects of toxin A or B. The absence
FMT by colonoscopy for recurrent C. diff disease of protection is attributed to the very weak intrin-
(Ianiro et al. 2017). sic affinities that the toxins exhibit toward the
HMOs (El-Hawiet et al. 2011). Prebiotics and
soluble starch may stimulate the growth of
3 Prevention of Clostridium lactobacilli and bifidobacteria strains that were
difficile Disease compatible with each other and exhibited high
growth and antimicrobial activity against clinical
The financial burden of a C. diff disease is C. diff strains and a hypervirulent NAPI/027
substantial. strain (Ambalam et al. 2015).
Restricting the use of antibiotics is of course Antibiotics often fail to reduce recurrence
the most effective prevention. However, (Surawicz et al. 2000). S. boulardii was shown
antibiotics are needed medication in bacterial in an open trial to stop recurrence of C. diff
142 M. Dinleyici and Y. Vandenplas

disease in 11/13 (85%) of patients (Surawicz et al. antibiotics and in hospital settings where the risk
1989a, b, 2000). The protective effects of of C. diff infection is 5% (Johnston et al. 2018).
S. boulardii on C. diff-induced inflammatory diar- The “oral probiotics” strategy improves quality of
rhea is due, at least in part, to proteolytic digestion life and reduces healthcare cost (Li et al. 2018).
of toxins A and B molecules by a protease that is Studies conducted in adults demonstrated that
secreted by the yeast (Castagliuolo et al. 1999), both probiotics and fermented products have a
resulting in blocking the activation of Erk1/ modest effect in preventing antibiotic-associated
2 MAP kinases (Chen et al. 2006). S. boulardii diarrhea with a number needed to treat of 6–7
is effective in preventing C. diff infection by (Goldenberg et al. 2015). Accordingly a
outbreak associated via inhibition of the cytotoxic Cochrane review concluded that the number
effects of C. difficile toxins (Koon et al. 2016). needed to treat in children is 10 (Goldenberg
Overall, probiotics seem to have been mainly et al. 2015; Johnston et al. 2016). According to
evaluated in the prevention of C. diff disease. the Cochrane review, the overall incidence of
Vernaya et al. reported no significant effect of antibiotic-associated diarrhea is 8% in the probi-
any probiotic in primary prevention of C. diff otic group versus 19% in the control group
infection in elderly (Vernaya et al. 2017), while (RR 0.46, 95% CI 0.35–0.61; I2 ¼ 55%), with a
Shen et al. reported a significant reduction by any moderate overall quality of the evidence
probiotic in adults (Shen et al. 2017a, b). (Goldenberg et al. 2015; Johnston et al. 2016).
According to the review by Sinclair et al., there Which probiotic? Lactobacillus (L),
was an inconclusive evidence of any Lactobacil- S. boulardii, and a mixture of probiotics were all
lus on C. diff infection prevention in adults beneficial in reducing the risk of developing
(Sinclair et al. 2016). Based on several systematic C. diff-associated diarrhea (63.7%, 58.5%, and
reviews and meta-analyses of randomized con- 58.2% reduction) (Lau and Chamberlain 2016).
trolled trials, very low-quality or moderate- The most common probiotic formulations
quality evidence suggests that probiotics are administered were S. boulardii (32% of patients
both safe and effective for preventing C. diff- receiving probiotics), L. acidophilus and
associated diarrhea and are associated with L. bulgaricus (30%), L. acidophilus (28%), and
fewer adverse events vs placebo or no treatment L. rhamnosus (11%). Probiotic use increased
(Goldenberg et al. 2013; Goldenberg et al. 2018; from 1.0% of 1,090,373 discharges in 2006 to
Esposito et al. 2015; Johnston et al. 2012). “Any 2.9% of 1,006,051 discharges in 2012
probiotics” was considered as beneficial in both (P < 0.0001) (Yi et al. 2016). In this sample of
adults and children (59.5% and 65.9% reduction), US hospitals, a sizable and growing number of
especially among hospitalized patients (Lau and inpatients received probiotics as part of their care
Chamberlain 2016). Also in adults and children, despite inadequate evidence to support their use
Mc Farland et al. concluded that there was a in this population. Additional research is needed
significant reduction of C. diff infection with any to guide probiotic use in the hospital setting
probiotic and by species (McFarland et al. 2016). (Yi et al. 2016). Adjusted subgroup analyses
Probiotics reduced the incidence of C. diff- suggested that, compared to no probiotics, multi-
associated diarrhea by 66% (pooled-relative risk, species probiotics were more beneficial than
0.34 [95% CI, 0.24 to 0.49]; I(2) ¼ 0%) (Johnston single-species probiotics, as was using probiotics
et al. 2012). In a population with a 5% incidence in clinical settings where the C. diff infection risk
of antibiotic-associated C. diff-associated diarrhea is 5% (Johnston et al. 2018). A few strains,
(median control group risk), probiotic prophy- namely, Lactobacillus rhamnosus GG (LGG)
laxis would prevent 33 episodes (CI, 25 to and S. boulardii, have been studied to some
38 episodes) per 1,000 persons (Johnston et al. extent (Valdés-Varela et al. 2018). S. boulardii
2012). Probiotic prophylaxis may be a useful and reduces the incidence of antibiotic-associated
safe C. diff disease prevention strategy, particu- diarrhea in hospitalized patients (Surawicz et al.
larly among patients taking two or more 1989a, b). A specific recommendation for the use
Clostridium difficile Colitis Prevention and Treatment 143

of LGG and S. boulardii emerges (Mantegazza associated diarrhea in hospitalized children and
et al. 2018). LGG’s sensitivity to penicillin might adolescents receiving a therapeutic course of
make this probiotic (theoretically) less effective. antibiotics reduced its risk and represent a cost-
Lactobacillus species and in particular LGG are saving strategy (Li et al. 2018). An economic
sensible to some antibiotics and in particular evaluation performed in Belgium concluded that
penicillins; therefore their ability to colonize the the administration of S. boulardii to prevent
gut and act as a probiotic may be limited when antibiotic-associated diarrhea would result in a
antibiotic therapy is administered (Goldstein et al. cost benefit of €15 to € 95 per hospitalized patient
2015). This is quite important especially in chil- treated with antibiotics from the societal and hos-
dren where oral penicillin is the most frequently pital perspectives (Vermeersch et al. 2018). For
administered antibiotic, with amoxicillin Belgium, based on 831,655 hospitalizations
accounting for more than 60% of antibiotic treated with antibiotics, the generalized use of
prescriptions in children aged less than 5 years S. boulardii would result in up to € 41.8 million
(Gibson et al. 2015). savings for the Belgian healthcare payer
Despite a demonstrated positive effect of spe- (Vermeersch et al. 2018). However, there is still
cific strains of probiotics on antibiotic-associated insufficient evidence to recommend the routine
diarrhea, safety issues still remain among which administration of probiotics to each hospitalized
the risk of associated severe infections and of patient on antibiotics.
antibiotic-resistant gene exchange (Mantegazza Probiotic result in a reduced cost compared no
et al. 2018). More efforts are needed to standard- probiotics. Probiotic may be a cost-effective strat-
ize the clinical studies evaluating probiotic egy to prevent C. diff infection in hospitalized
products in order to select the best candidate adults receiving antibiotics age 65 or older or
(Valdés-Varela et al. 2018). when the baseline risk of CDI exceeds 1.6%
The working group on prebiotics and (Shen et al. 2017a, b). In probabilistic sensitivity
probiotics of the European Society of Paediatric analysis, probiotic use may be a cost-effective
Gastroenterology, Hepatology, and Nutrition strategy to prevent C. diff infection in hospitalized
(ESPGHAN) recommends using LGG (moderate patients receiving antibiotics when the baseline
QoE, strong recommendation) or S. boulardii risk to develop C. diff infection exceeds 1.6%
(moderate QoE, strong recommendation) if (Shen et al. 2017a, b).
probiotics for preventing antibiotic-associated
diarrhea are considered because of the existence
of risk factors such as class of antibiotic(s), dura- 4 Conclusion
tion of antibiotic treatment, age, need for hospi-
talization, comorbidities, or previous episodes of C. diff infection is difficult and expensive to treat.
antibiotic-associated diarrhea (Szajewska et al. Oral metronidazole is recommended for a patient
2016). If the use of probiotics for preventing developing C. diff disease for the first time.
C. diff-associated diarrhea is considered, the Recurrent C. diff disease can be treated with
ESPGHAN working group suggests using (oral) vancomycin, but there is also an increasing
S. boulardii (low QoE, conditional recommenda- evidence for the efficacy of FMT. Well-
tion). Other strains or combinations of strains conducted trials comparing vancomycin vs FMT
have been tested, but sufficient evidence is still in pediatrics are needed. Although some
lacking (Lau and Chamberlain 2016; Szajewska probiotics have been shown to be effective in
et al. 2016). Despite a diversity of epidemiologi- the treatment of (recurrent) C. diff as well, their
cal, socioeconomical, and health system therapeutic use is not recommended in guidelines.
conditions, similar recommendations apply well In terms of prevention, however, probiotics can
to Asia-Pacific countries (Cameron et al. 2017). be used to prevent C. diff disease in high-risk
From the perspective of the medical system, populations and when high-risk antibiotics are
oral probiotics as a preventive strategy for C. diff- administered. Nevertheless, it must be stressed
144 M. Dinleyici and Y. Vandenplas

that avoidance of inappropriate use of antibiotics D’Ostroph AR, So TY (2017) Treatment of pediatric
or other medications increasing the risk for C. diff Clostridium difficile infection: a review on treatment
efficacy and economic value. Infect Drug Resist
disease such as proton pump inhibitors is the most 10:365–375
recommended prevention. Most meta-analyses Davidovics ZH, Michail S, Nicholson MR et al (2018)
tend to generalize and evaluate all probiotics FMT special interest group of the North American
together. However, only probiotic products for society of pediatric gastroenterology hepatology and
nutrition and the European society for pediatric gastro-
which there is evidence for efficacy should be enterology hepatology and nutrition. Fecal microbiota
used. In pediatrics, LGG and S. boulardii are transplantation for recurrent Clostridium difficile infec-
recommended to prevent antibiotic-associated tion and other potential applications in children: clini-
diarrhea and S. boulardii to prevent C. diff dis- cal report. J Pediatr Gastroenertol Nutr. submitted
Drekonja D, Reich J, Gezahegn S et al (2015) Fecal
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charides, may enhance the efficacy of probiotics, infection: a systematic review. Ann Intern Med
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future trials. El-Hawiet A, Kitova EN, Kitov PI et al (2011) Binding of
clostridium difficile toxins to human milk oligosac-
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El-Hawiet A, Kitova EN, Klassen JS (2015) Recognition
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Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2019) 1125: 147–150
https://doi.org/10.1007/978-3-030-14636-8
# Springer Nature Switzerland AG 2019

Index

A Community state types (CSTs), 4, 5

Acute infectious diarrhea (AID) Constipation
immunization programs, 110 colonization, 132
infectious diarrhea (see Diarrhea) intestinal microbiota, 131
pediatric age, 109 meta-analysis, 130
pre-school child, 110 methanogenic microorganisms, 132
probiotics, 110 proposed algorithm, 131
Amniotic fluid microbiome, 11, 12 treatment, 132–133
Antibiotic-associated diarrhea, 139, 142, 143 Cow’s milk allergy (CMA), 59, 62–64
Crohn’s disease (CD), 4, 70, 74, 75, 97, 101, 103
B
Bacterial vaginosis (BV), 15 D
Butyrate De Simone Formulation, 104, 105
intestinal eubiosis, 88 Diarrhea
PBMCs, 63 bacterial and viral toxins, 111
producing bacteria, 7, 51 Bifidobacterium, 112
SCFAs, 42, 60, 87, 90 host and probiotic metabolic network, 113
treatment, 63 immune modulation, 112
UC patients, 76 physiological conditions, 110
prevention, children
C day-care centers, 114
Celiac disease (CeD) hospital settings, 114
autoimmunity, 72 probiotic metabolites, 111–112
dysbiosis, 72 Saccharomyces boulardii, 113
G protein-coupled receptor CXCR3, 70–71 secretory diarrhea, 111
HLA DQ2/DQ8, 70, 72, 77 treatment
and IBD, 70, 73 AID management, 115
infant’s diet, 72 LGG, 115
pathogenesis, 70 low-income settings, 115–116
probiotics, 74–75 probiotics, 115, 117
Celiac Disease Genomic, Environmental, Microbiome and Ussing chamber system, 111
Metabolomic (CDGEMM), 76–77 Diet
Clostridium difficile (C. diff) FAP and IBS, 128
healthcare-associated pathogen, 139 fiber/SCFAs, 63
healthcare clinics, 140 interventions, 4, 63
low-risk populations, 139–140 maternal, 13
management, 140–141 microbiome development, 26
nucleic acid amplification, 140 microbiota modulation, 92, 94, 96
prevention, 141–143 milk and egg allergy, 60
Colitis, 139, 140 and nutritional counselling, 14, 15, 17
Colony forming units (CFU), 53, 63, 91, 104, 114, 115, during pregnancy, 14
126, 129, 130, 133 supplements, 31–33

147
148 Index

E Gestation
Enteric microbiota, 52, 126 gut microbiota, 7
microorganisms, 4
F oral microbiota, 7–8
Fecal microbiota transplantation (FMT) physiological changes, 6
diet and modulation, 92–96 placental microbiome, 9–10
gut-liver axis, 87 pre-gestational type 2 diabetes, 13, 32, 96
intestinal preterm births, 27
microbiome, 86–87 probiotics, 15–16
microbiota, 87–89 Gestational diabetes mellitus (GDM), 10, 11, 14–16
lifestyle and pharmacological interventions, 86 Gut-associated lymphoid tissue (GALT), 52, 53, 70
and NAFLD (see Nonalcoholic fatty liver disease Gut-brain axis, 38, 51, 53
(NAFLD)) Gut-liver axis, 86–89, 97
and NASH, 89–91 Gut microbiota
probiotics, 91–92 allergy, 59
symbiotics, 91–92 analysis, 62
Food allergy (FA) butyrate-producing, 64
allergenicity, 60 dysbiosis, 65
CMA (see Cow’s milk allergy (CMA)) ecology, 98
experimental therapies, 60 functions, 38
factors, 58 gestation, 7
global health problem, 58 in IBD, 102–103
gut microbiota, 58, 59 infant, 14
life-threatening allergic reaction, 58 liver damage, 86, 93
non-allergen specific therapies, 60 maternal microbiota, 7
preclinical evidence metabolites, 60
animal model, 62 modulation and promotion of barrier functions, 112
CMP, 62 NAFLD and microbiota, 93–94
cytokine IL-10, 61 probiotics, 113
HDAC, 63 target of intervention, 59
immune tolerance, 60 tolerogenic mucosal environment, 63
OVA, 62 vagina, 8
PMBCs, 60, 61
probiotics, 61 H
pro-tolerogenic effects, 62 Homeostasis, 41
SCFAs, 63 Human microbiota, 8, 26, 37
probiotics efficacy, 63–64
therapeutic strategies, 59 I
triggers, 58 Immune tolerance, 16, 52, 58, 60, 63
Food and Drug Administration (FDA), 30, 31, 33, 59 Immunomodulation activity, 42–43
Fructooligosaccharides (FOS), 15, 30, 32 Infantile colic
Functional abdominal pain anxiety, 50
children and adolescents, 123–124 crying, 49–50
and IBS (see Irritable bowel syndrome (IBS)) definition, 50
proposed algorithm, 131 epidemiology, 50
randomized controlled trials, 129 etiology, 50–52, 125
Functional gastrointestinal disorders (FGID) FGIDs, 50
in children and adolescents, 121, 123–124 inconsolable crying, 125
definition, 121 microbiota
neonates, infants and toddlers, 121, 122 classification, 50–51
CNS, 51
G enterobacteria, 51
Galactooligosaccharides (GOS), 32 GALT, 52
Gastroenteritis, 110, 112, 116, 117 gut inflammation, 52, 125
Gastrointestinal infections, 54, 114, 117 intestinal inflammatory processes, 52
Gastrointestinal microbiome microbial colonization, 51
external environment, 52 multifactorial etiology, 51
gut-brain interactions, 51 TLRs, 51
hospitalization, 114 proposed algorithm, 126, 127
infection, 54 Proteobacteria, 125
SCFA, 53 randomized controlled trials, 126
Index 149

symptoms, 124 in CIDs, 69–70

treatment, 126–128 dysbiosis, 4
Inflammatory bowel diseases (IBD) gut, 38
microbiome, 72–73 IBD (see Inflammatory bowel diseases (IBD))
pediatric (see Pediatric IBD) intestinal, 86–87
probiotics and NAFLD (see Nonalcoholic fatty liver disease
BFM, 76 (NAFLD))
CD and UC, 101 NEC (see Necrotizing enterocolitis (NEC))
gut microbiota, 102–103 placenta, 9–10
microbiota, 102 probiotics interventions, 73–74
pro-inflammatory and anti-inflammatory T cell, Microbiota
102 breastfeeding, 26
treatment, 76 diet (see Diet)
UC, 75–76 etiology and role, 50–52
pro-inflammatory pathways, 42 FA (see Food allergy (FA))
Intestinal barrier function, 40–42, 70, 72 FMT (see Fecal microbiota transplantation (FMT))
Interstitial cells of Cajal (ICC), 54 maternal, 4, 6–8
In vitro fertilization (IVF), 14–15 reproductive, 4–6
Irritable bowel syndrome (IBS) tools, modulation, 14–16
pathogenesis, 128 Mucin secretion (MUC2), 41, 42
pediatric patients, 128
treatment, 128–130 N
Necrotizing enterocolitis (NEC)
L bacterial diversity, 29
Lactobacillus rhamnosus GG (LGG) Bifidobacterium, 38, 74
and EHCF, 63–64 epidemiology, 38–40
infantile colic, 53, 126 gut microbiota, 38
and micronutrients, 114 human microbiota, 26, 37
murine model, 62 infant microbiome, 26–27, 32–33
probiotic strains, 117 intestinal homeostasis, 38
RV infection, 113 ischemic bowel necrosis, 25
Lactobacillus spp. late-onset sepsis, 16
and Bifidobacterium, 27, 32 microbiome, 28–29
biomarkers, 5 pathogenic factors, 38–40
CSTs, 4 preterm bacterial colonization patterns, 27–28
LGG (see Lactobacillus rhamnosus GG (LGG)) preventive strategies, 40–41
during pregnancy, 26 shaping microbiota, 41–42
strains, 11 Neonatal intensive care units (NICUs), 25, 27, 28, 31–33
Low birth weight (LBW) infant, 9, 27, 38 Nonalcoholic fatty liver disease (NAFLD)
chronic liver illness, 86
M and FMT (see Fecal microbiota transplantation (FMT))
Maternal microbiota and HFD, 98
gut, 7 intestinal microbiota, 87–89
oral, 7–8 lifestyle interventions, 86
vaginal and uterine, 6–7 microbiota, 93–94
Meconium and NASH, 89–91
bacteria, 12 obesity trial, 95–96
human microbiota, 8 probiotics, 91–92
immune system, 13 randomized controlled trials, 98
maternal-foetal-neonatal microbiota, 5 symbiotics, 91–92
microflora, 70 type 1 and type 2, 86
neonatal stool, 12 Nonalcoholic steatohepatitis (NASH)
placenta tissue, 11 antibiotics, 92
Microbiome microbiota, 89–91
amniotic fluid, 11 and NAFLD, 87–89
blood, 8 steatosis, 86
CeD (see Celiac disease (CeD)) Non-communicable diseases (NCDs), 4
150 Index

P R
Pediatric IBD Rotavirus (RV), 110–113, 115, 116
CD, 103
UC, 103–104 S
Perinatal microbial environment, 3–4 Semen, 6, 15, 16
Peripheral blood mononuclear cells (PBMCs), Short-chain fatty acids (SCFA), 38, 42, 53, 60, 62, 63, 72,
60–61, 63 87–90
Peyer’s patches, 52 Signaling
Placenta cytokine secretions, 52
and amniotic fluid, 5 gut microbiota, 38
exosomes, 8 host immune modulation, 111
maternal, 71 INF-beta, 112
microbiome, 8–10, 12 maternal placenta, 71
molecular mechanisms, 13 pathway, 51
Placental microbiome, 9–10 TLR4, 39, 112
Postbiotics TMEM16A and CFTR, 110
conception and pregnancy, 17 Synbiotics, 14, 15
intestinal barrier function, 41
NEC (see Necrotizing enterocolitis (NEC)) T
Postnatal bacterial exposition, 13–14 Toll-like receptors (TLRs)
Prebiotics, 14, 16, 32, 53, 92, 141, 143, 144 and APC, 52
Prenatal microbial exposition bacterial signaling, 39
amniotic fluid microbiome, 11 human models, 51
bacteria-immune system, 11 inflammation, 51, 87
bacterial DNA, 12 intestinal epithelial cells, 39
contaminant detection, 8 pathogenesis, 39
high-fat maternal diet, 13 pattern recognition receptors, 87
metabolites, 13 TLR-2, 87
microbial population, 12 TLR-3, 113
molecular and sequencing technologies, 8 TLR-4, 87, 89
placental microbiome, 8–10 TLR-5, 87
Preterm birth TLR-9, 87
cesarean delivery, 27 and TOLLIP, 30
foetal growth restriction, 15
foetal/neonatal microbial colonization, 7 U
molecular mechanisms, 12 Ulcerative colitis (UC), 75, 76, 101, 103–104
placental microbiome, 9–10 Urinary tract infections (UTIs), 6, 15
preeclampsia, 8
pregnancy, 6 V
Preventive strategy, 40, 43, 53, 54, 143 Vagina
Probiotics gut microbiota, 8
at-risk infants, 29–30 healthy non-pregnant women, 4
on fertility, 14–15 infections, 14, 16, 32
FGID (see Functional gastrointestinal disorders microbes, 28
(FGID)) microbiota, 5, 6, 15, 26
FMT (see Fecal microbiota transplantation (FMT)) pathogenic colonization, 4
gestation, 15–16 and uterine microbiota, 6–7
IBD (see Inflammatory bowel diseases (IBD)) Very low birth weight (VLBW) infant, 30, 31, 38
infectious diarrhea, 110–113 Vulvovaginal candidiasis (VVC), 15
postnatal period, 16
to premature infants, 30–31 W
preventative and therapeutic strategy, 52–54 World Health Organization (WHO), 52