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bm2018 0116
bm2018 0116
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
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).
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
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.
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.
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
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)..
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.
● ●●
0.0546
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Normalised bifidobacterial/total-bact. 16S rRNA gene count values
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ab ●●● 0.0742 ● ab ● ●●●●
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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.
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0.04 0.0474 ● ● ●
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● −−−−−−−− 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 ●
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Placebo Prebiotic Placebo Prebiotic Placebo Prebiotic
Ant. treatment day 14 Experimental end
P same 0.005 P same 0.048
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transformed values
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Placebo Prebiotic Placebo Prebiotic
Figure 4. Group comparison of the relative abundance of bifidobacteria levels at study start, study end and during antibiotic
treatment.
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).
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
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 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
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
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 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
analysis shows that prebiotic intake induces specific changes References
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