Wageningen Academic

Beneficial Microbes, 2019; 10(3): 253-263 P u b l i s h e r s

Prebiotic supplementation over a cold season and during antibiotic treatment

specifically modulates the gut microbiota composition of 3-6 year-old children

S. Soldi1, S. Vasileiadis2, S. Lohner3, F. Uggeri1, E. Puglisi4, P. Molinari4, E. Donner5, C. Sieland6, T. Decsi3, M. Sailer6
https://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0116 - Tuesday, January 21, 2020 11:39:54 AM - IP Address:148.204.6.250

and S. Theis6*

1AAT – Advanced Analytical Technologies Srl, Via P. Majavacca 12, 29017 Fiorenzuola d’Arda, Italy; 2Department of
Biochemistry & Biotechnology, University of Thessaly, Viopolis, 41500 Larissa, Greece; 3Department of Paediatrics, Clinical
Center of the University of Pécs, Medical School, University of Pécs, József Attila u. 7, 7623 Pécs, Hungary; 4Microbiology
Institute, Università Cattolica del Sacro Cuore in Piacenza, Via Emilia Parmense 84, 29122 Piacenza, Italy; 5Future
Industries Institute (FII), Mawson Lakes Campus, University of South Australia, 5095 Mawson Lakes, Australia; 6Beneo-
Institute, c/o Beneo GmbH, Wormser Straβe 11, 67283 Obrigheim, Germany; stephan.theis@beneo.com

Received: 24 September 2018 / Accepted: 21 November 2018

© 2019 Wageningen Academic Publishers

OPEN ACCESS RESEARCH ARTICLE

Abstract

Supplementing kindergarten children during a cold season with a prebiotic inulin-type fructans product with shorter
and longer fructan chains has been shown to reduce febrile episodes requiring medical attention and to lower
the incidence of sinusitis. These beneficial effects may be connected to the specific modulation of children’s gut
microbiota. By applying quantitative and qualitative microbiota analysis this study aimed at characterising the gut
microbiota composition and at exploring effects of prebiotic intervention on the gut microbiota during a 24-weeks
intervention and during antibiotic treatment in healthy children. The study was a randomised, placebo-controlled
trial with 258 healthy children aged 3 to 6 years consuming 6 g/day prebiotic inulin-type fructans or maltodextrin.
During the course of the study, faecal samples were collected and subject to targeted qPCR analysis and phylogenetic
profiling by multiplexed high throughput sequencing of the prokaryotic 16S rRNA gene PCR amplicons. The
microbiota composition of the cohort could be clustered into three distinct constellations (enterotypes). Prebiotic
intake resulted in a selective modulation of the gut microbiota composition. Relative abundance of Bifidobacterium
was significantly higher in the prebiotic group (n=104) compared to control group (n=105) and this effect was found
for all three enterotypes. Antibiotic administration decreased the relative abundance of Bifidobacterium in both
groups. Nonetheless, children of the prebiotic group receiving antibiotic treatment displayed significantly higher
levels of Bifidobacterium than children receiving the placebo control. Prebiotic supplementation induced specific
changes in the gut microbiota composition of children aged 3 to 6 years. Moreover, it attenuated antibiotic-induced
disturbances in the gut microbiota composition as shown by higher relative abundance of bifidobacteria at the end of
the antibiotic treatment in the prebiotic group. With the previously reported benefits on immune function, the study
contributes to the evidence on the immune-modulating effects of prebiotics through gut microbiota modifications.
The study was registered as NCT03241355 (https://clinicaltrials.gov/show/NCT03241355).

Keywords: fructans, children, gut microbiota, bifidobacteria

1. Introduction international expert panel recently defined a prebiotic in

their consensus statement as ‘a substrate that is selectively
Prebiotics are dietary ingredients that target human- utilised by host microorganisms conferring a health benefit’.
associated microbiota with the goal of improving health Recognised health effects of prebiotics include benefits to
and reducing risk of diseases (Gibson et al., 2017). An the gastrointestinal tract such as inhibition of pathogens

ISSN 1876-2883 print, ISSN 1876-2891 online, DOI 10.3920/BM2018.0116253

 S. Soldi et al.
and immune stimulation (Gibson et al., 2017; Lohner and
Decsi, 2014; Lohner et al., 2018; Moro and Boehm, 2012;
Roberfroid et al., 2010).
The role of prebiotics has been extensively investigated in
the field of infant nutrition (Boehm et al., 2004; Thomas
and Greer, 2010). Prebiotics mimic the beneficial functional
properties of human milk oligosaccharides with respect to
modulation of the gut microbiota. An immune-modulating
https://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0116 - Tuesday, January 21, 2020 11:39:54 AM - IP Address:148.204.6.250
effect of prebiotics through gut microbiota modifications
has been considered as principal mechanism for the
observed infection prevention early in life (Arslanoglu et
al., 2007). Corresponding data in children above 3 years of
age are more limited (Lohner et al., 2014). Therefore, we
recently conducted a randomised placebo-controlled trial
in 270 kindergarten children aged 3-6 years. We found that
daily intake of an inulin-type fructans product with shorter
and longer fructan chains reduces febrile episodes requiring
medical attention and lowers the incidence of sinusitis. This
finding might arise from a stimulated immune function
in prebiotic supplemented children and was connected
to a specific modulation of the gut microbiota (Lohner
et al., 2018).
It seems plausible that also children aged 3 to 6 years
benefit from prebiotic consumption and corresponding
modulation of the gut microbiota bearing the following
in mind: (1) it is well established that the gut microbiota
composition is important for the development of the
immune system (Martin et al., 2010). The immune system
is not fully established until about 10 to 15 years of age
(Martin et al., 2010; Weizman, 2015). Similarly, recent
data show that the gut microbiota continues to develop
until the teenage years (Agans et al., 2011; Cheng et al.,
2016; Hollister et al., 2015; Ringel-Kulka et al., 2013). (2)
the age group of 3-6 year old children frequently attends
day-care centres. This is associated with a 3-fold increased
risk to suffer from gastrointestinal or respiratory infections
(Weizman, 2015). (3) antibiotic therapies remain common
in this paediatric age group, too, and there is interest in
strategies to ameliorate or prevent negative repercussions
of antibiotic treatment on the gut microbiota composition,
and potential health implications linked to such a dysbiosis
also in the long run (Korpela and Vos, 2016; Shao et al.,
2017; Sturkenboom Miriam et al., 2008; Turta and Rautava,
2016; Vaz et al., 2014).
In order to further explore the potential benefits of prebiotic
interventions in this age group, there is interest to better
understand the effects on the gut microbiota composition.
Such data is currently scarce, as is data in general on the
gut microbiota of kindergarten children.
The aim of the current paper was to investigate the basal
microbiome composition and to examine the effects of
254
prebiotic inulin-type fructans supplementation on the
composition of the gut microbiota in healthy kindergarten
children aged 3 to 6 years. Moreover, the efficacy of
prebiotic inulin-type fructans supplementation in
preventing expected disturbances of faecal gut microbiota
composition during antibiotic treatment was determined
within a randomised, placebo-controlled trial (Lohner et al.,
2018) using advanced technologies of Illumina sequencing
together with targeted qPCR analysis.
2. Materials and methods
Study design
For an extensive overview of study design and procedures,
we refer to Lohner et al. (2018). In brief, the study was
designed as a randomised, placebo-controlled, double-
blind trial (Clinical Trials.gov ID: NCT03241355) and
aimed at exploring whether prophylactic dietary prebiotic
supplementation is able to influence the intestinal
microbiota and the frequency of infectious disease episodes
in kindergarten children during winter period. Healthy
children (3-6 years) were allocated to consume either 6 g/
day of an Orafti® inulin-type fructans product with shorter
and longer fructan chains (degree of polymerisation (DP)
≥11 approx. 25-30% (on g/100 g dry matter), average DP of
about 7 to 8) or maltodextrin as placebo for a period of 24
weeks. Children were considered eligible for the study when
fulfilling the following criteria: (1) over 3 years but under
7 years of age at the time of pre-examination, (2) healthy
at the time of inclusion, and (3) attending a kindergarten.
Faecal samples
Approximately 2 g of faeces were collected for microbiota
evaluation at baseline and week 24. In case of an antibiotic
treatment, additional stool samples were collected on the
1st day of antibiotic treatment and on the 7th and 14th day
after starting it. At home, samples were refrigerated at
4 °C and were collected within 24 h by the study personnel
and transported to the study centre for long-term storage
at -80 °C.
Bacterial community DNA extraction
Faecal bacterial DNA was extracted using the FastDNA
SPIN Kit and FastPrep Instrument (MP Biomedicals, Santa
Ana, CA, USA). The extracted DNA was quantified using
the picogreen method of the Quant-iT™ HS ds-DNA assay
kit in a Qubit™ fluorometer (Invitrogen, Carlsbad, CA, USA)
and verified. DNA extracts were diluted to a concentration 5
ng/µl for reducing template amount associated PCR biases.
Beneficial Microbes 10(3)
  Effect of prebiotics on gut microbiota composition of children
Quantitative real-time PCR amplification
All faecal samples were subject to targeted qPCR analysis.
As described in Lohner et al. (2018), qPCR reagents were
freshly prepared for each qPCR analytical session, according
to the number of samples in duplicates, including also a
positive control. The final qPCR reaction volume was 25
μl. The amplification mixtures were prepared using the
RealMasterMix Sybr Rox 2.5X (5 PRIME, Fisher Scientific,
https://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0116 - Tuesday, January 21, 2020 11:39:54 AM - IP Address:148.204.6.250
Hampton, NH, USA) (Qin et al., 2010), according to
manufacturer’s instructions. Different protocols and primer
pairs were applied for the detection of Bifidobacterium,
Lactobacillus, Clostridium perfringens, Clostridium
difficile and Enterobacteriaceae as previously documented
(Huijsdens et al., 2002; Malinen et al., 2005; Mutters et al.,
2009; Wise and Siragusa, 2005). Thermal cycling protocols
were performed as provided in the cited publications.
Multiplexed high throughput sequencing of the
prokaryotic 16S rRNA gene PCR amplicons
Baseline faecal samples and samples collected during
antibiotic treatment phases were assessed by microbiota
phylogenetic profiling. PCR amplicons of the V3-4
hypervariable region of the bacterial 16S rRNA gene were
screened using an Illumina platform multiplex approach.
Two nested PCR cycles were used to obtain the fragments
for sequencing: amplification, quantification and pooling
were published by Soldi et al. 2015 and indexed primers
are reported in the Supplementary Table S1. Details about
primer and linker design were previously published by
Vasileiadis et al. (2015).
The PCR amplicon pool was then purified using the SPRI
(solid phase reversible immobilisation) based method of
the Agencourt® AMPure® XP kit (Beckman Coulter, Milan,
Italy). Illumina sequencing with the V3 chemistry and
library preparation of the amplicons was performed by
Fasteris SA (Geneva, Switzerland).
Sequence data preparation
A total of 24,799,362 high quality paired-end sequence
reads (300 bp each read) of PCR amplicons was obtained
with the Illumina MiSeq platform (San Diego, CA, USA),
performed by Fasteris SA. Sequence cluster identification
on the Illumina MiSeq flow-cell and base calling of the
sequence reads were performed with the MiSeq Control
software v2.4.1.3, the RTA v1.18.54.0 software (Illumina)
and CASAVA v1.8.2 (Illumina). The sequencing data were
quality controlled for sequencing artefacts and experimental
biases, such as amplification of non-targeted taxa (archaea
and chloroplasts) and generation of chimeric sequences
during PCR amplification by bioinformatic elaboration
resulting in a final number of 22,741,948 reconstructed
amplicon sequences which were used in the analysis.
Beneficial Microbes 10(3)
Further details of quality control procedure are provided
in the supplementary materials.
The LotuS v1.461 (Hildebrand et al., 2014) pipeline was
used for the generation of the 0.03 sequence distance
operational taxonomic units (OTUs) using the combination
of the USEARCH v7.0.1090 (Edgar, 2004) as implemented
in the LotuS pipeline. The Naïve Bayesian classifier (Wang
et al., 2007) with a 0.8 bootstrap cut-off value was used
for classifying sequences into taxa using the SILVA v115
sequence database (https://www.arb-silva.de) as reference.
Achieved classification depth along with the sequence
numbers per taxon level show that about 85% of the
sequences (19,338,291) were classified at the genus level.
Statistics
Data were normalised via Box-Cox transformation (power
transformed to obtain normal value distributions per
analysed marker) as described in Osborne (2010) and
expressed as ratio of taxon per total bacteria. Data are
displayed as mean ± standard deviation. The statistical
analysis was performed using the R software v3.3.1 (R Core
Team, 2017) and the packages Vegan v2.0-10 (Dixon, 2003)
for the multivariate analyses, the MetagenomeSeq v1.6.0
(Paulson et al., 2013) implementation of the linear model
fitting of the Limma v3.20.9 (Smyth, 2005) R package for
the performance of pairwise comparisons. Enterotyping was
performed as previously described (Arumugam et al., 2011).
In order to identify the core microbiomes among the three
putative enterotypes the Corbata v20130611 suite of R
scripts (Li et al., 2013) was used. Cut-off values of min 1%
for the relative abundance and 80% for the ubiquity were
applied. The core taxa were determined with bootstrapping
by sampling with replacement of both the number of
samples used in the analysis and the distribution of taxa
in each sample for an alpha value of 0.05. In all bar plot
cases the error bars represent the standard error.
3. Results
209 children of the per protocol population (Lohner et
al., 2018) provided a full faecal sample set and were thus
considered for the qPCR and sequencing analysis. The
basic characterisation of the microbiota composition by
phylogenetic profiling was performed with samples of all
children who provided a faecal sample at baseline (n=233).
Basic characterisation of the microbiota composition
Basic information on the gut microbiota composition of 3
to 6 years old children was obtained from the sequencing
data of faecal samples collected from 233 children at the
start of the study. This baseline characterisation included
255
 S. Soldi et al.

the subject classification according to putative enterotypes
and their corresponding key microbial taxa.
As reported previously for adults (Arumugam et al., 2011),
three distinct enterotypes (namely Enterotype 1, Enterotype
2 and Enterotype 3) could be identified in the present cohort
of 3 to 6 years old healthy children (Figure 1). Most of the
faecal samples (n=100) were allocated to Enterotype 2,
while Enterotype 1 was represented by 62 samples and
https://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0116 - Tuesday, January 21, 2020 11:39:54 AM - IP Address:148.204.6.250
Bacteroides representing almost 25% of the taxa. Enterotype
3 was also characterised by the absence of Bifidobacterium
from the core microbiome and the Bifidobacterium levels
(1.8%) were significantly lower compared to Enterotype 1
(5.6%) and 2 (5.7%) (Supplementary Figure S1).
Prebiotic intake stimulates the growth of bifidobacteria in
kindergarten children

Enterotype 3 by 71 samples. The specific effects of regular prebiotic intake on the

Bacteroides, Anaerostipes, Alistipes, Roseburia and
Ruminococcaceae were among the dominant taxa
shared by all enterotypes but with different amounts
specific for each enterotype. Samples of Enterotype 1
had a significantly higher percentage (14.0 and 6.2%) of
Roseburia and Lachnospiracea incertae sedis compared
to Enterotype 2 (5.2 and 3.6%) and 3 (9.4 and 3.2%), while
the percentage of Alistipes was lowest (4.5%). Alistipes
(15.0%) and Ruminococcaceae (13.1%) were representative
for Enterotype 2 with significantly higher levels compared to
Enterotype 1 and 3, while the percentage of Roseburia (5.2%)
and Faecalibacterium (2.4%) levels were lowest. The core
microbiota of Enterotype 3 was found to be dominated by
microbiota composition in 3 to 6 years old children during
the 6 months intervention period was assessed by using a
targeted qPCR approach, as reported previously (Lohner
et al., 2018). Intake of the prebiotic fructans resulted in a
significantly higher relative abundance of bifidobacteria at
week 24 compared to children receiving the placebo control
(P=1.228×10-05). Likewise, children of the prebiotic group
had a significantly higher relative abundance of lactobacilli
as compared to children of the placebo group at week 24
(P=0.014). There was no difference between the groups for
total bacteria, or the relative abundance of C. perfringens, C.
difficile and Enterobacteriaceae. Faecal pH values tended to
be lower in the prebiotic group compared with the placebo
group (P=0.11) (Lohner et al., 2018).

Enterotype 1 Enterotype 2 Enterotype 3

unclass. Bacteria Clostridiales Clostridium IV

Bifidobacterium Anaerostipes Faecalibacterium
Bacteroides Blautia Ruminococcus
Parabacteroides Lachnospiracea IS Escherichia Shigella
Alistipes Roseburia Akkermansia
Streptococcus Ruminococcaceae other 1

x var. 18.5% , y var. 16.3%

Figure 1. Baseline characterisation and enterotyping of the gut microbiota of children as shown at the streamplot (upper panel)
and the Principal Coordinates Analysis (PCoA) plots (lower right panel)..

256 Beneficial Microbes 10(3)

  Effect of prebiotics on gut microbiota composition of children

Prebiotic intake stabilises the gut microbiota composition
during antibiotic treatment
The pronounced and selective effect of regular consumption
of prebiotic on bifidobacteria were also observed during
antibiotic administration as confirmed by qualitative and
quantitative analysis with qPCR and high-throughput
sequencing.
https://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0116 - Tuesday, January 21, 2020 11:39:54 AM - IP Address:148.204.6.250
Total bacteria remained unchanged over the course of the
study in both groups (Supplementary Figure S2). Relative
abundance of bifidobacteria in children of the placebo
group remained significantly reduced compared to baseline
conditions also 14 days after starting antibiotic intake and
even at the end of the 6 months intervention period. In
children receiving the prebiotic, the relative abundance of
bifidobacteria was significantly higher compared to placebo
control already at the onset of antibiotic treatment and

Children of both groups, i.e. those allocated to the prebiotic
or the placebo control did not differ significantly at baseline
with respect to relative abundance of individual bacterial
taxa. In contrast, during antibiotic treatment differences
were observed. Children of the prebiotic group receiving
antibiotic treatment displayed significantly higher levels of
Bifidobacterium than children receiving the placebo control.
In addition, relative abundance of Roseburia and Blautia
genera significantly differed with higher levels in antibiotic
treated children receiving the placebo control (Figure 2).
As illustrated in Figure 3 showing qPCR data, antibiotic
treatment significantly reduced the relative abundance of
bifidobacteria in children of the placebo, yet not of the
prebiotic group.
remained significantly higher at each sampling time point
during antibiotic treatment as well as at week 24 (Figure 4).
Consistent with that, sequencing data reveal that
bifidobacteria are more abundant in the prebiotic group at
the onset of antibiotic treatment (11.8% relative abundance)
compared to the placebo group (4.2% relative abundance)
(Figure 5).
Antibiotic treatment led in both groups to decreased relative
Bifidobacterium abundance (placebo group: from 4.2 to
2.1%; prebiotic group: from 11.8 to 5.5%). Nonetheless,
the relative abundance of Bifidobacterium in the prebiotic
group after antibiotic treatment was even higher than that
appearing in the placebo group at the onset of antibiotic

A B
other other
unclassified Bacteria unclassified Bacteria
Streptococcus Streptococcus
prebiotic
Clostridium IV Clostridium IV
placebo
Clostridiales Clostridiales
Parabacteroides Ruminococcus
Akkermansia Lachnospiracea IS
Ruminococcus Akkermansia
Escherichia Shigella Parabacteroides
Faecalibacterium Faecalibacterium
Blautia Escherichia Shigella
Lachnospiracea IS Bifidobacterium*
Bifidobacterium Roseburia*
Roseburia Blautia*
Ruminococcaceae Ruminococcaceae
Alistipes Alistipes
Anaerostipes Bacteroides
Bacteroides Anaerostipes

0 4 8 12 0 4 8 12 16
% %

Figure 2: Barplot showing differences in microbiota composition of children at baseline (A) and during antibiotic treatment (B)
for prebiotic and placebo.

Beneficial Microbes 10(3) 257

 S. Soldi et al.

A Product A samples. P same 0

●
0.10 ●
●
●●●
● ●
0.08 ●
● ●
●●● ● ●
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a ●
● ●●● ●● ●
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0.06 ●
−−−−−−−− a ●
0.0594 ●●●
●●● ●
●●●●
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0.056−−−−−−−− ● ● ●
0.0554 ●●●
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https://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0116 - Tuesday, January 21, 2020 11:39:54 AM - IP Address:148.204.6.250

● ●●
0.0546
●●●●● ●●
●● b
●
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0.04 ●●
● ●●● ●●●● b ●●● ●
−−−−−−−−
●
b
●●●●
●●●
Normalised bifidobacterial/total-bact. 16S rRNA gene count values

−−−−−−−− 0.0403 −−−−−−−−

0.0388 ●
● 0.0367 ●● ●
● 0.0443 0.0425
0.0419 ● ● ●
0.02 ●● ●
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B Product B samples. P same 0.017

●

0.12
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0.08 ● a ●●
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−−−−−−−− ●
●● ●
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● 0.0739 ●
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● ●
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ab ●●● 0.0742 ● ab ● ●●●●
●
●
●●●●
−−−−−−−− ● ●● ab ● ●●●●●
0.0582 ●●● ●
● ●●●●
0.057−−−−−−−−
●● 0.0552 ●●●
−−−−−−−− b ●
●
−−−−−−−−
●
0.0576
●● 0.0578 0.07 ● 0.05 ●●●●●●
0.04 ●● ●
●
● 0.0539
●
●
● ●
●
● ● ●●● ●●
●●
0.00 ● ●● ●●
baseline 1st 7th 14th exp. end
mean
median Ant. treatment day

Figure 3. Relative abundance of bifidobacteria over the course of the study. (A) Placebo group; (B) prebiotic group.

treatment or study start. This effect of prebiotic fructans 4. Discussion

on the gut microbiota composition occurred irrespective
of the enterotype (Supplementary Figure S3). In all three To the best of our knowledge, this is the first study
enterotypes, bifidobacteria levels during and without conducted in the age group of 3 to 6 years old children
antibiotic treatment were higher in children receiving that characterised in depth the gut microbiota composition
the prebiotic than in placebo children (Supplementary combined with investigating the effect of inulin-type
Figure S3). This is particularly remarkable for Enterotype fructans consumption and antibiotic treatment. The study
3, in which the abundance of Bifidobacterium at baseline demonstrates that regular consumption of an inulin-type
was significantly lower compared to Enterotype 1 and 2 fructans product results in a selective modulation of the gut
and bifidobacteria were found to be absent from the core microbiota composition and a pronounced and sustained
microbiome. These results supported the positive influence bifidogenic effect in children aged 3 to 6 years. Children
exerted by the administered prebiotic regardless of the receiving the prebiotic displayed higher relative abundance
different conditions at baseline. No additional significant of bifidobacteria compared to children in the placebo
differences were observed for other bacterial taxa. control group, an effect confirmed by both qPCR and high-

258 Beneficial Microbes 10(3)

  Effect of prebiotics on gut microbiota composition of children

Baseline samples Ant. treatment day 1 Ant. treatment day 7

P same 0.829 P same 0 P same 0.002
● 0.12 ● ●
●
0.08 ●
● 0.08
●
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0.06 ●
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● ● ●●●● ●
Normalised ratio Bifidob/total bact. 16S rRNA gene copies

●
● ●
● ●●●● ● ●●●
●
−−−−−−−−
● ●●● ●●● ●
●●●●● ●●●●● ●●●●●●●
−−−−−−−−
●
0.0507 ● ●
●●●
● −−−−−−−−
●
0.0497 ● ● ●
● ● ●●●
−−−−−−−−
● ●
●●● 0.0468
● ● 0.0605 ●● ● 0.04 ● ● 0.0472 ● ●●
0.04 0.0474 ● ● ●
0.0492 ●●● ●●
●●
●●●●●●
●●●●●●● ● ●●● ●
●●
●●●● ●
●
●●●
●●● ●
● −−−−−−−− 0.0624 ● ● −−−−−−−−
● 0.04 0.0415 ●●●●●●● ● 0.0311 ● ●
● ●●●●● ● 0.0343 ● ● ● ●
https://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0116 - Tuesday, January 21, 2020 11:39:54 AM - IP Address:148.204.6.250

0.0433 ● ● 0.02 ●
0.02 ●●● ●
●
●●
● ● ●●
●● ● ●
●
●
●● ●● ●●
●●
●●
●●●
0.00 ● 0.00 ●
Placebo Prebiotic Placebo Prebiotic Placebo Prebiotic
Ant. treatment day 14 Experimental end
P same 0.005 P same 0.048
● ● mean
●●●
● ● ● median
●●● ● ●
● ●●●●● 0.06 ●
0.06 ● ●●●● ●
●
● ● ● ●●●
●●●● ●●
●●● ●●●
● ●
●
●●●●●
●●●
●●
transformed values

−−−−−−−−
●●● ● ●●
●●● 0.0471 ●●●●●● −−−−−−−−
●
●●●
0.04 ●● ● 0.04 0.0428 ● ●●
●●
● ●● 0.0543 ● ●●●●
●●
●●●
●●
−−−−−−−− ●●
−−−−−−−− 0.0461
0.0332 ●●●● 0.0334 ● ●
0.0373 ● ● 0.0365 ●
0.02 ● 0.02
●
●
●
●●●● ●● ●●
●● ●●
●●●●● ●
●●● ●● ●●
0.00 0.00
Placebo Prebiotic Placebo Prebiotic

Figure 4. Group comparison of the relative abundance of bifidobacteria levels at study start, study end and during antibiotic
treatment.

Baseline Product A Product B

other other other
unclassified Bacteria unclassified Bacteria unclassified Bacteria
Streptococcus Streptococcus Streptococcus
Clostridium IV Clostridium IV ant. treat day 14 Clostridium IV ant. treat day 14
Clostridiales product B Clostridiales Ruminococcus
ant. treat day 7 ant. treat day 7
product A
Parabacteroides Ruminococcus ant. treat day 1 Clostridiales ant. treat day 1
Akkermansia Akkermansia Lachnospiracea IS
Ruminococcus Parabacteroides Parabacteroides
Escherichia Shigella Escherichia Shigella Akkermansia
Faecalibacterium Bifidobacterium Roseburia
Blautia Lachnospiracea IS Faecalibacterium
Lachnospiracea IS Faecalibacterium Blautia
Bifidobacterium Blautia Ruminococcaceae
Roseburia Ruminococcaceae Alistipes
Ruminococcaceae Roseburia Escherichia Shigella
Alistipes Bifidobacterium b ab
Alistipes a
Anaerostipes Bacteroides Anaerostipes
Bacteroides Anaerostipes Bacteroides

0 4 8 12 0 4 8 12 16 0 4 8 12 16 20
% % %

Figure 5. Gut microbiota composition in children at study start and during antibiotic treatment for both treatments (A indicates
placebo and B prebiotic group).

Beneficial Microbes 10(3) 259

 S. Soldi et al.
throughput Illumina sequencing. The observed increase in
bifidobacteria counts was irrespective of the individual’s
enterotype, hence independent from individual differences
in the microbiota composition and also sustained during
antibiotic therapy. The findings suggest that prebiotic
consumption stabilises the faecal microbiota composition
and attenuates the usual decrease of bifidobacteria observed
during antibiotic treatment. In this way, the prebiotic inulin-
type fructans product could attenuate antibiotic-related
https://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0116 - Tuesday, January 21, 2020 11:39:54 AM - IP Address:148.204.6.250
microbiota disturbances and may help to restore the initial
microbiota composition in young children after antibiotic
treatment.
Enterotyping of the current cohort allowed the identification
of three distinct microbiota constellations. These were
differently characterised by the presence of a specific species
composition despite a common pattern of shared bacterial
families among all the three enterotypes. Enterotype 1
displayed a high presence of Anaerostipes and Roseburia,
Enterotype 2 was characterised by the presence of Alistipes
and Ruminococcaceae, Enterotype 3 was strongly dominated
by Bacteroides and depleted of Bifidobacterium. To our
knowledge, such an enterotype classification is described
for the first time in children aged 3 to 6 years. A previous
study reported an enterotype classification of school-aged
children in Asian population (Nakayama et al., 2015). The
establishment of the enterotypes seems to start between
18 and 36 months of age, but is considered to be still
susceptible to shifting (Bergström et al., 2014).
It was previously thought that a stable, adult-like gut
microbiota is established by the age of 3 years. However,
more and more evidence is accumulating that the gut
microbiota composition continues to develop beyond the
age of three years. Children at the age of four years show
clear differences when compared to the adult microbiota
(Ringel-Kulka et al., 2013) and even the gut microbiota
of adolescent children can be distinguished from the
composition of adults, in particular when considering the
relative abundances of bacterial taxa (Agans et al., 2011).
Similarly, there are distinct compositional and functional
qualities in the healthy gut microbiome of school aged
children that differ from the adult one (Hollister et al.,
2015). Besides factors like geography, cultural traditions
including nutrition and lifestyle, age is an important
factor that determines the gut microbiota composition
(Yatsunenko et al., 2012). Hence, it can be speculated that
the microbiota composition of the current cohort is not
yet fully established and is still subject to dietary as well as
other external influences. Future longitudinal studies may
thus address the change in the enterotype composition
during childhood and adolescence.
The vast amount of data for prebiotic modulation of the
microbiota in children with inulin-type fructans comes
from studies in infants and toddlers (Closa-Monasterolo
260
et al., 2013; Veereman-Wauters et al., 2011; Wernimont
et al., 2015) as fructans derived from chicory root are
commonly added to infant and follow-on formula. Due
to their prebiotic properties fructans modulate the gut
microbiota composition closer to a composition of breast-
fed infants, in particular by increasing bifidobacteria levels.
This study adds to these previous reports and extends the
database to children aged 3 to 6 years. The sequencing
approach applied here allowed to demonstrate that prebiotic
inulin-type fructans exert a specific growth-stimulating
effect on bifidobacteria. This result is in agreement with
the findings obtained from targeted qPCR analysis (Lohner
et al., 2018). The observed specific and selective influence
of supplementation with prebiotic inulin-type fructans on
the microbiota composition is furthermore well in line with
recent studies in children aged 7 to 12 years (Nicolucci et
al., 2017) and adults (Vandeputte et al., 2017), also applying
high-throughput sequencing.
The present study confirms that antibiotic treatment alters
the abundance of bifidobacteria. The depletion observed in
children of the placebo control group was remarkable with
lower levels of bifidobacteria even retained after antibiotic
treatment and at study end. In contrast, typical reduction
in abundance of bifidobacteria was attenuated in children
supplemented with the prebiotic inulin-type fructans
product. Moreover, with prebiotic intake bifidobacteria
levels remained even higher than in the control group prior
to the antibiotic treatment. In this way, inulin-type fructans
could attenuate the potential negative effect of antibiotic
treatment on the microbiota composition by stabilising
it. Disturbances in the gut microbiota composition are
believed to also affect health later in life, in particular, as
the maturation of the immune system is not finalised by the
end of the first year and as the gut microbiota composition
is considered as driver for immune development (Martin
et al., 2010; Weizman, 2015). A stabilising effect on the
microbiota composition as observed in the current study
may thus provide a benefit also in the long-term.
When considering the antibiotic treatment, we found
significant differences for Bifidobacterium, Blautia, and
Roseburia while all other taxa were not affected. There
were also no changes in total bacteria counts throughout
the study, neither in the prebiotic nor in the placebo
group. The magnitude of antibiotic-induced disturbance
might thus appear rather limited. However, disturbance
of the microbiota composition depends on the type of
antibiotic (Korpela et al., 2016). Thus, it cannot be ruled
out that for specific antibiotics the effects were even more
pronounced for the individual child. In our study, children
were prescribed different types of antibiotics and we could
not distinguish between the diverse antibiotic compounds
in the data analysis which might be a limitation of the study.
Beneficial Microbes 10(3)
  Effect of prebiotics on gut microbiota composition of children
That early disturbances of the microbiota composition
due to antibiotic intake may affect health later in life is
supported by a study in healthy children aged 2 to 7 years.
Antibiotic intake resulted in long-lasting shifts in microbiota
composition as well as metabolism. The use of macrolide
was shown to reduce levels of Bifidobacterium and
Bacteroides which took 24 months to normalise, associated
with increased risk of asthma. Treatment with penicillins
also affected the gut microbiota composition, yet less
https://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0116 - Tuesday, January 21, 2020 11:39:54 AM - IP Address:148.204.6.250
pronounced (Korpela et al., 2016). Furthermore, changes in
the microbiota composition induced by macrolide have been
linked to BMI, adiposity, obesity and metabolic diseases
in children and adults (Korpela and Vos, 2016; Korpela et
al., 2016). In line, data from animal studies suggest that
early-life antibiotic-related disturbances of the microbiota
composition affect host metabolism (Cox et al., 2014; Nobel
et al., 2015). Hence, for long-term metabolic programming
of infants and children the microbiota composition as such,
but in particular bifidobacteria, is discussed as being crucial
(Korpela and Vos, 2016; Korpela et al., 2016). Accordingly,
it can be assumed that a modulation of the gut microbiota
composition by regular inulin-type fructans intake during
childhood may be promising to support adequate health
status also in the long-term.
5. Conclusions
The present in-depth analysis of the gut microbiota
composition using Illumina sequencing and targeted qPCR
analysis shows that prebiotic intake induces specific changes
in the gut microbiota composition of kindergarten children
aged 3 to 6 years. Furthermore, our data demonstrate
that the inulin-type fructans product with shorter and
longer fructan chains diminishes antibiotic-induced
disturbances in the gut microbiota composition as shown
by the attenuation of the decrease in bifidobacteria levels.
These changes are accompanied by beneficial effects in
clinical outcome parameters, as shown by reduced febrile
episodes requiring medical attention reported previously.
The current study thus provides coherent evidence that a
selective and sustained modulation of the gut microbiota
composition in young children can be achieved and that
children of this age group benefit from prebiotic inulin-type
fructans supplementation.
Supplementary material
Supplementary material can be found online at https://doi.
org/10.3920/BM2018.01116.
Table S1. List of indexed primers used in the study.
Methods. Sequence data preparation.
Figure S1. Barplot showing differential abundance of
bacterial groups among the three Enterotypes.
Beneficial Microbes 10(3)
Figure S2. Total bacteria over the course of the study in
both groups.
Figure S3. Changes in bacterial composition of the three
enterotypes during antibiotic intake for treated and control
children.
Conflict of interest
The authors declare no other conflicts of interest.
Acknowledgements
The study was funded by BENEO GmbH, Mannheim,
Germany, a member of the Suedzucker Group. The study
results and data contained in the publication have been
developed for BENEO GmbH. BENEO GmbH reserves
the exclusive right to use the results and data for possible
Health Claim requests. S. Theis, C. Sieland and M. Sailer
are/were employees of BENEO/Suedzucker Group. T. Decsi
received research funding from the National Research,
Development and Innovation Office, Hungary (OTKA
120193) to conduct a study on the role of prebiotics in
preventing childhood infections. The authors are grateful
to the families participating in this study and the clinical
investigators involved. The authors also thank Professor
Günther Boehm for critical review and comments on earlier
drafts of the manuscript.
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