Effects of Early Prebiotic and Probiotic Supplementation on

Development of Gut Microbiota and Fussing and Crying in Preterm

Infants: A Randomized, Double-Blind, Placebo-Controlled Trial*
Anna Partty, MD1,2, Raakel Luoto, MD, PhD1,2, Marko Kalliomaki, MD, PhD1,2,
Seppo Salminen, PhD3, and Erika Isolauri, MD, PhD1,2
Objective To evaluate the impact of early prebiotic and probiotic intervention on preterm infants well-being,
crying, growth, and microbiological programming.

Study design Ninety-four preterm infants (gestational age 32-36 weeks and birth weight >1500 g) randomized to
receive prebiotics (mixture of galacto-oligosaccharide and polydextrose 1:1), probiotics (Lactobacillus rhamnosus
GG), or placebo during the first 2 months of life were followed up for 1 year. Infants were categorized based on the
extent of crying and irritability during the first 2 months of life, and their gut microbiota was investigated by fluorescence in situ hybridization (n = 66) and quantitative polymerase chain reaction (n = 63).
Results A total of 27 of 94 infants (29%) infants were classified as excessive criers, significantly less frequently in
the prebiotic and the probiotic groups than in the placebo group (19% vs 19% vs 47%, respectively; P = .02). The
placebo group had a higher percentage of Clostridium histolyticum group bacteria in their stools than did the probiotic group (13.9% vs 8.9%, respectively; P = .05). There were no adverse events related to either supplementation.
Conclusions Early prebiotic and probiotic supplementation may alleviate symptoms associated with crying and
fussing in preterm infants. This original finding may offer new therapeutic and preventive measures for this common
disturbance in early life. (J Pediatr 2013;163:1272-7).
See editorial, p 1250

uring early infancy, balanced hostmicrobe interaction is essential for healthy intestinal and immunological development.1 Microbiological programming begins in utero and proceeds gradually during birth and infancy.2 The process is
determined by environmental influences such as mode of delivery, perinatal antibiotic exposure, mode of feeding, and
amount of skin-to-skin contact.3 Risk factors associated with perturbation of the compositional development of the gut microbiota tend to accumulate in infants born preterm. As a consequence, their encounter with beneficial bacteria is often delayed.
Thus far, deviations in gut microbiota composition in term infants have been associated with various inflammatory and functional gastrointestinal disorders such as colicky crying, cows milk allergy, infantile diarrhea, and even celiac disease.4 In such
clinical conditions, the use of probiotics (ie, live microorganisms that, when administered in adequate amounts, confer a health
benefit on the host) and prebiotics (ie, nondigestible food ingredients that stimulate the growth and/or activity of the indigenous intestinal bacteria) provide promising tools to alleviate specific disorders
and hence enhance infant well-being.5,6
In preterm infants, one of the most promising targets of probiotic intervention
From the Department of Pediatrics, Turku University
is prevention of necrotizing enterocolitis, a devastating immunoinflammatory
Hospital; Department of Clinical Sciences, and
Functional Foods Forum, University of Turku, Turku,
intestinal disease often resulting in severe impairment.2 However, in everyday
Finland
life, much more frequent sources of frustration to parents of preterm infants
ivikki
Supported by the Juho Vainio Foundation, the Pa
and Sakari Sohlberg Foundation, the Foundation for
are benign functional gastrointestinal disorders related to feeding, crying, and irPediatric Research, an EVO grant from Turku University
Hospital and Satakunta Central Hospital, and Mead
ritability. Data on the effects of prebiotic and probiotic interventions on the preJohnson Nutrition (covered the costs of the prebiotic and
probiotic products [prepared by Turku University Hospivention or treatment of these common problems are needed.
tal Pharmacy] and part of the salary for R.L.). The sponTo improve our understanding of the impact of prebiotics and probiotics
sors had no influence on the design or conduct of the
study, data management and analysis, writing of the
on preterm infant well-being and gut microbiota composition, we conducted a
report, or the decision to submit the manuscript for
1

publication. The authors declare no conflicts of interest.

Registered with ClinicalTrials.gov: NCT00167700.

Cy3
FISH
FITC
PCR
qPCR

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Carbocyanine 3
Fluorescence in situ hybridization
Fluorescein isothiocyanate
Polymerase chain reaction
Quantitative polymerase chain reaction

0022-3476/$ - see front matter. Copyright 2013 The Authors.

All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2013.05.035
*

This is an open-access article distributed under the

terms of the Creative Commons AttributionNonCommercial-No Derivative Works License, which
permits non-commercial use, distribution, and reproduction in any medium, provided the original author and
source are credited.

Vol. 163, No. 5  November 2013

randomized, double-blind, prebiotic and probiotic intervention study in preterm infants. We hypothesized that early
modification of the gut microbiota with specific prebiotics
and probiotics can enhance infant well-being by reducing
the risk of infant crying and fussing. The overall aims were
to balance the gut microbiota composition with prebiotic
or probiotic supplementation and consequently improve infant well-being and reduce their disease risk. We describe the
impacts of prebiotic and probiotic intervention on infant
crying, fussing, and irritability and on their gut microbiota
development.

Methods
This randomized, double-blind placebo-controlled study
involved 94 infants who were recruited between days of life
1 and 3 in the Department of Pediatrics, Turku University
Hospital, between June 2008 and May 2011. To be eligible
for enrollment, infants had to meet the following criteria:
gestational age between 32 + 0 and 36 + 6 weeks, birth weight
>1500 g, and absence of any congenital defects in the gastrointestinal system or other defects preventing enteral nutrition. The study was approved by the Ethics Committee of
the Hospital District of South-West Finland. Written
informed consent was obtained from the infants parents.
The participants were randomly assigned to 1 of the 3
study groups according to computer-generated block
randomization by an independent statistician (J.M.). To
avoid disproportionate numbers of patients in each group,
randomization was performed in blocks of 6 subjects.
Randomization was implemented with SAS software package
PROC PLAN (SAS Institute, Cary, North Carolina). To
ensure allocation concealment, a member of a group not
involved in the conduct or reporting of this study was
responsible for the packaging and labeling of the study products. Study personnel, staff in the neonatal ward, and parents
were blinded to the randomization. The study nurse allocated the next available product on entry to the trial, and
each patient received the study product directly from the
department. The code was revealed to the investigator once
recruitment, data collection, and analysis were completed.
The study data were collected on printed case record forms,
and the members of the research group entered the data. All
data were kept confidential.
Preterm infants were randomly assigned to receive orally a
prebiotic mixture (polydextrose [Danisco Sweeteners, Surrey, United Kingdom] and galacto-oligosaccharides [Friesland Foods Domo, Zwolle, Netherlands] 1:1; 600 mg/d in 1
dose from day 1 to day 30 and 600 mg twice daily from day
31 to day 60), probiotic (Lactobacillus rhamnosus GG
[ATCC 53103; Mead Johnson & Co, Evansville, Indiana]
109 colony-forming units/d in 1 dose from day 1 to day 30
and 109 colony-forming units twice daily from day 31 to 60
day), or placebo (microcrystalline cellulose and dextrose anhydrate [Chr. Hansen, Hoersholm, Denmark]). All study
products were prepared by the Turku University Hospital
Pharmacy. The products were identical in appearance, taste,

and smell and were stored in identical closed capsules. The

viability of the probiotic was confirmed by regular blinded
analysis in the Functional Foods Forum, Turku University.
Parents were taught by the study nurse to mix the products
immediately before administration to the infant with
10
mL of breast milk or formula given by spoon or bottle
once a day. The parents were instructed to inspect the product for any signs of damage before use and to keep it in a
refrigerator when not in use.
After the enrollment day, follow-up consultations were
conducted by the same study nurse at the ages of 1, 2, 4, 6,
and 12 months. The infants were clinically examined by a
physician (R.L. or A.P.) at the age of 12 months and otherwise as needed. During all study visits, parents reported the
infants behavior patterns, including sleeping patterns, fussing, crying, irritability, feeding, vomits, stools (consistency
[normal, loose, firm, hard, atypical] and frequency [1/wk,
2-3/wk, daily, 1-2/d, 2-3/d, >3/d], infection and other diseases, and medication use. An infant was classified as an
excessive crier if parents and study nurse reported excessive
crying and irritability (>3 h/d) causing clinical concern
without underlying medical causes during the 1- and 2month study visits and resolving by the age of 6 months.
The parents kept a structured diary on each childs infectious
disease symptoms and pharmacological treatment during the
follow-up year. Weight, height, and head circumference were
measured, and growth charts were drawn at each visit.
Adverse events were asked from the parents during all visits.
Fecal samples were collected from diapers after defecation at
the age of 1 month, immediately frozen to 20 C, and delivered to the study clinic within 24 hours.
Homogenized fecal samples were fixed overnight in 4%
paraformaldehyde and stored in phosphate-buffered saline:ethanol (1:1) at 20 C until analyzed (maximum 3
months). Fluorescence in situ hybridization (FISH) with a
flow cytometer was performed as previously described.7,8
Samples were hybridized at specific temperatures in hybridization buffer (containing 26 mM Tris-HCl, 1.2 M NaCl,
and 0.1% sodium dodecyl sulfate, pH 7.2) with specific
probes at a concentration of 5 ng/mL. After overnight
hybridization, samples were washed with buffer without
sodium dodecyl sulfate, pelleted, and resuspended in
phosphate-buffered saline. The EUB 338 probe was covalently linked at the 50 end with fluorescein isothiocyanate
(FITC) and other probes with carbocyanine 3 (Cy3). The sequences of the probes are presented in Table I (available at
www.jpeds.com). All oligonucleotides were purchased from
Thermo Electron Corporation (Thermo Bioscienses, Ulm,
Germany).
Data acquisition was performed with an LSR II flow cytometer equipped with an HTS 96-well-plate reader (Becton
Dickinson, San Jose, California). The 15-mV argon ion laser
(488 nm) was used to measure forward and side scatter (488
 10 nm)green fluorescent for FITC (530  30 nm) and
red fluorescent for Cy3 (575  26 nm); 40 mL of specimens
of each sample was collected in duplicate. Data were analyzed
with use of BD FACSDiva software (Becton Dickinson). The
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total amount of bacteria was determined with use of the EUB

338 FITC probe. Determination of specific bacteria was made
by combining each of the group-specific Cy3 probes with the
EUB 338 FITC probe and counting double-positive cells.9
For the quantitative polymerase chain reaction (PCR)
(qPCR) analysis, fecal samples were stored at 80 C until
analyzed. Samples were pretreated and DNA was extracted using an automated KingFisher DNA extraction system
(Thermo Fisher Scientific Oy, Vantaa, Finland) and InviMag
Stool DNA kit (Stratec Molecular, Berlin, Germany), as previously described.10 The standard DNA for qPCR was prepared
as previously described.10 All DNA samples were stored at
20 C until analyzed. qPCRs were conducted as previously
described.7,11 PCR amplification and detection were
performed with an ABI PRISM 7300-PCR sequence detection
system (Applied Biosystems, Foster City, California).
Statistical Analyses
Univariate associations between clinical characteristics and
the dichotomous outcome variable of excessive crying were
studied using logistic regression analysis. All continuous variables such as microbiota variables and stool frequency were
compared between the groups using Kruskal-Wallis test.
Associations between study group and categorical clinical
characteristics were studied using c2 test. All analyses were
performed on data from the intention-to-treat population.
P values <.05 were considered statistically significant.

Results
A total of 94 infants were eligible for inclusion and were equally
distributed between the intervention groups (Figure; available
at www.jpeds.com). All participants were white. The mean
(range) gestational age of the infants was 34.6 (32-36) weeks
and the mean (range) gestational weight was 2393 (15503965) g. Clinical characteristics were comparable among the
study groups (Table II). The 1-year study was completed by
68 of the 94 infants (72%) (Figure). At the age of 1 year, the

Table II. Clinical characteristic of the study subjects

Characteristic

Prebiotics
(n = 31)

Probiotics
(n = 31)

Placebo
(n = 32)

Male
16 (52)
19 (61)
23 (72)
Vaginal delivery
20 (65)
24 (77)
20 (63)
Apgar score at 5 min
8 (2)
8 (1)
8 (1)
Maternal peripartal antibiotic
6 (19)
10 (32)
10 (31)
treatment
Antibiotic treatment during the
20 (65)
18 (58)
17 (53)
first 2 mo of age
Exclusively breast-fed, mo*
1.3 (1.9)
1.6 (2.2)
1.9 (2.1)
7.2 (4.4)
5.9 (4.5)
Total duration of breast-feeding, 5.5 (3.3)

mo
9873 (1083) 9717 (1415) 10 107 (1633)
Weight at age of 1 y, gz
75 (2)
75 (3)
76 (3)
Length at age of 1 y, cmz
Results are given as mean (SD) or as number (%) of subjects.
All clinical characteristics were comparable among the study groups (P > .05).
*n = 24 for prebiotic group, n = 22 for probiotic group, and n = 25 for placebo group.
n = 23 for prebiotic group, n = 21 for probiotic group, and n = 23 for placebo group.
zn = 22 for prebiotic group, n = 21 for probiotic group, and n = 23 for placebo group.

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mean weight and height were comparable among the study
groups (Table II). There were no withdrawals attributable to
any adverse effects related to the study products or their
administration regimen reported by the parents (Figure).
During the first 2 months of life, 27 of the 94 infants (29%)
were classified as excessive criers, significantly less frequently
among the prebiotic and probiotic groups than in the placebo
group (19% vs 19% vs 47%, respectively; P = .02) (Table III).
The frequency of stools (>3 stools/d) tended to be higher in the
prebiotic than in the probiotic and placebo groups at the age of
1 month (71% vs 50% vs 57%, respectively; P = .08), but there
was no difference between the groups in consistency of stool.
The frequency of perinatal antibiotic treatment (42% vs 22%,
respectively; P = .07) and the number of vaginal deliveries
(81% vs 63%, respectively; P = .07) tended to be higher in
excessive criers than among contented infants. Other possible
factors influencing gut microbiota colonization, such as
postnatal treatment in a neonatal intensive care unit,
duration of breastfeeding, and antibiotic treatment during
the first 2 months of life, were comparable between excessive
criers and contented infants (Table III).
In analysis of the gut microbiota composition by use of
FISH, a significant difference between the study groups
was detected in the proportion of Clostridium histolyticumtype bacteria to total bacterial count. This proportion
was significantly lower in the probiotic group compared
with the prebiotic and the placebo group (8.9% vs 10.5%
vs 13.9%, respectively; P = .047). There were no statistically
significant differences in gut microbiota composition among
the study groups in qPCR analyses (Table IV; available at
www.jpeds.com).
Finally, we compared the gut microbiota composition between excessive criers and contented infants by FISH and
qPCR at the age of 1 month. The proportion of LactobacillusLactococcus-Enterococcus group to total bacterial count was

Table III. Clinical characteristics of contented infants

and excessive criers
Excessive
Contented
crier
(n = 67) (n = 27) P
Study group
.02
Prebiotic
25 (37)
6 (22)
Probiotic
25 (37)
6 (22)
Placebo
17 (25)
15 (56)
Male
38 (57)
20 (74) .16
Vaginal delivery
42 (63)
22 (81) .07
Gestational age at birth, wk
35 (0.1)
35 (0.3) .92
Birth weight, g
2380 (60) 2424 (76) .68
Birth length, cm
47 (0.3)
47 (0.5) .86
Apgar score at 5 min
8 (0.2)
9 (0.2) .29
Maternal perinatal antibiotic treatment
15 (22)
11 (41) .07
Postnatal treatment in NICU
52 (78)
18 (67) .28
Antibiotic treatment during the first 2 mo of life
41 (61)
14 (52) .40
Exclusively breast-fed over 2 mo*
20 (42)
5 (22) .09
39 (85)
15 (71) .21
Total duration of breast-feeding over 2 mo
NICU, neonatal intensive care unit.
Results are given as mean (SD) or as numbers (%) of subjects.
*n = 48 for contented infants and n = 23 for excessive criers.
n = 46 for contented infants and n = 21 for excessive criers.

rtty et al
Pa

ORIGINAL ARTICLES

November 2013

Table V. The mean (SD) proportion of different

bacterium count to total bacterial count by FISH at the
age of 1 month in content infants and excessive criers

Bifidobacterium
Bacteroides-Prevotella
Clostridium Histolyticum
Lactobacillus-Enterococcus
Akkermansia muciniphila

Contented
(n = 45)

Excessive crier
(n = 21)

P-value

23.03 (21.54)
11.85 (10.99)
10.36 (5.01)
10.51 (4.23)
9.39 (3.79)

23.46 (22.07)
10.60 (5.97)
13.23 (6.61)
14.52 (5.5)
10.33 (4.53)

.38
.58
.11
.005
.33

higher in excessive criers than in contented infants (14.5% vs

10.5%, respectively; P = .005) (Table V). Moreover, the
former tended to have an increased percentage of Clostridium
histolyticum in their stools compared with the latter group
(13.2% vs 10.4%, respectively; P = .11) (Table V). The species
composition of Bifidobacterium also differed between these 2
groups: the number of Bifidobacterium infantis by qPCR was
found to be decreased among excessive criers compared with
contented infants (1.3  107 vs 2.5  108, respectively; P =
.035). There were no other statistically significant differences
in gut microbiota composition between these two groups.

Discussion
During the first weeks of life, irritability and crying of a preterm infant coincide with diverse maturational processes that
take place in the immature gastrointestinal tract in response
to massive antigen challenges by microbial colonization and
food intake. Consequently, infant crying has been related to
cows milk allergy and deviating compositional development
of the gut microbiota,4,12,13 and the principal attempts to
control irritability in full-term infants have focused on
various dietary supplementations such as prebiotics and probiotics.4,14-16 In the present study, specific prebiotics and
probiotics, administered during the first 2 months of life to
infants born preterm, not only were well tolerated, also in
terms of normal growth, but also concomitantly provided relief to their crying and fussing. The clinical benefit was paralleled by a lowering of the relative number of pathogenic
bacteria, C histolyticum, in stools.
Our results support earlier demonstrations in term infants
on deviations in gut microbiota composition in colicky infants4,13,17,18-20 and clinical benefit from gut microbiota
modification with specific probiotics.4,14 We extend these
data in 3 respects: to infants born preterm, to specific microbial species, and to interventions with both probiotics and
prebiotics.
In contrast to these previous studies, we chose to use questionnaires and interviews rather than cry diaries to identify
those infants who cry excessively. Because our participant
families were encountering extra stress due to the prematurity of their infant, we selected the former methods in the
hope of enhancing adherence to the study regimen. Indeed,
we consider our classification of infants into contented and
excessive criers reliable, because both parents and our

experienced study nurse, blinded to the randomization and

in close contact with the families from the outset and
throughout the follow-up period, participated in the process.
In the same vein, we found that irritable preterm infants had
higher numbers of Lactobacillus-Lactococcus-Enterococcus
group than did the contented infants. Increased numbers of
both Enterococcus and Lactobacillus, 2 of the first families
colonizing the intestine, have been related to prematurity
per se, thus suggesting that the preterm infant irritability
found here reflects a less mature gut microbiota colonization
process compared with contented infants.21 Our molecular
assays did not allow characterization of the Lactobacillus-Lactococcus-Enterococcus group of bacteria in more detail, to
specify whether the mentioned difference was related to specific lactobacilli, enterococci, or both. Future research is thus
needed to clarify this issue.
In the case of Bifidobacterium microbiota, typifying the microbiota of the healthy, term breast-fed infant, we have previously shown that different species may exert distinct effects
on infant crying.20 Our present findings suggest that delayed
colonization by B infantis is linked to the risk of irritability in
preterm infants. This species has been found to secrete bioactive factors capable of normalizing permeability defects in the
intestinal mucosa.22,23 Early B infantis colonization has also
been associated with appropriate development of immune
tolerance.22,24 Furthermore, according to 1 recent probiotic
study, supplementation of B infantis in untreated celiac patients significantly alleviated gastrointestinal symptoms,
including constipation, indigestion, and reflux.25 In light of
the aforesaid and our findings here, we would consider a
decreased number of B infantis and further delayed gut microbiota colonization combined with immature gut barrier
function to be potential risk factors for the development of
crying and irritability in preterm infants.
In this preterm population, we demonstrated a relative
abundance of C histolyticum in the placebo group, suggesting
that L rhamnosus GG promotes gut health through a reduction
of C histolyticum colonization via a variety of mechanisms
such as competitive exclusion.26,27 Beyond the incompletely
developed immune exclusion functions in preterm infants, it
is suggested that high numbers of Clostridia spp might be associated with degradation of antigen-specific immunoglobulin A
in the gut, which further impairs the immature gut barrier
function.28,29 Furthermore, C histolyticum has been shown
to evoke proinflammatory cytokines such as tumor necrosis
factor-a, which might already be overexpressed in antigen
challenges in preterm infants.30,31 Thus, imbalance between
proinflammatory and anti-inflammatory cytokines might be
one of the mechanisms behind infant crying, the symptom
associated with increased amounts of calprotectin, a gut inflammatory marker.13,31 Insufficient gut barrier function and
aberrant immune responses, typical features of premature infants, could lead to an increased risk of oral tolerance failure
and further excessive irritability and crying.
Notwithstanding differences in gut microbiota in the probiotic and prebiotic groups, the clinical outcomes appeared
similar. Earlier studies using supplementation of formula

Effects of Early Prebiotic and Probiotic Supplementation on Development of Gut Microbiota and Fussing and
Crying in Preterm Infants: A Randomized, Double-Blind, Placebo-Controlled Trial

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with polydextrose/galacto-oligosaccharide have been demonstrated to lessen abdominal discomfort, soften stools, and increase defecation frequency compared with placebo11,32-34
but not fussiness or gassiness in term infants.15,16 In our
study, stool frequency tended to be higher in the prebiotic
than in the probiotic or placebo groups, although the consistency of stools was comparable between the groups. Frequent
defecation may lessen abdominal distention and flatulence,
and thus constitute one of the possible reasons why the prebiotic intervention reduced infant irritability in our study.
In conclusion, breast-feeding is the first choice of nutrition
for all infants, especially preterm, because it constitutes a key
factor in the metabolic, immunological, and microbiological
programming of the infants health. However, the composition
of breast milk varies and reflects maternal health status and diet
and, consequently, influences the compositional development
of the gut microbiota in the offspring.2 The results presented
here suggest that redirecting the deviating colonization process
in the preterm infant gut microbiota by prebiotic and probiotic
supplementation may offer a safe and well-tolerated means to
optimize preterm infant well-being and to facilitate the
development of novel preventive and therapeutic options
against infant irritability and excessive crying. n
We would like to thank the families participating in our study, Jaakko
Matomaki, MSc, for statistical consultation, Robert MacGilleon, MA,
for language review of the manuscript, and Ulla-Maija Eriksson, RN,
and Anne Yrjana, RN, for help with the follow-up of participants.
Submitted for publication Mar 4, 2013; last revision received Apr 15, 2013;
accepted May 14, 2013.

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23. Chichlowski M, De Lartigue G, German JB, Raybould HE, Mills DA. Bifidobacteria isolated from infants and cultured on human milk oligosaccharides affect intestinal epithelial function. J Pediatr Gastroenterol Nutr
2012;55:321-7.
24. Sudo N, Sawamura S, Tanaka K, Aiba Y, Kubo C, Koga Y. The requirement of intestinal bacterial flora for the development of an IgE production system fully susceptible to oral tolerance induction. J Immunol
1997;159:1739-45.
25. Smecuol E, Hwang HJ, Sugai E, Corso L, Chernavsky AC, Bellavite FP,
et al. Exploratory, randomized, double-blind, placebo-controlled study
on the effects of Bifidobacterium infantis Natren Life Start Strain Super
Strain in active celiac disease. J Clin Gastroenterol 2013;47:139-47.
26. Nylund L, Satokari R, Nikkila J, Rajilic-Stojanovic M, Kalliomaki M,
Isolauri E, et al. Microarray analysis reveals marked intestinal microbiota
aberrancy in infants having eczema compared to healthy children in atrisk for atopic disease. BMC Microbiol 2013;13:12.
27. Ji YS, Kim HN, Park HJ, Lee JE, Yeo SY, Yang JS, et al. Modulation of the
murine microbiome with a concomitant anti-obesity effect by Lactobacillus rhamnosus GG and Lactobacillus sakei NR28. Benef Microbes
2012;3:13-22.
28. Kobayashi K, Fujiyama Y, Hagiwara K, Kondoh H. Resistance of normal
serum IgA and secretory IgA to bacterial IgA proteases: evidence for the
presence of enzyme-neutralizing antibodies in both serum and secretory
IgA, and also in serum IgG. Microbiol Immunol 1987;31:1097-106.

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29. Bakker-Zierikzee AM, Tol EA, Kroes H, Alles MS, Kok FJ, Bindels JG.
Faecal SIgA secretion in infants fed on pre- or probiotic infant formula.
Pediatr Allergy Immunol 2006;17:134-40.
30. Claud EC, Walker WA. Hypothesis: inappropriate colonization of the
premature intestine can cause neonatal necrotizing enterocolitis. FASEB
J 2001;15:1398-403.
31. Tuovinen E, Keto J, Nikkila J, Matto J, Lahteenmaki K. Cytokine
response of human mononuclear cells induced by intestinal Clostridium
species. Anaerobe 2013;19:70-6.
32. Costabile A, Fava F, Roytio H, Forssten SD, Olli K, Klievink J, et al.
Impact of polydextrose on the faecal microbiota: a double-blind, cross-

over, placebo-controlled feeding study in healthy human subjects. Br J

Nutr 2012;108:471-81.
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blends of prebiotics grow normally and have soft stools similar to those
reported for breast-fed infants. J Pediatr Gastroenterol Nutr 2007;44:
359-64.
34. Ribeiro TC, Costa-Ribeiro H Jr, Almeida PS, Pontes MV, Leite ME,
Filadelfo LR, et al. Stool pattern changes in toddlers consuming a
follow-on formula supplemented with polydextrose and galactooligosaccharides. J Pediatr Gastroenterol Nutr 2012;54:288-90.

50 Years Ago in THE JOURNAL OF PEDIATRICS

The Nature and Origin of Fluid in the Fetal Lamb Lung
Adams FH, Fujiwara T, Rowshan G. J Pediatr 1963;63:881-8

ifty years ago, it must have been such an exciting time for researchers in the field of lung biology! Surfactant, a novel
surface active material in the lung, had just been discovered. Avery and Meads observation1 that preterm infants
afflicted with hyaline membrane disease lacked surfactant had spurred excitement around a potential remedy for this
deadly disease. Yet, vexing questions remained: Where is surfactant made? How does it reach amniotic fluid? What
kind of fluid fills fetal lungs, and where does it come from?
Fifty years ago, Adams et al also made the astute observation that certain characteristics of fetal lung fluid point to an
active secretory process. They directed our attention to its low pH, low protein, low bicarbonate, and high chloride
content; they also used the Gibbs-Donnan equation to make a strong case for an active secretory process, pointing
to a big disparity in sodium and chloride ratios between the tracheal fluid and plasma. Years later, a Na-K-2Cl cotransport mechanism was found to be the primary source of fetal lung fluid secretion, a process that is readily suppressed by
diuretics such as bumetanide.
Yet, the question of how the fetus reverses this process at birth and clears a large amount of lung fluid had to wait
until 1994 for a definitive answer, when Hummer et al2 showed that mice pups lacking a key epithelial sodium channel
(ENaC) died within the first day or 2 of life, all from an inability to clear lung fluid. The subsequent cloning of ENaC
allowed studies of its regulation by key endogenous hormones such as catecholamines and glucocorticoids. We now
have a better understanding of how the alveolar surface fluid layer is regulated, balancing secretory forces (to which
Adams et al eluded 50 years ago) and fluid absorption by ENaC and its related ion channels. Therapeutic interventions
based on translation of this knowledge are currently in use or under investigation for conditions such as high-altitude
pulmonary edema, cystic fibrosis, and respiratory distress in the newborn. The calf lung extract first introduced by
Dr Fujiwara as a surfactant is still in use today. We have come a long way!
Lucky Jain, MD

Richard Blumberg Professor

Department of Pediatrics
Emory University School of Medicine
Atlanta, Georgia
http://dx.doi.org/10.1016/j.jpeds.2013.04.007

References
1. Avery ME, Mead J. Surface properties in relation to atelectasis and hyaline membrane disease. AMA J Dis Child 1959;97:517.
2. Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, et al. Early death due to defective neonatal lung liquid clearance in
alpha-ENaC-deficient mice. Nat Genet 1996;12:325-8.

Effects of Early Prebiotic and Probiotic Supplementation on Development of Gut Microbiota and Fussing and
Crying in Preterm Infants: A Randomized, Double-Blind, Placebo-Controlled Trial

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www.jpeds.com

Vol. 163, No. 5

Table I. Sequences of the probes used in FISH9

Probe

Target

Sequence from 50 to 30

Hybridization temperature

EUB 338
Bif 164
Bac 303
Chis 150
Lab 158
Muc 1437

Total bacteria
Bifidobacterium
Bacteroides-Prevotella
Clostridium histolyticum
Lactobacillus-Enterococcus
Akkermancia muciniphila

GCT GCC TCC CGT AGG AGT

CAT CCG GCA TTA CCA CCC
CCA ATG TGG GGG ACC TT
TTA TGC GGT ATT AAT CTY CCT TT
GGT ATT AGC AYC TGT TTC CA
CCT TGC GGT TGG CTT CAG AT

50 C
50 C
45 C
50 C
45 C
50 C

Y represents a (C/T) wobble nucleotide.

Figure. Flow chart of the procedure (N = 94).

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Table IV. Proportion of different bacterium count to total bacterial count by FISH at the age of 1 month in placebo,
probiotic, and prebiotic groups
Bacterium

Prebiotic (n = 25)

Probiotic (n = 19)

Placebo (n = 22)

Bifidobacterium
Bacteroides-Prevotella
C histolyticum
Lactobacillus-Enterococcus
Akkermansia muciniphila

18.26 (18.24)
14.46 (13.87)
10.46 (5.15)
11.67 (5.51)
9.73 (3.86)

30.56 (26.76)
9.75 (5.96)
8.90 (2.92)
10.83 (4.48)
8.66 (2.80)

22.78 (19.50)
9.50 (4.89)
13.88 (6.81)
12.37 (4.74)
10.41 (4.90)

.17
.59
.047
.64
.61

Results are given as mean (SD).

Effects of Early Prebiotic and Probiotic Supplementation on Development of Gut Microbiota and Fussing and
Crying in Preterm Infants: A Randomized, Double-Blind, Placebo-Controlled Trial

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