PII: S0378-1135(16)30504-1
DOI: http://dx.doi.org/doi:10.1016/j.vetmic.2016.10.020
Reference: VETMIC 7420
Please cite this article as: Han, Geon Goo, Lee, Jun-Yeong, Jin, Gwi-
Deuk, Park, Jongbin, Choi, Yo Han, Chae, Byung Jo, Kim, Eun Bae, Choi,
Yun-Jaie, Evaluating the association between body weight and the intestinal
microbiota of weaned piglets via 16S rRNA sequencing.Veterinary Microbiology
http://dx.doi.org/10.1016/j.vetmic.2016.10.020
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Evaluating the association between body weight and the intestinal microbiota of weaned
Geon Goo Hana, Jun-Yeong Leea, Gwi-Deuk Jinb, Jongbin Parkc, Yo Han Choib, Byung Jo
a
Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of
Korea
b
Department of Animal Life Science, Kangwon National University, Chuncheon, Gangwon-
c
Department of Animal Life System, Kangwon National University, Chuncheon, Gangwon-
d
Division of Applied Animal Science, Kangwon National University, Chuncheon, Gangwon-
e
Research Institute for Agriculture and Life Science, Seoul National University, Seoul,
Republic of Korea
*
Corresponding authors with equal contribution
1
Highlights
Microbial richness was higher in the heavier piglets than in the lighter piglets.
FB ratio was higher in the heavier piglets than in the lighter piglets.
Several genera were significantly different in the lighter and heavier piglets.
Several metabolic pathways were different in the lighter and heavier piglets.
ABSTRACT
Due to the ban on the use of antimicrobial growth promoters in livestock feeds,
understanding the relationship between intestinal microbiota and the physiology of the host
has become very important for improving livestock performance. In this study, we
investigated the relationship between intestinal microbiota and body weights of weaned
piglets. Lighter (n=9) and heavier (n=9) 9-week-old weaned piglets were selected from
approximately one-hundred individuals based on their body weights. Their fecal microbial
communities were analyzed by sequencing the V4 region of the 16S rRNA gene. The
microbial richness estimators of the heavier piglets, were significantly higher than those of
the lighter piglets. At the phylum level, the microbiota of the heavier group had significantly
higher levels of Firmicutes and a higher Firmicutes-to-Bacteroidetes ratio than that of the
lighter group. At the genus level, the levels of several genera, such as Anaerococcus and
Lactococcus, were significantly different in the two groups. In particular, the lighter group
2
and Bacteroides, compared with those of the heavier group. Moreover, the levels of bacteria
expressing the components of several metabolic pathways were significantly different in the
two groups. The microbiota of the heavier group had a significantly higher involvement in
three KEGG pathways concerned with xenobiotic degradation than that of the lighter group.
These results may provide insights into host-microbe interactions occurring in the piglet
intestine and will be useful in establishing a strategy for improving growth performance in
Keywords: Fecal microbiota, Metagenome, Weaned piglet, Body weight, 16S rRNA gene,
High-throughput sequencing
1. Introduction
Using antibiotics as antimicrobial growth promoters (AGPs) in livestock feeds has been
banned since 1986 in Sweden, since 2006 in the EU, and since July 2011 in the Republic of
Korea due to the emergence of bacteria that are resistant to a broad range of antibiotics
vancomycin-resistant enterococci (VRE). After the ban on the use of AGPs, various problems
were observed in the livestock industry, such as a reduction in growth performance and the
increasing therapeutic use of antibiotics (Heo et al., 2013; Wierup, 2001). The development
of alternatives to antibiotics to solve these problems has been an important issue in the
livestock industry, including the swine industry; however, effective strategies have not yet
been developed. Given this situation, modulating the intestinal microbiota of livestock
particularly a relationship between the composition of the intestinal microbiota and body
weight. For example, Yang et al. discovered obesity-associated bacteria in three distinct gut
locations in pigs (Yang et al., 2016). Other researchers have found body weight-related
bacterial groups in mice and humans (Everard et al., 2011; Turnbaugh et al., 2009). In
particular, studies of the gut microbiota of livestock in their youth are needed because it is
during this crucial period that the intestinal microbiota is stabilized. However, there is
The aim of this study was to investigate the relationship between the intestinal microbiota
and body weights of weaned piglets under a commercial environment. To investigate the
relationship, we analyzed the microbial communities and inferred the functions of the fecal
4
2. Materials and methods
Republic of Korea) were used in this study, and the experimental protocols followed in this
study were approved by the Institutional Animal Care and Use Committee of Kangwon
National University (IACUC #: KW-140509-1). The piglets were fed a commercial diet
designed for weaned piglets (Table 1) and had access to feed and water ad libitum. The
weaned piglets were individually weighed, and a total 18 piglets were selected from
approximately one-hundred individuals based on their body weights, with 9 piglets being
placed in the lighter group and 9 piglets placed in the heavier group (Table 2). The body
weights of the members of the lighter group ranged from 8.09 to 11.89 kg (mean values SD
= 10.20 1.33 kg), and the body weights of the members of the heavier group ranged from
16.70 to 22.75 kg (mean values SD = 19.00 2.38 kg), with the total body weights ranging
from 8.09 to 22.75 kg (mean values SD = 14.60 4.90 kg) (Table 3). The mean body
weights of the lighter and heavier groups were significantly different (P < 0.001). Fecal
samples were collected from each piglet and were stored at -70 C until DNA extraction was
performed.
DNA was extracted from 250 mg of each fecal sample using a NucleoSpinSoil Kit
stored at -20 C until further analysis. The V4 region of the bacterial 16S rRNA gene, which
communities (Caporaso et al., 2011; Zhao et al., 2013), was amplified from the total extracted
5
DNA. PCR amplification was performed using Takara Ex-taq polymerase (Takara Bio, Shiga,
94 C for 3 min, followed by 40 cycles of 94 C for 45 sec, 55 C for 1 min, and 72 C for
1.5 min and finally, 1 cycle of 72 C for 10 min. The amplicons were separated by agarose
gel electrophoresis and were purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia,
CA, USA).
The DNA libraries were constructed using a NEBNext Ultra DNA Library Prep Kit for
Illumina (New England Biolabs, Ipswich, MA, USA), with some modifications of the
manufacturers instructions. The size selection steps for the adaptor-ligated DNAs and the
cleanup steps were replaced by PCR product purification using a QIAquick PCR Purification
Kit (Qiagen, Valencia, CA, USA). The adaptor and index primers were added to the
amplicons using the NEBNext Multiplex Oligos for Illumina Kit (New England Biolabs,
Ipswich, MA, USA). The construction of the DNA libraries was confirmed by agarose gel
electrophoresis, and the libraries were purified using a QIAquick Gel Extraction Kit. The
components of the libraries were then sequenced using an Illumina MiSeq platform (NICEM,
SNU, Seoul, Republic of Korea). The 16S rRNA gene sequences determined in this study
were deposited in the NCBI Sequence Read Archive (SRA) database with the accession
number SRP080895.
The microbial communities were analyzed using Quantitative Insights Into Microbial
Ecology (QIIME) version 1.9.0 (http://qiime.org) software. The raw sequence reads were
quality trimmed and demultiplexed. The sequence reads were then clustered into operational
6
taxonomic units (OTUs) by de novo OTU picking at a 97% level of sequence similarity. The
taxonomic assignments for each representative sequence were obtained using the uclust
consensus taxonomic classifier using the GreenGenes 13_8 database, and the representative
The microbial diversity indices of the samples (alpha diversity) were determined using the
(PD), Shannon, and Simpson methods, and these indices were calculated from 5,000
Principal component analysis (PCA) was performed at the phylum and the genus level, and
the results were visualized using the STAMP version 2.1.3 program (Parks et al., 2014). The
abundance of the microbial taxa was expressed as a percentage of the total 16S rRNA gene
sequences, and the differences between the groups were compared. A two-sided Welchs t-test
was used to identify significant differences in the microbial taxa represented and the alpha
diversity of the microbiota of the two groups, with P < 0.05 considered significant. The
statistical analyses were performed using Microsoft Excel 2013 and STAMP software.
functional profile of the microbial communities based on the 16S rRNA gene sequences
obtained. Closed reference OTU picking against the GreenGenes 13_5 database was
conducted at 97% sequence similarity using the QIIME and OTU tables, and the data were
normalized for 2,627 reads per sample by single rarefaction. The resulting biom files were
normalized according to known/predicted 16S rRNA gene copy numbers, and the
7
metagenomes were predicted using precalculated Kyoto Encyclopedia of Genes and
Genomes (KEGG) orthologs. The predicted metagenomes were collapsed into a specified
level in a hierarchy using the KEGG pathway metadata and were analyzed using the STAMP
program. Unclassified functional categories were eliminated from the analysis. A two-sided
Welchs t-test was used to identify significant differences in the microbial taxa represented
and the alpha diversity of the microbiota of the two groups, with P < 0.05 considered
significant.
8
3. Results
3.1. Differences in the alpha diversity of the intestinal microbiota of the lighter and
heavier piglets
A total of 337,534 (mean value = 18,752 11,675) 16S rRNA gene sequence reads were
generated, with an average of 23,067 ( 13,261) reads per piglet in the lighter group and
ACE, Chao1, observed OTUs, PD, Shannon index, and Simpson index values were used as
parameters of the alpha diversity of intestinal microbiota in this study (Table 3). The ACE
metric was 16,243.60 ( 1,720.96) for the lighter group and 18,642.29 ( 2,605.18) for the
heavier group. The Chao1 metric was 15,628.56 ( 1,309.44) for the lighter group and
17,485.92 ( 1,887.86) for the heavier group. The number of observed OTUs was 2,257.08 (
100.19) for the lighter group and 2,452.78 ( 180.69) for the heavier group. The PD value
was 174.82 ( 10.70) for the lighter group and 181.56 ( 10.95) for the heavier group. The
Shannon index value was 9.41 ( 0.41) for the lighter group and 9.63 ( 0.29) for the heavier
group. The Simpson index value was 0.99 ( 0.01) for the lighter group and 0.99 ( 0.00) for
the heavier group. The richness estimators (ACE, Chao1, and observed OTUs) were
significantly higher for the heavier group than for the lighter group (P < 0.05). The diversity
indices (PD, Shannon, and Simpson) were also higher for the heavier group than for the
3.2. Differences in the microbial taxa represented in the intestinal microbiota of the
At the 97% similarity level, all of the OTUs observed were classified into 26 phyla and
9
258 genera (Fig. 1). At the phylum level, the microbiota of the lighter and heavier group
identified in that of the lighter group and the heavier group, respectively. The three dominant
phyla detected in both groups were Firmicutes (48.42% in the lighter group and 53.59% in
the heavier group), Bacteroidetes (39.31% in the lighter group and 34.15% in the heavier
group), and Proteobacteria (5.25% in the lighter group and 4.23% in the heavier group). At
the genus level, the intestinal microbiota of the two groups shared 180 genera (69.77%), with
41 (15.89%) and 37 genera (14.34%) uniquely identified in the lighter group and the heavier
group, respectively. Three dominant genera, Prevotella (20.08%), Lactobacillus (6.36%), and
Bacteroides (4.54%), were found in the intestinal microbiota of the lighter group, whereas the
three dominant genera in the intestinal microbiota of the heavier group were Prevotella
overall composition of the microbiota of the two groups at the phylum and genus levels using
PCA. In the resulting PCA plot, the microbial communities were clustered into two groups by
body weight at the phylum level (Fig. 2A), although they were not separated at the genus
Next, we compared the relative abundance of the members of microbial taxa in the lighter
and heavier groups and found that some phyla and genera were represented at significantly
different levels in the groups (Table 4). At the phylum level, the levels of Firmicutes and
Planctomycetes members were significantly higher in the heavier group than in the lighter
group (Fig. 2B). In contrast, the level of Bacteroidetes members was significantly higher in
the lighter group than in the heavier group (Fig. 2B). The Firmicutes-to-Bacteroidetes ratio
(FB ratio) was significantly higher in the heavier group than in the lighter group (Table 4).
10
At the genus level, the levels of Anaerococcus, Sediminibacterium, and Butyrivibrio were
significantly higher in the intestinal microbiota of the heavier group than in that of the lighter
lighter group than the heavier group (Fig. 3B). Streptococcus was more abundant in the
heavier group than in the lighter group, although the difference was not significant (P =
0.056).
3.3. Comparison of the KEGG pathways of the fecal microbiota of the lighter and
heavier piglets
We compared the KEGG pathways predicted for the fecal microbiota of the lighter and
heavier piglets and found that various KEGG pathways were significantly differentially
identified in the microbiota of the two groups (Fig. 4). Nucleotide-binding oligomerization
domain (NOD)-like receptor (NLR) signaling pathway, alanine, aspartate and glutamate
levels in the microbiota of the lighter group, (0.06%, 1.28%, and 0.17%, respectively) than in
that of the heavier group (0.05%, 1.26%, and 0.16%, respectively). In contrast, dioxin
degradation, xylene degradation, and benzoate degradation pathways that are involved in
xenobiotics biodegradation and metabolism at the higher KEGG pathway hierarchical level,
were predicted at significantly higher levels in the microbiota of the heavier group (0.06%,
0.05%, and 0.24%, respectively) than in that of the lighter group (0.05%, 0.05%, and 0.23%,
respectively).
11
4. Discussion
In this study, the levels of the microbial richness estimators (ACE, Chao1, and observed
OTUs) were significantly higher for the heavier group than for the lighter group. However,
many studies have shown that intestinal microbial diversity and richness are negatively
related to body weight. Turnbaugh et al. reported that the level of intestinal microbial
diversity was reduced in obese mice and humans compared with those of lean individuals
(Turnbaugh et al., 2008; Turnbaugh et al., 2009). Le Chatelier et al. reported that individuals
with a low body mass index (BMI) showed a higher level of gut bacterial richness than did
high BMI individuals (Le Chatelier et al., 2013). Our previous study revealed that the number
of OTUs observed in the cecum of broiler chickens is negatively related to their body weights
(Han et al., 2016). However, several studies have suggested that the level of intestinal
microbial diversity is not related to obesity or to the existence of obesity-related disorders (de
Goffau et al., 2014; Mejia-Leon et al., 2014; Walters et al., 2014). The relationship between
intestinal microbial diversity and body weight or obesity remains controversial issues.
In this study, we observed differences in the levels of several different phyla in the
microbiota of the lighter and heavier groups. The microbiota of the heavier group had a
significantly higher Firmicutes level and FB ratio compared with that of the lighter group,
whereas a significantly higher Bacteroidetes level was observed in the microbiota of the
lighter group, consistent with the results observed in many previous studies (Delzenne and
Cani, 2011; Hildebrandt et al., 2009; Kasai et al., 2015; Ley et al., 2006). However, some
researchers have reported contrary results (Clarke et al., 2014), and other researchers have
reported that there was no relationship between the FB ratio and BMI (Hu et al., 2015; Louis
et al., 2016). More studies of the host-microbe interactions in various animals in various
12
environments are required to explain these results.
In this study, the relative abundance of members of the genera Bacteroides and
Anaerotruncus were significantly lower in the microbiota of the heavier group than they were
in that of the lighter group. Hu et al. reported that Bacteroides species are more abundant in
the microbiota of normal adolescents than they are in that of obese adolescents (Hu et al.,
2015). Haro et al. reported that Bacteroides species are more abundant in the microbiota of
low BMI men than in that of high BMI men, although the abundance of Bacteroides species
in the microbiota of women was not affected by their BMI values (Haro et al., 2016). Some
species of Bacteroides are opportunistic pathogens, although many Bacteroides species are
normally mutualistic in the mammal intestine. For example, Bacteroides fragilis is associated
with anaerobic bacteremia and sepsis, and infection with this species causes malignancy,
inflammatory bowel disease, inflammatory diarrhea, and heart disease (Choi et al., 2016; Ngo
et al., 2013; Redondo et al., 1995; Rhee et al., 2009; Robert et al., 2008; Zhu et al., 2013).
Infection with Anaerotruncus colihominis, the only known species of the genus
Anaerotruncus, is associated with bacteremia, bloating, and cachexia (Bindels et al., 2016;
Jalanka-Tuovinen et al., 2011; Lau et al., 2006). Based on these data, we can hypothesize that
the presence of certain pathogenic bacteria in the intestine causes the relatively reduced body
weight of the host animal. More studies of the relationship between infections with
opportunistic pathogens and body weight are required to clarify our hypothesis.
In this study, the fecal metagenomes were predicted using PICRUSt and several different
KEGG pathways were observed in the lighter and heavier piglets. NLR signaling pathway-
related genes were more abundant in the fecal microbiota of the lighter group than in that of
the heavier group. This pathway is associated with the host innate immune response, and its
13
activation affects the activity of alternative signaling pathways, such as the caspase activation
and cell death pathways (Wells et al., 2011). NLR is a member of the pattern recognition
receptor family, which recognizes bacterial cell wall components, such as meso-
pattern. We hypothesize that the activation of NLR signaling induces a host immune response
and would lead to the expenditure of more energy to maintain immune homeostasis. The
immune response is very energy intensive, as are the febrile processes of an animal and the
various subsequent responses, such as the production of cytokines, the reduction of amylase
activity, and antibody formation (Carroll and Forsberg, 2007; Liu et al., 2015). Therefore, the
body weight gain of the piglets with a microbiota that had a more activated NLR signaling
pathway might be less than that of the piglets in whose microbiota that pathway was less
activated.
We observed that three KEGG pathway- (dioxin degradation, xylene degradation, and
metabolism pathway were significantly more abundant in the microbiota of the heavier
group than in that of the lighter group, consistent with the results of other studies. Bahr et al.
reported that the administration of the atypical antipsychotic risperidone was related to
weight gain and the level of xenobiotics biodegradation and metabolism pathway-related
genes and the FB ratio in the gut microbiome were higher in the risperidone-treated group
than in the controls (Bahr et al., 2015). Yang et al. found that the benzoate degradation
pathway-related genes were significantly enriched in the cecal microbiome of high fatness
pigs compared with that of lean fatness pigs (Yang et al., 2016). Xenobiotics are foreign
chemical substances, and this term is commonly used in the context of harmful substances.
14
For example, dioxin is a well-known carcinogen, and long-term exposure to xylene is toxic to
the central nervous system and liver. These toxic substances are harmful to a host and may
inhibit its growth. In the microbiota of the heavier group, microbial degradation pathways for
these toxic substances were more activated than they were in that of the lighter group, which
may have led to the body weight gain of the former group.
15
5. Conclusion
In this study, we compared the fecal microbiota of lighter and heavier piglets and their
predicted metagenomes. The level of microbial richness was higher in the microbiota of the
heavier group than in that of the lighter group, and several different bacterial phyla and
genera that were differentially represented in the two groups were identified (e.g., Firmicutes,
several metabolic pathways were significantly different in the microbiota of the two groups,
such as NLR signaling pathway and xenobiotics biodegradation and metabolism pathway-
related genes. Based on these results, we can infer that the intestinal microbiota affects the
immune response and xenobiotic degradation. These results may provide insights into
understanding the host-microbe interactions occurring in the piglet intestine and will be
useful in establishing a strategy for improving growth performance in the swine industry.
16
Conflict of interest
Acknowledgements
This study was supported by the Strategic Initiative for Microbiomes in Agriculture and
Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (grant number
17
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Figure 1. Compositions of the fecal microbiota of the lighter and heavier piglets. The number
of phyla (A) and genera (B) shared by the two groups are shown in Venn diagrams. The
overall compositions of the fecal microbiota of the lighter and heavier groups were
represented as bar plots at the phylum level (C) and the genus level (D).
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Figure 2. Comparison of the compositions of the fecal microbiota of the lighter and heavier
piglets at the phylum level. (A) Principal component analysis (PCA) plot. (B) The phyla
represented at significantly different levels in the microbiota of two groups are shown in an
extended error bar plot. The differences in the phylum level compositions were tested using a
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Figure 3. Comparison of the compositions of the fecal microbiota of the lighter and heavier
piglets at the genus level. (A) Principal component analysis (PCA) plot. (B) The genera
represented at significantly different levels in the microbiota of the two groups are shown in
an extended error bar plot. The differences in the genus level compositions were tested using
The microbial functions were predicted using PICRUSt at the third level of the KEGG
pathway and were expressed as relative abundances. The differences between the levels of
the predicted functions were tested using a two-sided Welchs t-test, and P < 0.05 was
considered significant.
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Tables
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Table 2. Characteristics of the weaned piglets used in this study
Piglet Body weight (kg) Group Age (week)
L1 8.09 Lighter 9
L2 8.22 Lighter 9
L3 9.70 Lighter 9
L4 10.18 Lighter 9
L5 10.23 Lighter 9
L6 11.05 Lighter 9
L7 11.17 Lighter 9
L8 11.23 Lighter 9
L9 11.89 Lighter 9
H1 16.70 Heavier 9
H2 17.01 Heavier 9
H3 17.13 Heavier 9
H4 17.45 Heavier 9
H5 17.98 Heavier 9
H6 18.78 Heavier 9
H7 20.70 Heavier 9
H8 22.50 Heavier 9
H9 22.75 Heavier 9
The piglets were sorted in ascending order by their body weights
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Table 3. Differences in the fecal microbial diversity of the two groups
Item Lighter Heavier P value a
Body Weight (kg) 10.20 1.33 19.00 2.38 < 0.001 ***
Alpha diversity b
ACE 16,243.60 1,720.96 18,642.29 2,605.18 0.037 *
Chao1 15,628.56 1,309.44 17,485.92 1,887.86 0.029 *
Observed OTUs 2,257.08 100.19 2,452.78 180.69 0.014 *
PD 174.82 10.70 181.56 10.95 0.205
Shannon 9.41 0.41 9.63 0.29 0.208
Simpson 0.99 0.01 0.99 0.00 0.935
The data were expressed as the mean values standard deviation (SD)
a
The P values were determined using Welchs t-test (* P < 0.05; ** P < 0.01; *** P < 0.001)
b
The alpha diversity indices were calculated from 5,000 sequence reads with 10 iterations
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Table 4. Differences in the fecal microbiota of the two groups
Taxon Lighter Heavier P value a
Phylum (%)
Firmicutes 48.42 2.38 53.59 4.42 0.009 **
Planctomycetes 0.01 0.01 0.03 0.02 0.019 *
Bacteroidetes 39.31 3.39 34.15 4.88 0.020 *
FB ratio 1.24 0.16 1.61 0.32 0.010 **
Genus (%)
Anaerococcus 0.01 0.01 0.02 0.01 0.024 *
Lactococcus 0.01 0.01 0.00 0.00 0.025 *
Anaerotruncus 0.03 0.02 0.01 0.01 0.034 *
[Eubacterium] 0.25 0.13 0.14 0.05 0.035 *
Sediminibacterium 0.00 0.00 0.01 0.01 0.038 *
Bilophila 0.02 0.02 0.00 0.00 0.040 *
Bacteroides 4.54 1.75 2.65 1.84 0.040 *
Butyrivibrio 0.05 0.03 0.14 0.12 0.045 *
[Prevotella] 3.13 0.67 2.21 1.09 0.049 *
Corynebacterium 0.07 0.03 0.05 0.03 0.049 *
Streptococcus 0.36 0.13 0.52 0.19 0.056
The data were expressed as the mean values standard deviation (SD)
a
The P values were determined using Welchs t-test (* P < 0.05; ** P < 0.01)
31