Accepted Manuscript

Title: Evaluating the association between body weight and the

intestinal microbiota of weaned piglets via 16S rRNA
sequencing

Author: Geon Goo Han Jun-Yeong Lee Gwi-Deuk Jin Jongbin

Park Yo Han Choi Byung Jo Chae Eun Bae Kim Yun-Jaie Choi

PII: S0378-1135(16)30504-1
DOI: http://dx.doi.org/doi:10.1016/j.vetmic.2016.10.020
Reference: VETMIC 7420

To appear in: VETMIC

Received date: 19-8-2016

Accepted date: 14-10-2016

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

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
Evaluating the association between body weight and the intestinal microbiota of weaned

piglets via 16S rRNA sequencing

Geon Goo Hana, Jun-Yeong Leea, Gwi-Deuk Jinb, Jongbin Parkc, Yo Han Choib, Byung Jo

Chaeb, Eun Bae Kimb,d,* and Yun-Jaie Choia,e,*

a
Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of

Korea

b
Department of Animal Life Science, Kangwon National University, Chuncheon, Gangwon-

do, Republic of Korea

c
Department of Animal Life System, Kangwon National University, Chuncheon, Gangwon-

do, Republic of Korea

d
Division of Applied Animal Science, Kangwon National University, Chuncheon, Gangwon-

do, Republic of Korea

e
Research Institute for Agriculture and Life Science, Seoul National University, Seoul,

Republic of Korea

*
Corresponding authors with equal contribution

Yun-Jaie Choi: E-mail: cyjcow@snu.ac.kr

Eun Bae Kim: E-mail: itanimal@kangwon.ac.kr

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

had significantly higher levels of opportunistic pathogenic bacteria, such as Anaerotruncus

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

the swine industry.

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

(known as super bacteria), such as methicillin-resistant Staphylococcus aureus (MRSA) and

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

through administering prebiotics and probiotics is considered an effective strategy by many

researchers. To develop an effective strategy for modulating the intestinal microbiota,

3
understanding the relationship between the intestinal microbial composition and the

physiology of the host, particularly regarding growth performance, is very important.

The development of high-throughput sequencing technologies, such as 454 pyrosequencing

and Illumina sequencing, has enabled culture-independent analysis of the composition of

microbial communities, and many researchers have reported host-microbe interactions,

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

insufficient knowledge of this topic at present.

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

microbiota of lighter and heavier piglets.

4
2. Materials and methods

2.1. Sample preparation

Nine-week-old weaned LYD piglets raised on a local commercial farm (Gangneung,

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.

2.2. DNA extraction and sequencing

DNA was extracted from 250 mg of each fecal sample using a NucleoSpinSoil Kit

(Macherey-Nagel, Dren, Germany), according to the manufacturers protocol, and was

stored at -20 C until further analysis. The V4 region of the bacterial 16S rRNA gene, which

provides sufficient information regarding the phylogenetic diversity of microbial

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,

Japan) and universal primers (forward: 5-GGACTACHVGGGTWTCTAAT-3 and reverse:

5-GTGCCAGCMGCCGCGGTAA-3). The amplification program consisted of 1 cycle of

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.

2.3. Microbial community analysis

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

sequences were aligned using the PyNAST program.

The microbial diversity indices of the samples (alpha diversity) were determined using the

abundance-based coverage estimator (ACE), Chao1, observed OTUs, phylogenetic diversity

(PD), Shannon, and Simpson methods, and these indices were calculated from 5,000

sequence reads through rarefaction, with 10 iterations.

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.

2.4. Prediction of the functions of the microbial communities

The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States

(PICRUSt) version 1.0.0 (http://picrust.github.io/picrust/) program was used to predict the

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

14,437 ( 8,480) reads per piglet in the heavier group.

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

lighter group, although the differences were not significant.

3.2. Differences in the microbial taxa represented in the intestinal microbiota of the

lighter and heavier piglets

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

shared 24 phyla (92.31%), with members of Acidobacteria and Armatimonadetes uniquely

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

(18.86%), Lactobacillus (7.66%), and Faecalibacterium (3.01%). We then compared the

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

level (Fig. 3A).

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

group (Fig. 3B). In contrast, the levels of Lactococcus, Anaerotruncus, [Eubacterium],

Bilophila, Bacteroides, [Prevotella], and Corynebacterium were significantly higher in the

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

metabolism, and novobiocin biosynthesis pathways were predicted at significantly higher

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-

diaminopimelic acid and muramyl dipeptide, as part of a pathogen-associated molecular

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

benzoate degradation) related genes belonging to the xenobiotics biodegradation 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,

Bacteroidetes, Anaerotruncus, and Bacteroides). Additional, the levels of genes related to

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

growth performance of piglets through host-microbe interactive processes, such as 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

The authors declare that they have no conflicts 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

914005-04). Geon Goo Han was supported by the BK21 program.

17
References

Bahr, S.M., Tyler, B.C., Wooldridge, N., Butcher, B.D., Burns, T.L., Teesch, L.M., Oltman,

C.L., Azcarate-Peril, M.A., Kirby, J.R., Calarge, C.A., 2015. Use of the second-

generation antipsychotic, risperidone, and secondary weight gain are associated with

an altered gut microbiota in children. Transl. Psychiatr. 5, e652.

doi:10.1038/tp.2015.135.

Bindels, L.B., Neyrinck, A.M., Claus, S.P., Le Roy, C.I., Grangette, C., Pot, B., Martinez, I.,

Walter, J., Cani, P.D., Delzenne, N.M., 2016. Synbiotic approach restores intestinal

homeostasis and prolongs survival in leukaemic mice with cachexia. ISME J. 10,

1456-1470. doi:10.1038/ismej.2015.209.

Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Lozupone, C.A., Turnbaugh,

P.J., Fierer, N., Knight, R., 2011. Global patterns of 16S rRNA diversity at a depth of

millions of sequences per sample. Proc. Natl. Acad. Sci. U. S. A. 108 Supplement 1,

4516-4522. doi:10.1073/pnas.1000080107.

Carroll, J.A., Forsberg, N.E., 2007. Influence of stress and nutrition on cattle immunity. Vet.

Clin. N. Am.-Food. Anim. Pract. 23, 105-+. doi:10.1016/j.cvfa.2007.01.003.

Choi, V.M., Herrou, J., Hecht, A.L., Teoh, W.P., Turner, J.R., Crosson, S., Wardenburg, J.B.,

2016. Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes

to anaerobic sepsis in mice. Nat. Med. 22, 563-567. doi:10.1038/nm.4077.

Clarke, S.F., Murphy, E.F., O'Sullivan, O., Lucey, A.J., Humphreys, M., Hogan, A., Hayes, P.,

O'Reilly, M., Jeffery, I.B., Wood-Martin, R., Kerins, D.M., Quigley, E., Ross, R.P.,

O'Toole, P.W., Molloy, M.G., Falvey, E., Shanahan, F., Cotter, P.D., 2014. Exercise

and associated dietary extremes impact on gut microbial diversity. Gut 63, 1913-1920.

18
 doi:10.1136/gutjnl-2013-306541.

de Goffau, M.C., Fuentes, S., van den Bogert, B., Honkanen, H., de Vos, W.M., Welling,

G.W., Hyoty, H., Harmsen, H.J.M., 2014. Aberrant gut microbiota composition at the

onset of type 1 diabetes in young children. Diabetologia 57, 1569-1577. doi:

10.1007/s00125-014-3274-0.

Delzenne, N.M., Cani, P.D., 2011. Interaction between obesity and the gut microbiota:

Relevance in nutrition. Annu. Rev. Nutr. 31, 15-31. doi:10.1146/annurev-nutr-072610-

145146.

Everard, A., Lazarevic, V., Derrien, M., Girard, M., Muccioli, G.G., Neyrinck, A.M.,

Possemiers, S., Van Holle, A., Francois, P., de Vos, W.M., Delzenne, N.M., Schrenzel,

J., Cani, P.D., 2011. Responses of gut microbiota and glucose and lipid metabolism to

prebiotics in genetic obese and diet-induced leptin-resistant mice. Diabetes 60, 2775-

2786. doi:10.2337/db11-0227.

Han, G.G., Kim, E.B., Lee, J., Lee, J.Y., Jin, G., Park, J., Huh, C.S., Kwon, I.K., Kil, D.Y.,

Choi, Y.J., Kong, C., 2016. Relationship between the microbiota in different sections

of the gastrointestinal tract, and the body weight of broiler chickens. SpringerPlus 5,

911. doi:10.1186/s40064-016-2604-8.

Haro, C., Rangel-Zuniga, O.A., Alcala-Diaz, J.F., Gomez-Delgado, F., Perez-Martinez, P.,

Delgado-Lista, J., Quintana-Navarro, G.M., Landa, B.B., Navas-Cortes, J.A., Tena-

Sempere, M., Clemente, J.C., Lopez-Miranda, J., Perez-Jimenez, F., Camargo, A.,

2016. Intestinal microbiota is influenced by gender and body mass index. PLoS One

11, e0154090. doi:10.1371/journal.pone.0154090.

Heo, J.M., Opapeju, F.O., Pluske, J.R., Kim, J.C., Hampson, D.J., Nyachoti, C.M., 2013.

Gastrointestinal health and function in weaned pigs: a review of feeding strategies to

19
 control post-weaning diarrhoea without using in-feed antimicrobial compounds. J.

Anim. Physiol. Anim. Nutr. 97, 207-237. doi:10.1111/j.1439-0396.2012.01284.x.

Hildebrandt, M.A., Hoffmann, C., Sherrill-Mix, S.A., Keilbaugh, S.A., Hamady, M., Chen,

Y.Y., Knight, R., Ahima, R.S., Bushman, F., Wu, G.D., 2009. High-fat diet determines

the composition of the murine gut microbiome independently of obesity.

Gastroenterology 137, 1716-1724. doi:10.1053/j.gastro.2009.08.042.

Hu, H.J., Park, S.G., Jang, H.B., Choi, M.G., Park, K.H., Kang, J.H., Park, S.I., Lee, H.J.,

Cho, S.H., 2015. Obesity alters the microbial community profile in Korean

adolescents. PLoS One 10, e0138015. doi:10.1371/journal.pone.0138015.

Jalanka-Tuovinen, J., Salonen, A., Nikkila, J., Immonen, O., Kekkonen, R., Lahti, L., Palva,

A., de Vos, W.M., 2011. Intestinal microbiota in healthy adults: Temporal analysis

reveals individual and common core and relation to intestinal symptoms. PLoS One 6,

e23035. doi:10.1371/journal.pone.0023035.

Kasai, C., Sugimoto, K., Moritani, I., Tanaka, J., Oya, Y., Inoue, H., Tameda, M., Shiraki, K.,

Ito, M., Takei, Y., Takase, K., 2015. Comparison of the gut microbiota composition

between obese and non-obese individuals in a Japanese population, as analyzed by

terminal restriction fragment length polymorphism and next-generation sequencing.

BMC Gastroenterol. 15, 100. doi:10.1186/s12876-015-0330-2.

Lau, S.K.P., Woo, P.C.Y., Woo, G.K.S., Fung, A.M.Y., Ngan, A.H.Y., Song, Y., Liu, C.,

Summanen, P., Finegold, S.M., Yuen, K., 2006. Bacteraemia caused by Anaerotruncus

colihominis and emended description of the species. J. Clin. Pathol. 59, 748-752.

doi:10.1016/jcp.2005.031773.

Le Chatelier, E., Nielsen, T., Qin, J.J., Prifti, E., Hildebrand, F., Falony, G., Almeida, M.,

Arumugam, M., Batto, J.M., Kennedy, S., Leonard, P., Li, J.H., Burgdorf, K., Grarup,
20
 N., Jorgensen, T., Brandslund, I., Nielsen, H.B., Juncker, A.S., Bertalan, M., Levenez,

F., Pons, N., Rasmussen, S., Sunagawa, S., Tap, J., Tims, S., Zoetendal, E.G., Brunak,

S., Clement, K., Dore, J., Kleerebezem, M., Kristiansen, K., Renault, P., Sicheritz-

Ponten, T., de Vos, W.M., Zucker, J.D., Raes, J., Hansen, T., Bork, P., Wang, J.,

Ehrlich, S.D., Pedersen, O., Consortium, M., 2013. Richness of human gut

microbiome correlates with metabolic markers. Nature 500, 541-+.

doi:10.1038/nature12506.

Ley, R.E., Turnbaugh, P.J., Klein, S., Gordon, J.I., 2006. Microbial ecology: human gut

microbes associated with obesity. Nature 444, 1022-1023.

doi:10.1038/nature4441022a.

Liu, L., Qin, D.K., Wang, X.F., Feng, Y., Yang, X.J., Yao, J.H., 2015. Effect of immune stress

on growth performance and energy metabolism in broiler chickens. Food Agric.

Immunol. 26, 194-203. doi:10.1080/09540105.2014.882884.

Louis, S., Tappu, R.M., Damms-Machado, A., Huson, D.H., Bischoff, S.C., 2016.

Characterization of the gut microbial community of obese patients following a

weight-loss intervention using whole metagenome shotgun sequencing. PLoS One 11,

e0149564. doi:10.1371/journal.pone.0149564.

Mejia-Leon, M.E., Petrosino, J.F., Ajami, N.J., Dominguez-Bello, M.G., de la Barca, A.M.,

2014. Fecal microbiota imbalance in Mexican children with type 1 diabetes. Sci. Rep.

4, 3814. doi:10.1038/srep03814.

Ngo, J.T., Parkins, M.D., Gregson, D.B., Pitout, J.D.D., Ross, T., Church, D.L., Laupland,

K.B., 2013. Population-based assessment of the incidence, risk factors, and outcomes

of anaerobic bloodstream infections. Infection 41, 41-48. doi:10.1007/s15010-012-

0389-4.
21
Parks, D.H., Tyson, G.W., Hugenholtz, P., Beiko, R.G., 2014. STAMP: statistical analysis of

taxonomic and functional profiles. Bioinformatics 30, 3123-3124.

doi:10.1093/bioinformatics/btu494.

Redondo, M.C., Arbo, M.D.J., Grindlinger, J., Snydman, D.R., 1995. Attributable mortality

of bacteremia associated with the Bacteroides-fragilis group. Clin. Infect. Dis. 20,

1492-1496.

Rhee, K.J., Wu, S.G., Wu, X.Q., Huso, D.L., Karim, B., Franco, A.A., Rabizadeh, S., Golub,

J.E., Mathews, L.E., Shin, J., Sartor, R.B., Golenbock, D., Hamad, A.R., Gan, C.M.,

Housseau, F., Sears, C.L., 2009. Induction of persistent colitis by a human commensal,

enterotoxigenic Bacteroides fragilis, in wild-type C57BL/6 mice. Infect. Immun. 77,

1708-1718. doi:10.1128/IAI.00814-08.

Robert, R., DeRaignac, A., Le Moal, G., Ragot, S., Grollier, G., 2008. Prognostic factors and

impact of antibiotherapy in 117 cases of anaerobic bacteraemia. Eur. J. Clin.

Microbiol. Infect. Dis. 27, 671-678. doi:10.1007/s10096-008-0487-5.

Turnbaugh, P.J., Baeckhed, F., Fulton, L., Gordon, J.I., 2008. Diet-induced obesity is linked

to marked but reversible alterations in the mouse distal gut microbiome. Cell Host

Microbe 3, 213-223. doi:10.1016/j.chom.2008.02.015.

Turnbaugh, P.J., Hamady, M., Yatsunenko, T., Cantarel, B.L., Duncan, A., Ley, R.E., Sogin,

M.L., Jones, W.J., Roe, B.A., Affourtit, J.P., Egholm, M., Henrissat, B., Heath, A.C.,

Knight, R., Gordon, J.I., 2009. A core gut microbiome in obese and lean twins. Nature

457, 480-484. doi:10.1038/nature07540.

Walters, W.A., Xu, Z., Knight, R., 2014. Meta-analyses of human gut microbes associated

with obesity and IBD. FEBS Lett. 588, 4223-4233. doi:10.1016/j.febslet.2014.09.039.

Wells, J.M., Rossi, O., Meijerink, M., van Baarlen, P., 2011. Epithelial crosstalk at the
22
 microbiota-mucosal interface. Proc. Natl. Acad. Sci. U. S. A. 108 Supplement 1,

4607-4614. doi:10.1073/pnas.1000092107.

Wierup, M., 2001. The Swedish experience of the 1986 year ban of antimicrobial growth

promoters, with special reference to animal health, disease prevention, productivity,

and usage of antimicrobials. Microb. Drug Resist. 7, 183-190.

doi:10.1089/10766290152045066.

Yang, H., Huang, X.C., Fang, S.M., Xin, W.S., Huang, L.S., Chen, C.Y., 2016. Uncovering

the composition of microbial community structure and metagenomics among three gut

locations in pigs with distinct fatness. Sci. Rep. 6, 27427. doi:10.1038/srep27427.

Zhao, L., Wang, G., Siegel, P., He, C., Wang, H., Zhao, W., Zhai, Z., Tian, F., Zhao, J., Zhang,

H., Sun, Z., Chen, W., Zhang, Y., Meng, H., 2013. Quantitative genetic background of

the host influences gut microbiomes in chickens. Sci. Rep. 3, 1163.

doi:10.1038/srep01163.

Zhu, Q., Gao, R., Wu, W., Qin, H., 2013. The role of gut microbiota in the pathogenesis of

colorectal cancer. Tumor Biol. 34, 1285-1300. doi:10.1007/s13277-013-0684-4.

23
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).

24
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

two-sided Welchs t-test, and P < 0.05 was considered significant.

25
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

a two-sided Welchs t-test, and P < 0.05 was considered significant.

26
Figure 4. The different functions of the fecal microbiota of the lighter and heavier piglets.

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.

27
Tables

Table 1. Composition of the commercial diet of the weaned piglets

Item Contents, %
Corn 39.04
Oat 5.00
Wheat bran 2.00
Dried sweets by-products 4.00
Glucose 3.00
Whey powder 10.00
Lactose 4.00
Fish meal 4.00
Dehulled soybean meal 21.70
Soy oil 3.00
Monocalcium phosphate 0.65
Limestone 1.60
Salt 0.25
Lysine 0.26
Methionine 0.09
Threonine 0.06
Vitamin 0.30
Mineral 0.20
Choline chloride 0.10
Enzyme complex 0.10
Organic acids 0.20
Phytase 0.05
Emulsifier 0.10
Zinc oxide 0.30

28
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

29
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

30
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