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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 prebiotic inulin-type fructans supplementation on the
Decsi, 2014; Lohner et al., 2018; Moro and Boehm, 2012; composition of the gut microbiota in healthy kindergarten
Roberfroid et al., 2010). children aged 3 to 6 years. Moreover, the efficacy of
prebiotic inulin-type fructans supplementation in
The role of prebiotics has been extensively investigated in preventing expected disturbances of faecal gut microbiota
the field of infant nutrition (Boehm et al., 2004; Thomas composition during antibiotic treatment was determined
and Greer, 2010). Prebiotics mimic the beneficial functional within a randomised, placebo-controlled trial (Lohner et al.,
properties of human milk oligosaccharides with respect to 2018) using advanced technologies of Illumina sequencing
modulation of the gut microbiota. An immune-modulating together with targeted qPCR analysis.
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 2. Materials and methods
observed infection prevention early in life (Arslanoglu et
al., 2007). Corresponding data in children above 3 years of Study design
age are more limited (Lohner et al., 2014). Therefore, we
recently conducted a randomised placebo-controlled trial For an extensive overview of study design and procedures,
in 270 kindergarten children aged 3-6 years. We found that we refer to Lohner et al. (2018). In brief, the study was
daily intake of an inulin-type fructans product with shorter designed as a randomised, placebo-controlled, double-
and longer fructan chains reduces febrile episodes requiring blind trial (Clinical Trials.gov ID: NCT03241355) and
medical attention and lowers the incidence of sinusitis. This aimed at exploring whether prophylactic dietary prebiotic
finding might arise from a stimulated immune function supplementation is able to influence the intestinal
in prebiotic supplemented children and was connected microbiota and the frequency of infectious disease episodes
to a specific modulation of the gut microbiota (Lohner in kindergarten children during winter period. Healthy
et al., 2018). children (3-6 years) were allocated to consume either 6 g/
day of an Orafti® inulin-type fructans product with shorter
It seems plausible that also children aged 3 to 6 years and longer fructan chains (degree of polymerisation (DP)
benefit from prebiotic consumption and corresponding ≥11 approx. 25-30% (on g/100 g dry matter), average DP of
modulation of the gut microbiota bearing the following about 7 to 8) or maltodextrin as placebo for a period of 24
in mind: (1) it is well established that the gut microbiota weeks. Children were considered eligible for the study when
composition is important for the development of the fulfilling the following criteria: (1) over 3 years but under
immune system (Martin et al., 2010). The immune system 7 years of age at the time of pre-examination, (2) healthy
is not fully established until about 10 to 15 years of age at the time of inclusion, and (3) attending a kindergarten.
(Martin et al., 2010; Weizman, 2015). Similarly, recent
data show that the gut microbiota continues to develop Faecal samples
until the teenage years (Agans et al., 2011; Cheng et al.,
2016; Hollister et al., 2015; Ringel-Kulka et al., 2013). (2) Approximately 2 g of faeces were collected for microbiota
the age group of 3-6 year old children frequently attends evaluation at baseline and week 24. In case of an antibiotic
day-care centres. This is associated with a 3-fold increased treatment, additional stool samples were collected on the
risk to suffer from gastrointestinal or respiratory infections 1st day of antibiotic treatment and on the 7th and 14th day
(Weizman, 2015). (3) antibiotic therapies remain common after starting it. At home, samples were refrigerated at
in this paediatric age group, too, and there is interest in 4 °C and were collected within 24 h by the study personnel
strategies to ameliorate or prevent negative repercussions and transported to the study centre for long-term storage
of antibiotic treatment on the gut microbiota composition, at -80 °C.
and potential health implications linked to such a dysbiosis
also in the long run (Korpela and Vos, 2016; Shao et al., Bacterial community DNA extraction
2017; Sturkenboom Miriam et al., 2008; Turta and Rautava,
2016; Vaz et al., 2014). Faecal bacterial DNA was extracted using the FastDNA
SPIN Kit and FastPrep Instrument (MP Biomedicals, Santa
In order to further explore the potential benefits of prebiotic Ana, CA, USA). The extracted DNA was quantified using
interventions in this age group, there is interest to better the picogreen method of the Quant-iT™ HS ds-DNA assay
understand the effects on the gut microbiota composition. kit in a Qubit™ fluorometer (Invitrogen, Carlsbad, CA, USA)
Such data is currently scarce, as is data in general on the and verified. DNA extracts were diluted to a concentration 5
gut microbiota of kindergarten children. ng/µl for reducing template amount associated PCR biases.

The aim of the current paper was to investigate the basal


microbiome composition and to examine the effects of

254 Beneficial Microbes 10(3)


 Effect of prebiotics on gut microbiota composition of children

Quantitative real-time PCR amplification Further details of quality control procedure are provided
in the supplementary materials.
All faecal samples were subject to targeted qPCR analysis.
As described in Lohner et al. (2018), qPCR reagents were The LotuS v1.461 (Hildebrand et al., 2014) pipeline was
freshly prepared for each qPCR analytical session, according used for the generation of the 0.03 sequence distance
to the number of samples in duplicates, including also a operational taxonomic units (OTUs) using the combination
positive control. The final qPCR reaction volume was 25 of the USEARCH v7.0.1090 (Edgar, 2004) as implemented
μl. The amplification mixtures were prepared using the in the LotuS pipeline. The Naïve Bayesian classifier (Wang
RealMasterMix Sybr Rox 2.5X (5 PRIME, Fisher Scientific, et al., 2007) with a 0.8 bootstrap cut-off value was used
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 for classifying sequences into taxa using the SILVA v115
manufacturer’s instructions. Different protocols and primer sequence database (https://www.arb-silva.de) as reference.
pairs were applied for the detection of Bifidobacterium, Achieved classification depth along with the sequence
Lactobacillus, Clostridium perfringens, Clostridium numbers per taxon level show that about 85% of the
difficile and Enterobacteriaceae as previously documented sequences (19,338,291) were classified at the genus level.
(Huijsdens et al., 2002; Malinen et al., 2005; Mutters et al.,
2009; Wise and Siragusa, 2005). Thermal cycling protocols Statistics
were performed as provided in the cited publications.
Data were normalised via Box-Cox transformation (power
Multiplexed high throughput sequencing of the transformed to obtain normal value distributions per
prokaryotic 16S rRNA gene PCR amplicons analysed marker) as described in Osborne (2010) and
expressed as ratio of taxon per total bacteria. Data are
Baseline faecal samples and samples collected during displayed as mean ± standard deviation. The statistical
antibiotic treatment phases were assessed by microbiota analysis was performed using the R software v3.3.1 (R Core
phylogenetic profiling. PCR amplicons of the V3-4 Team, 2017) and the packages Vegan v2.0-10 (Dixon, 2003)
hypervariable region of the bacterial 16S rRNA gene were for the multivariate analyses, the MetagenomeSeq v1.6.0
screened using an Illumina platform multiplex approach. (Paulson et al., 2013) implementation of the linear model
Two nested PCR cycles were used to obtain the fragments fitting of the Limma v3.20.9 (Smyth, 2005) R package for
for sequencing: amplification, quantification and pooling the performance of pairwise comparisons. Enterotyping was
were published by Soldi et al. 2015 and indexed primers performed as previously described (Arumugam et al., 2011).
are reported in the Supplementary Table S1. Details about
primer and linker design were previously published by In order to identify the core microbiomes among the three
Vasileiadis et al. (2015). putative enterotypes the Corbata v20130611 suite of R
scripts (Li et al., 2013) was used. Cut-off values of min 1%
The PCR amplicon pool was then purified using the SPRI for the relative abundance and 80% for the ubiquity were
(solid phase reversible immobilisation) based method of applied. The core taxa were determined with bootstrapping
the Agencourt® AMPure® XP kit (Beckman Coulter, Milan, by sampling with replacement of both the number of
Italy). Illumina sequencing with the V3 chemistry and samples used in the analysis and the distribution of taxa
library preparation of the amplicons was performed by in each sample for an alpha value of 0.05. In all bar plot
Fasteris SA (Geneva, Switzerland). cases the error bars represent the standard error.

Sequence data preparation 3. Results

A total of 24,799,362 high quality paired-end sequence 209 children of the per protocol population (Lohner et
reads (300 bp each read) of PCR amplicons was obtained al., 2018) provided a full faecal sample set and were thus
with the Illumina MiSeq platform (San Diego, CA, USA), considered for the qPCR and sequencing analysis. The
performed by Fasteris SA. Sequence cluster identification basic characterisation of the microbiota composition by
on the Illumina MiSeq flow-cell and base calling of the phylogenetic profiling was performed with samples of all
sequence reads were performed with the MiSeq Control children who provided a faecal sample at baseline (n=233).
software v2.4.1.3, the RTA v1.18.54.0 software (Illumina)
and CASAVA v1.8.2 (Illumina). The sequencing data were Basic characterisation of the microbiota composition
quality controlled for sequencing artefacts and experimental
biases, such as amplification of non-targeted taxa (archaea Basic information on the gut microbiota composition of 3
and chloroplasts) and generation of chimeric sequences to 6 years old children was obtained from the sequencing
during PCR amplification by bioinformatic elaboration data of faecal samples collected from 233 children at the
resulting in a final number of 22,741,948 reconstructed start of the study. This baseline characterisation included
amplicon sequences which were used in the analysis.

Beneficial Microbes 10(3) 255


S. Soldi et al.

the subject classification according to putative enterotypes Bacteroides representing almost 25% of the taxa. Enterotype
and their corresponding key microbial taxa. 3 was also characterised by the absence of Bifidobacterium
from the core microbiome and the Bifidobacterium levels
As reported previously for adults (Arumugam et al., 2011), (1.8%) were significantly lower compared to Enterotype 1
three distinct enterotypes (namely Enterotype 1, Enterotype (5.6%) and 2 (5.7%) (Supplementary Figure S1).
2 and Enterotype 3) could be identified in the present cohort
of 3 to 6 years old healthy children (Figure 1). Most of the Prebiotic intake stimulates the growth of bifidobacteria in
faecal samples (n=100) were allocated to Enterotype 2, kindergarten children
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

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


microbiota composition in 3 to 6 years old children during
Bacteroides, Anaerostipes, Alistipes, Roseburia and the 6 months intervention period was assessed by using a
Ruminococcaceae were among the dominant taxa targeted qPCR approach, as reported previously (Lohner
shared by all enterotypes but with different amounts et al., 2018). Intake of the prebiotic fructans resulted in a
specific for each enterotype. Samples of Enterotype 1 significantly higher relative abundance of bifidobacteria at
had a significantly higher percentage (14.0 and 6.2%) of week 24 compared to children receiving the placebo control
Roseburia and Lachnospiracea incertae sedis compared (P=1.228×10-05). Likewise, children of the prebiotic group
to Enterotype 2 (5.2 and 3.6%) and 3 (9.4 and 3.2%), while had a significantly higher relative abundance of lactobacilli
the percentage of Alistipes was lowest (4.5%). Alistipes as compared to children of the placebo group at week 24
(15.0%) and Ruminococcaceae (13.1%) were representative (P=0.014). There was no difference between the groups for
for Enterotype 2 with significantly higher levels compared to total bacteria, or the relative abundance of C. perfringens, C.
Enterotype 1 and 3, while the percentage of Roseburia (5.2%) difficile and Enterobacteriaceae. Faecal pH values tended to
and Faecalibacterium (2.4%) levels were lowest. The core be lower in the prebiotic group compared with the placebo
microbiota of Enterotype 3 was found to be dominated by 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 Total bacteria remained unchanged over the course of the
during antibiotic treatment study in both groups (Supplementary Figure S2). Relative
abundance of bifidobacteria in children of the placebo
The pronounced and selective effect of regular consumption group remained significantly reduced compared to baseline
of prebiotic on bifidobacteria were also observed during conditions also 14 days after starting antibiotic intake and
antibiotic administration as confirmed by qualitative and even at the end of the 6 months intervention period. In
quantitative analysis with qPCR and high-throughput children receiving the prebiotic, the relative abundance of
sequencing. bifidobacteria was significantly higher compared to placebo
control already at the onset of antibiotic treatment 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

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

●● ●

a ●
● ●●● ●● ●

0.06 ●
−−−−−−−− a ●
0.0594 ●●●
●●● ●
●●●●
●● ●
●●●
●●
●●● ●
0.056−−−−−−−− ● ● ●
0.0554 ●●●
●●● ●●● ●
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


●●

●●


●●●●●●
●●●●
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 ●● ●

● ● ●●
●●●●
●●●● ●●● ●
●●●
0.00 ●●

B Product B samples. P same 0.017


0.12

● ●
● ●

●●● ●●
0.08 ● a ●●
●●●● ●●
●● ●●●
●● ●


● ●
●●●●
−−−−−−−− ●
●● ●
●● ●
● 0.0739 ●
●●●
● ●
●● ● ●●
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

● ●
● ●●
● ● ●

● ● ● ● ●●●●●
● ● ● ●
● ● ●● 0.06 ●● ●●●
0.06 ●
●●● ● 0.08 ● ●●● ●
● ● ●●●● ●
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).

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S. Soldi et al.

throughput Illumina sequencing. The observed increase in et al., 2013; Veereman-Wauters et al., 2011; Wernimont
bifidobacteria counts was irrespective of the individual’s et al., 2015) as fructans derived from chicory root are
enterotype, hence independent from individual differences commonly added to infant and follow-on formula. Due
in the microbiota composition and also sustained during to their prebiotic properties fructans modulate the gut
antibiotic therapy. The findings suggest that prebiotic microbiota composition closer to a composition of breast-
consumption stabilises the faecal microbiota composition fed infants, in particular by increasing bifidobacteria levels.
and attenuates the usual decrease of bifidobacteria observed This study adds to these previous reports and extends the
during antibiotic treatment. In this way, the prebiotic inulin- database to children aged 3 to 6 years. The sequencing
type fructans product could attenuate antibiotic-related approach applied here allowed to demonstrate that prebiotic
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microbiota disturbances and may help to restore the initial inulin-type fructans exert a specific growth-stimulating
microbiota composition in young children after antibiotic effect on bifidobacteria. This result is in agreement with
treatment. the findings obtained from targeted qPCR analysis (Lohner
et al., 2018). The observed specific and selective influence
Enterotyping of the current cohort allowed the identification of supplementation with prebiotic inulin-type fructans on
of three distinct microbiota constellations. These were the microbiota composition is furthermore well in line with
differently characterised by the presence of a specific species recent studies in children aged 7 to 12 years (Nicolucci et
composition despite a common pattern of shared bacterial al., 2017) and adults (Vandeputte et al., 2017), also applying
families among all the three enterotypes. Enterotype 1 high-throughput sequencing.
displayed a high presence of Anaerostipes and Roseburia,
Enterotype 2 was characterised by the presence of Alistipes The present study confirms that antibiotic treatment alters
and Ruminococcaceae, Enterotype 3 was strongly dominated the abundance of bifidobacteria. The depletion observed in
by Bacteroides and depleted of Bifidobacterium. To our children of the placebo control group was remarkable with
knowledge, such an enterotype classification is described lower levels of bifidobacteria even retained after antibiotic
for the first time in children aged 3 to 6 years. A previous treatment and at study end. In contrast, typical reduction
study reported an enterotype classification of school-aged in abundance of bifidobacteria was attenuated in children
children in Asian population (Nakayama et al., 2015). The supplemented with the prebiotic inulin-type fructans
establishment of the enterotypes seems to start between product. Moreover, with prebiotic intake bifidobacteria
18 and 36 months of age, but is considered to be still levels remained even higher than in the control group prior
susceptible to shifting (Bergström et al., 2014). to the antibiotic treatment. In this way, inulin-type fructans
could attenuate the potential negative effect of antibiotic
It was previously thought that a stable, adult-like gut treatment on the microbiota composition by stabilising
microbiota is established by the age of 3 years. However, it. Disturbances in the gut microbiota composition are
more and more evidence is accumulating that the gut believed to also affect health later in life, in particular, as
microbiota composition continues to develop beyond the the maturation of the immune system is not finalised by the
age of three years. Children at the age of four years show end of the first year and as the gut microbiota composition
clear differences when compared to the adult microbiota is considered as driver for immune development (Martin
(Ringel-Kulka et al., 2013) and even the gut microbiota et al., 2010; Weizman, 2015). A stabilising effect on the
of adolescent children can be distinguished from the microbiota composition as observed in the current study
composition of adults, in particular when considering the may thus provide a benefit also in the long-term.
relative abundances of bacterial taxa (Agans et al., 2011).
Similarly, there are distinct compositional and functional When considering the antibiotic treatment, we found
qualities in the healthy gut microbiome of school aged significant differences for Bifidobacterium, Blautia, and
children that differ from the adult one (Hollister et al., Roseburia while all other taxa were not affected. There
2015). Besides factors like geography, cultural traditions were also no changes in total bacteria counts throughout
including nutrition and lifestyle, age is an important the study, neither in the prebiotic nor in the placebo
factor that determines the gut microbiota composition group. The magnitude of antibiotic-induced disturbance
(Yatsunenko et al., 2012). Hence, it can be speculated that might thus appear rather limited. However, disturbance
the microbiota composition of the current cohort is not of the microbiota composition depends on the type of
yet fully established and is still subject to dietary as well as antibiotic (Korpela et al., 2016). Thus, it cannot be ruled
other external influences. Future longitudinal studies may out that for specific antibiotics the effects were even more
thus address the change in the enterotype composition pronounced for the individual child. In our study, children
during childhood and adolescence. were prescribed different types of antibiotics and we could
not distinguish between the diverse antibiotic compounds
The vast amount of data for prebiotic modulation of the in the data analysis which might be a limitation of the study.
microbiota in children with inulin-type fructans comes
from studies in infants and toddlers (Closa-Monasterolo

260 Beneficial Microbes 10(3)


 Effect of prebiotics on gut microbiota composition of children

That early disturbances of the microbiota composition Figure S2. Total bacteria over the course of the study in
due to antibiotic intake may affect health later in life is both groups.
supported by a study in healthy children aged 2 to 7 years.
Antibiotic intake resulted in long-lasting shifts in microbiota Figure S3. Changes in bacterial composition of the three
composition as well as metabolism. The use of macrolide enterotypes during antibiotic intake for treated and control
was shown to reduce levels of Bifidobacterium and children.
Bacteroides which took 24 months to normalise, associated
with increased risk of asthma. Treatment with penicillins Conflict of interest
also affected the gut microbiota composition, yet less
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pronounced (Korpela et al., 2016). Furthermore, changes in The authors declare no other conflicts of interest.
the microbiota composition induced by macrolide have been
linked to BMI, adiposity, obesity and metabolic diseases Acknowledgements
in children and adults (Korpela and Vos, 2016; Korpela et
al., 2016). In line, data from animal studies suggest that The study was funded by BENEO GmbH, Mannheim,
early-life antibiotic-related disturbances of the microbiota Germany, a member of the Suedzucker Group. The study
composition affect host metabolism (Cox et al., 2014; Nobel results and data contained in the publication have been
et al., 2015). Hence, for long-term metabolic programming developed for BENEO GmbH. BENEO GmbH reserves
of infants and children the microbiota composition as such, the exclusive right to use the results and data for possible
but in particular bifidobacteria, is discussed as being crucial Health Claim requests. S. Theis, C. Sieland and M. Sailer
(Korpela and Vos, 2016; Korpela et al., 2016). Accordingly, are/were employees of BENEO/Suedzucker Group. T. Decsi
it can be assumed that a modulation of the gut microbiota received research funding from the National Research,
composition by regular inulin-type fructans intake during Development and Innovation Office, Hungary (OTKA
childhood may be promising to support adequate health 120193) to conduct a study on the role of prebiotics in
status also in the long-term. preventing childhood infections. The authors are grateful
to the families participating in this study and the clinical
5. Conclusions investigators involved. The authors also thank Professor
Günther Boehm for critical review and comments on earlier
The present in-depth analysis of the gut microbiota drafts of the manuscript.
composition using Illumina sequencing and targeted qPCR
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