Académique Documents
Professionnel Documents
Culture Documents
of Microbiology
of Medicine
3Center for Health Informatics and Bioinformatics
4Department of Radiology
5Departments of Population Health (Biostatistics) and Environmental Medicine
NYU Langone Medical Center, New York, NY 10016, USA
6Department of Biomedical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA 01536, USA
7Department of Biology, NYU Abu Dhabi, Abu Dhabi, United Arab Emirates
8New York Genome Center, New York, NY 10013, USA
9New York Harbor Department of Veterans Affairs Medical Center, New York, NY 10010, USA
*Correspondence: martin.blaser@nyumc.org
http://dx.doi.org/10.1016/j.cell.2014.05.052
2Department
SUMMARY
LDP-w
5 6
LDP-b
-3
5 4
0
12
16
108
106
104
102
20
100
Age (weeks)
F
Lean Mass
G
Fat Mass
**
grams
20
18
14
Male
Female
0.40
0.35
16
0.30
Male
Female
Male
16
Female
Male
Bone Area
Female
9.0
24
20
0.2
9.5
0.45
0.4
8.5
**
8.0
* **
0.050
7.5
Male
0.045
7.0
Female
Male
Male
Female
Female
MRI n = 3 males
30
20
10
0.18
0.12
0.06
0.00
Total body
M 12
Abdominal slice
fat volume (cc)
grams
22
0.6
28
0.8
0.0
Female
0.50
grams
24
Male
1.0
Total Mass
32
Control
LDP-b
grams
Control
Control
LDP-w
LDP-b
Day 21-28
1.2
g/cm2
# Mice
M F
6 5
copies/g sample
Total bacteria
1010
B
No Antibiotics
Low-dose penicillin at weaning
cm2
0.052
10
8
6
4
2
0
Liver
PPAR /GAPDH
*
CD36/GAPDH
3 10-05
2 10-05
1
10-05
5.0 10-02
0.0
APOA4/GAPDH
SREBP/GAPDH
Lean
Fat
Volume of Interest
1.5 10-01
1.0 10-01
3 10-04
2 10-04
1
10-04
0
4 10-04
FABP2/GAPDH
4 10-05
8 10-01
6 10-01
4 10-01
2 10-01
0
5 10-02
4 10-02
3 10-02
2 10-02
1 10-02
0
DGAT2/GAPDH
2.5 10-01
2.0 10-01
1.5 10-01
1.0 10-01
5.0 10-02
0.0
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from the earliest time point studied (3 weeks) to the end of the
experiment (28 weeks) (Figures 6A6C, ADONIS test, Table
S4). However, after antibiotic cessation, community structure
progressively reverted to normal in the fecal, cecal, and ileal
samples. To assess community structure patterns according to
age, we examined intragroup phylogenetic distances. In infancy,
the microbiota of control mice are highly diverse, becoming more
homogeneous as mice age, a pattern that is also observed in all
LDP groups (Figure 6D and Table S5). After antibiotic cessation,
microbiota from the 4-LDP and 8-LDP mice become no more
distant from the control mice than the intragroup control variation, demonstrating recovery; however, the 28- (continuous)
LDP microbiota divergence never normalized (Figure 6E and
Table S5), and in the complementary analyses, as expected,
the opposite pattern was observed (Figure 6F). All told, the
community analyses demonstrate early-life individual host
microbiota variability that decreases during maturation and
that microbial populations are only transiently disrupted by
limited duration LDP, yet the effects on phenotype are lifelong
(Figure 5B).
LDP induced numerous compositional changes in the microbiota (Figure 6G), and HFD had further effects. After HFD was
started, control mouse Allobaculum markedly increased with
concomitant decreases in Akkermansia mucinophila and an
unidentified Bacteroidales S24-7 family member. After antibiotic
cessation in the 4-LDP and 8-LDP mice, the patterns associated
with HFD exposure of the control mice began to emerge but were
never present in the 28-LDP mice. The persistent phenotypes
after LDP cessation, despite microbiota normalization, provide
evidence that the early-life microbiota influences adult body
composition. LDP suppressed early-life Lactobacillus, Allobaculum, Rikenellaceae, and Candidatus Arthromitus (SFB) (Figures
6H and 6I) as observed in the prior experiments (Table S1),
further implicating them as candidate protective taxa; these
organisms were positively correlated with markers of ileal immunity (Figure 5J).
LDP-Altered Microbiota Are Sufficient to Induce
Metabolic Changes
Although the above observations suggest that the change in the
microbiota is driving the metabolic and immunologic effects,
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No antibiotics
Low-dose penicillin
Microbiome sample
35
Mass (g)
Control
4-LDP
15
28-LDP
0
-3
12
16
20
24
28
16
**
** *
20
Fat
* *
*
*
10
12
10
30
10
20
30
10
20
30
Age (week)
*
*
14
10
15
Lean
18
4-LDP, n = 9
8-LDP, n = 12
28-LDP, n = 8
Control, n = 13
10
Age (week)
0, none
200
2, moderate
Ileal Atrophy
3, severe
*
Atrophy Score
kcal
** *
**
*
20
20
*
* *
*
*
25
8-LDP
Total
30
High
Fat
Diet
Normal Chow
Nursing
Gestation
180
160
140
120
100
Cd74
H2Aa
Ikzf2
Il7
Cd226
Cd8a
Runx3
Cd274
Icos
Ccl22
Spn
Thy1
Irf4
Cd3e
Cd6
Sh2d1a
Cd5
Il2ra
Klrk1
Pdcd1
Tnfsf8
Prf1
Marco
Il2rb
Cd3d
Cd7
Pou2f2
Lck
Slamf7
C4a
Fyn
Psmb9
Syk
Irf8
H2DMa
H2Eb1
H2Ab1
Control
LDP
Female
1.5 10-03
1.0 10-03
0.11
5.0 10-04
LDP
10-04
1 10-04
LDP
2 10-03
0.003
0.21
2
1
0
Ctl
LDP
Female
Ctl
LDP
Male
0.45
Ctl
LDP
Male
LDP
Female
Ctl
LDP
Male
LDP
Ctl
LDP
Female
Male
0.10
0.0002
N
1.5 10-03
6 10-05
0.016
0.06
1.0 10-03
5.0 10-04
4 10-05
2 10-05
0.0
Ctl
Ctl
LDP
Ctl
Female
LDP
Ctl
Male
LDP
Female
Ctl
LDP
Male
Colon
LDP
IL-17
*
4
3
2
1
0
LDP
Female
10-06
Control
0
Ctl
Ctl
Ileum
4
3
LDP
Male
5.0 10-06
Control
LDP
Ctl
LDP
Male
IL-22
0.8
0.6
0.4
0.2
0.0
Control LDP
IL-17
0.8
Control LDP
3
2
1
0
Control LDP
% CD4+ production
0.31
4 10-03
Ctl
Female
LDP
Female
Ctl
1.0 10-05
0.0
Ctl
0.50
0.015
1.5 10-05
IL-17A
P
5
LDP
6 10-06
4
2 10-05
Male
10-06
2 10-06
Ctl
1 10-05
8
3 10-04
2
LDP
Female
0.97
0.93
0.052
2.0 10-05
Ctl
6 10-05
4 10-05
Th17
2.5 10-05
8 10-05
% CD4+ production
6 10-03
LDP
0.055
10-04
% CD4+ production
0.013
Reg3 /GAPDH
Defb1/GAPDH
8 10-03
2 10-05
Male
5 10-04
Antimicrobial peptides
Ctl
Female
1 10-04
0.007
4 10-05
0.0
Ctl
0.30
6 10-05
% CD4+ production
Control
LDP
Male
2.0 10-03
GATA3/GAPDH
T-bet/GAPDH
IFN /GAPDH
LDP
J
0.28
ROR t/GAPDH
0.01
2.5 10-03
2 1
Treg
I
Foxp3/GAPDH
Row ZScore
Th2
IL-17F/GAPDH
Th1
TGF 1/GAPDH
IL-4/GAPDH
Control
LDP
IL-22
Control
IL-22
*
0.6
0.4
0.2
0.0
Control LDP
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involvement in initial immunologic programming, other organisms also stimulate Th17 responses because SFB were absent
from both control and LDP-microbiota recipients, which show
altered Th17 markers. Two (Lactobacillus and Allobaculum) of
the three other candidate protective taxa positively correlate
with RORgT and IL-17 expression, suggesting roles in intestinal
Th17 differentiation. However, although there are fewer Th17
cells and diminished expression of genes involved in intestinal
immune responses after LDP, our observations do not provide
causal evidence that LDP is directly altering intestinal immune
responses. Nevertheless, these findings are consistent with prior
studies that have identified impaired intestinal barrier function
and immunity as important factors in the development of metabolic syndromes (Cani et al., 2008; Henao-Mejia et al., 2012;
Vijay-Kumar et al., 2010).
The metabolic consequences observed indicate that microbiota disruption during a critical developmental window can
explain later life phenomena, including adult obesity. LDP may
decrease particular early-life-protective bacterial populations,
which is consistent with coevolution with microbes having specific interactions promoting metabolic fitness (Blaser, 2006). As
keystone species, their reduction could lead to broad intestinal ecologic changes, permitting increases in taxa having alternate roles in metabolic or immunologic development (Chung
et al., 2012). That the microbiota recovers, yet the phenotype remains, suggests that microbiota changes can affect metabolic
programming; studies examining epigenetic regulation could
help elucidate mechanisms of host-microbe interaction.
Epidemiologic studies provide evidence that antibiotic exposures in human infancy can lead to increased weight later in
life (Ajslev et al., 2011; Murphy et al., 2013; Trasande et al.,
2013). Combining multiple mouse models with high-throughput
microbial 16S rRNA profiling, we identified key microbiota members impacted by antibiotic treatment and linked with host physiologic changes. If humans have analogous organisms, their loss
in early life due to collateral antibiotic effects may have life-long
consequences. Restoration of lost taxa following early-life antibiotic exposures is a potential strategy to reverse MIO and related
sequelae. Nevertheless, humans have greater dietary and lifestyle variation than do experimental mice, which introduces
factors that affect recovery from metabolic disruption or susceptibility to weight gain. Further studies are needed to examine
specific metabolic interactions of key members of the early-life
microbiota and to determine whether these changes are important in humans.
EXPERIMENTAL PROCEDURES
Animals
All animal experiments were performed according to IACUC-approved protocols. LDP was delivered via drinking water as described (Cho et al., 2012).
Control mice did not receive antibiotics. Diets used in the studies were either
NC (Purina Mills International Diet 5001, 13.5% kcal from fat), or HFD (Rodent
Diet D12451, Research Diets, 45% kcal from fat). For the microbiota transfer
experiments, cecal contents were collected from three microbiota donors
from each group and placed in a prereduced anaerobically sterilized liquid
dental transport medium (Anaerobe Systems, Morgan Hill), pooled in sterile
prereduced saline under anaerobic conditions, and then transferred to 3- to
4-week-old germ-free Swiss Webster mice via oral gavage (control recipients
n = 7; LDP recipients n = 8). The microbiota recipient mice were housed in
autoclaved cages under specific pathogen-free conditions and fed irradiated
HFD, as the donor mice had been. See Extended Experimental Procedures
for details on individual experiments.
Body Composition Analysis
Body composition was determined using dual energy X-ray absorptiometry
(DEXA) with a Lunar PIXImus II mouse densitometer (GE Medical Systems,
Waukesha) and by MRI on a 7-tesla MRI system (Bruker Biospin MRI,
Ettlingen). For the MRI, fat and lean tissues were determined using the iterative
decomposition of water and fat with an echo asymmetry and least-squares
(IDEAL) method (Reeder et al., 2004, 2005).
Hepatic and Ileal Gene Expression Profiling
Total RNA was extracted by the RNeasy Mini Kit (QIAGEN, Valencia). For
microarrary analysis, expression profiling was performed using the Affymetrix
Genechip Chip Mouse 430_2 system (Affymetrix, Santa Clara). The raw microarray data were normalized using the Robust Multi-Array (RMA) method
(Irizarry et al., 2003). The Limma package in R was used to fit a linear
model to the expression data using empirical Bayes moderated t statistics
(Smyth, 2004). RNA-seq libraries were generated using the TruSeq v2 RNA
sample prep (Illumina, San Diego); libraries were sequenced with 2 3 50 bp
paired-end reads in an Illumina HiSeq 2500. Fastq files were processed as
follows: alignment was done with STAR (Dobin et al., 2013) with an index
build from the mm10 reference sequence. The genes annotated in UCSC
mm10 were quantified with HTSeq-count v0.5.3p9 (Anders et al., 2014).
Differential expression analysis was performed with the Bioconductor package DESeq (Anders and Huber, 2010). Ileal gene expression was measured
by the nCounter GX Mouse Immunology Kit (Nanostring Technologies,
Seattle).
Flow Cytometry
Lamina propria mononuclear cells were isolated from the ileum and colon.
Cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA)
and 500 ng/ml Ionomycin for 4 hr at 37 C in the presence of brefeldin A
(GolgiPlug, Becton Dickinson, Franklin Lakes). Following this in vitro stimulation, cells were stained with LIVE/DEAD Fixable Aqua (Life Technologies),
anti-CD3, and anti-CD4 and fixed in 4% paraformaldehyde in PBS and then
permeabilized in Perm/Wash buffer (Becton Dickinson) and stained with
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ACKNOWLEDGMENTS
We acknowledge assistance with sequencing from Argonne National Laboratory and NYULMC Genome Technology Center and help from the NYULMC
Histology Core and the New York Genome Center. These studies were supported by NIH grants R01 DK 090989 and 1UL1RR029893, the Diane Belfer
Program in Human Microbial Ecology, Knapp Family Foundation, and Daniel
Ziff Foundation.
Received: December 19, 2013
Revised: April 21, 2014
Accepted: May 27, 2014
Published: August 14, 2014
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Supplemental Information
EXTENDED EXPERIMENTAL PROCEDURES
Animals
All animal experiments were performed according to an IACUC-approved protocol, with mice housed in specific pathogen-free
conditions, and maintained on a 12 hr light/dark cycle. In all experimental variations, low-dose penicillin (LDP) was delivered via
penicillin in the drinking water at a dose of 6.67 mg/l, to deliver approximately 1 mg per kg body weight (LDP, low dose penicillin)
based on the expected fluid consumption of mice, which is in the middle of the FDA approved range of sub-therapeutic antibiotic
treatment (http://www.fda.gov/animalveterinary/products/approvedanimaldrugproducts/default.htm). Control mice did not receive
antibiotics. Diets used in the studies were either normal chow (NC, Purina Mills International Diet # 5001, 13.5% kcal from fat), or
high fat diet (HFD, Rodent Diet D12451, Research Diets, 45% kcal%). Breeder C57BL/6 mice (Jackson Labs, Bar Harbor ME)
were obtained at 6-8 weeks of age and experimental animals were bred in the NYU facility, with 2-4 litters per experimental group.
Germ-free Swiss-Webster mice (Taconic Farms, Germantown NY) were obtained at 3-4 weeks of age and colonized with defined
microbiota in the NYU animal facility. At the end of each experiment, mice were humanely euthanized with CO2 narcosis for specimen
collection.
Time-LDP: The Effect of Timing of Initial LDP Exposure, Related to Figure 1
C57BL/6J mice received low-dose penicillin (LDP) either at weaning at 4 weeks of age (LDP-w, n = 5 males, 6 females) or their
mothers were started on LDP 1 week prior to birth and continuously maintained on penicillin (LDP-b, n = 5 males, 4 females). Control
mice did not receive antibiotics (n = 6 males, 5 females) and all mice were fed NC. Body composition was measured by dual energy
X-ray absorptiometry at 20 weeks of age, and by MRI at 18 weeks of age.
Dyna-LDP: Dynamics of Phenotype Development, Related to Figures 2 and S1
C57BL/6J mice were exposed to LDP at birth through 30 weeks of age (n = 10 males, 10 females), control mice did not receive
antibiotics (n = 9 males, 10 females), and all mice were fed NC. DEXA scanning was done at 8, 12, 16, 20, 26, and 30 weeks of
age. At 30 weeks of age, at sacrifice, cecal and ileal samples and serum samples were collected and stored at 80 C.
Fat-LDP: The Effect of LDP and High-Fat Diet on Growth Promotion and Adiposity, Related to Figures 3, 4, and S1S3
C57BL/6J mice were exposed to LDP at birth through 30 weeks of age. All mice were weaned onto NC at 4 weeks of age and half the
mice were switched to HFD at 17 weeks of age (n = 8, 16 for control NC; 9, 10 for control HFD; 10, 10 for LDP NC; and 10, 10 for LDP
HFD for male and female mice, respectively). Body composition was serially measured by DEXA scanning from 6 to 30 weeks for all
mice, and by MRI at 26 weeks of age for the median 5-6 mice on HFD. Fasting serum samples and liver samples preserved in RNA
later were collected at sacrifice at 30 weeks of age. Liver gene expression at 30 weeks of age was analyzed by microarray (n = 3 from
each group).
Dura-LDP: Durability of LDP Phenotype with Limited Antibiotic Exposure, Related to Figures 5, 6, S1, S4, and S6
C57BL/6J mice received LDP for 4 weeks (4-LDP, n = 15 males, 9 females), 8 weeks (8-LDP, n = 14 males, 12 females), or continuous (28 weeks (28-LDP, n = 8) for females, 32 weeks (32-LDP, n = 13) for males). Controls did not receive LDP (n = 20 males,
13 females). Body composition was measured by DEXA scanning. Mice were weaned at 4 weeks of age onto NC then switched
to HFD at 6 weeks of age. Mice sacrificed: 5 control and continuous-LDP male mice at 4 weeks of age, 4 male and female, control
and continuous-LDP mice at 8 weeks of age, and 3 control and continuous-LDP female mice at 18-weeks of age, and the remaining
mice at 28 or 32 weeks for and males respectively. Cecal contents from 18-week old female control and LDP mice were used to
colonize germ-free mice.
Trans-LDP1: Transmissibility of LDP Phenotypes via Microbiota Transplant, Related to Figures 7, S6, and S7
Female C57BL/6J mice (from Dura-LDP) either received no antibiotics (control) or continuous LDP. At 18 weeks of age, after a change
in body composition was observed, 3 microbiota donors from each group (control n = 9, LDP n = 8) were selected based on median
weight. Donor mice were humanely euthanized, and the proximal third of the cecum was aseptically removed and immediately
placed in a pre-reduced anaerobically sterilized liquid dental transport medium (Anaerobe Systems, Morgan Hill CA) and brought
into an anaerobic chamber (Sheldon, Cornelius OR). The three cecal samples from each group (LDP or control) were pooled, and
pre-reduced saline was added to a final volume of 8 ml, of which 5 ml was frozen, and 3 ml was used for microbiota transfer.
Germ-free Swiss Webster mice (3- to 4- week old) were anesthetized using isoflurane, then 250 ml of either the control or LDP microbiota suspensions were placed in their stomachs (control n = 7, LDP n = 8) by oral gavage. To maintain viability of anaerobic organisms, vials containing the inoculum were not opened. Instead, using a sharp needle, the suspension was drawn through a rubber
Hungate cap, and then was replaced with a soft 20-gauge feeding tube (Fisher Science, Pittsburgh PA) prior to oral gavage. Recipients were chosen randomly, and the inocula alternated between control- and LDP-exposed microbiota recipients. Gloves were
changed between every inoculation. Mice awoke from anesthesia within minutes and no mouse exhibited ill effects from the microbiota transfer. The microbiota-recipient mice were housed in autoclaved cages, under specific pathogen-free conditions, and fed
HFD, as the donor mice had been. Fecal pellets and scale weights were taken 2-3 times per week. Body composition was assessed
Cell 158, 705721, August 14, 2014 2014 Elsevier Inc. S1
by DEXA scanning at 6, 23, 28, and 34 days post-transfer. Ileal tissues were collected at sacrifice and gene expression was measured
by qPCR. Cecal contents were collected for a sequential microbiota transplant.
Trans-LDP2: Transmissibility of LDP Phenotypes in a Successive Transfer, Related to Figure S7
Cecal contents were recovered from 3 control-microbiota recipients and 3 LDP-microbiota recipients from Trans-LDP1, and transferred to new 3- to 4-week old germ-free Swiss Webster mice (n = 6 each), as described above. Mice were fed HFD, longitudinal fecal
pellets were collected, and body composition was measured by serial DEXA scans. Mice were sacrificed 70 days post-transfer and
cecal contents were collected for microbiota analysis.
Statistical Comparison of Growth Rates
The longitudinal group means of total, fat, and lean mass were fitted with the following linear spline model with common knots at
week c.
EY = b0 + b1 group + b2 week + b3 week c + + b4 group 3 week + b5 group 3 week c +
where group = 0 indicates the control group and group = 1 indicates the LDP group, and (x)+ is defined as a function that equals x
when x is positive and is equal to zero otherwise. With this model, we performed group comparisons at baseline, before week c and to
examine patterns of change over time through tests H0: b1 = 0, H0: b4 = 0 and H0: b4 + b5 = 0, respectively. The value of c varies in
different data sets. Some data sets were fitted using a multiple knot model which was constructed similarly. The MIXED procedures
of SAS software (version 9.2; SAS Institute, Inc.) were used to perform the tests and calculate the estimates.
Body Composition Measurements
Body composition was determined using dual energy X-ray absorptiometry (DEXA) with a Lunar PIXImus II mouse densitometer (GE
Medical Systems, Waukesha WI).
Metabolic Cage Measurements
Mice were singly housed in metabolic cages (Techniplast, Exton PA) and had unrestricted access to powdered HFD and either LDP or
control water, as appropriate. After a 2-day acclimation period, there were 12 days of measurement in 6-week old male and female
mice (n = 4 each). Measurements included body weight, weight of food and water consumed, and feces and urine produced. Calories
consumed were calculated by weight of food consumed as 4,057 cal/g as per the manufacturer (45% kcal from fat, D12451,
Research Diets, New Brunswick, NJ).
Hormone Measurements
Blood cells and serum were separated through centrifugation (3000 g for 10 min at 4 C) and the serum frozen at 80 C. Insulin, leptin,
and peptide YY were measured using the Millipore Mouse Gut Hormone Panel (Millipore Corp., St. Charles MO) using a Luminex 200
(Millipore) analyzer.
MRI
MRI experiments were performed on a 7-tesla MRI system (Bruker Biospin MRI, Ettlingen, Germany) with a custom-made volume
transmit and receive coil. During the scan, anesthesia was maintained with 1.5% isoflurane in oxygen. Each animals body temperature was maintained at 37 (1) C during the scan by directing a thermostatically controlled warm air source. For 3-point Dixon
imaging, T1-weighted images without fat-suppression were acquired using a three-dimensional spoiled gradient echo pulse
sequence with repetition time = 25 ms, flip angle = 10 , spatial resolution = 0.29 3 0.27 3 0.27 mm, and respiratory gating. Three
echo times were 3.4, 3.7, and 4.0 ms and the total scan time was 16 min. The water and fat images were reconstructed using iterative
decomposition of water and fat with an echo asymmetry and least-squares (IDEAL) method (Reeder et al., 2004, 2005) with read-out
chemical shift correction in the fat image. MRI data were coded prior to analysis and the observer was blinded to their experimental
status. The total body fat percentage (volume/volume) was calculated by merging the fat and lean images using MRIcron software
(http://www.mccauslandcenter.sc.edu/mricro/mricron/). Abdominal fat was quantified in 3 consecutive slices mid-way between the
inferior pole of the kidneys and the upper margin of the hind legs (James et al., 2009), and liver adiposity was quantified in 5 consecutive slices.
Histopathology
Ileal tissue was collected from 4-week old control and LDP male mice (n = 5 each), fixed in neutral-buffered formalin, routinely
processed and stained with hemotoxylin and eosin (H&E). From each mouse, four regions were examined. Samples were coded
and scored on a semiquantitative basis for atrophy by a board-certified veterinary pathologist (A.B.R.) blinded to experimental status.
The following criteria were used for villus atrophy: (0) none; (1) mild, villous height 75% normal; (2) moderate, villous height 50%
normal; (3) severe, villous height % 25% normal; scoring was based on the regions showing the most severe lesion to avoid underestimating histological changes. Four independent scores (technical replicates) were averaged for each mouse, the group mean
S2 Cell 158, 705721, August 14, 2014 2014 Elsevier Inc.
computed, and then tested for statistical significance by the Mann-Whitney U test. Liver tissue was collected from 30-week old control and LDP male and female mice, and fixed in neutral-buffered formalin, routinely processed and stained with hemotoxylin and
eosin (H&E) and Masons trichrome. Samples were coded and scored by 3 individuals on a semiquantitative basis for steatosis,
lobular inflammation, hepatocyte ballooning, and fibrosis as described previously (Kleiner et al., 2005) in 10 fields per slide. Average
scores were computed for each mouse, and statistical significance was calculated by the Mann-Whitney U test.
Isolation of Lamina Propria Mononuclear Cells
The ileum and colon were collected at sacrifice, all fat and mesentery removed, and Peyers patches excised from the ileum. Tissues
were first cut longitudinally, then into 2-cm pieces and washed with calcium and magnesium-free phosphate buffered saline (PBS;
LifeTechnologies, Grand Island NY). The mucus was removed with a 10 min wash with 1 mM dithiothreitol (Sigma-Aldrich, St. Louis
MO) in PBS. Epithelial cells were separated from the underlying lamina propria by two 10 min washes with 30 mM EDTA and 10 mM
HEPES in PBS at 37 C. The tissues were then washed with complete RPMI 1640 (LifeTechnologies) containing 10% fetal calf serum,
2 mM L-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.05 mM 2-mercaptoethanol and digested for 1 hr in
100 units/ml type VIII collagenase (Sigma-Aldrich) and 150 mg/ml DNase I (Sigma-Aldrich) at 37 C in complete RPMI. After digestion,
cells were eluted from the tissue by shaking, passed through a 50-micron filter, washed with PBS, and then pelleted. Lamina propria
mononuclear cells (LPMCs) were isolated using a percoll (GE Healthcare, Piscataway NJ) gradient separation method. Cells were
resuspended in 40% Percoll in complete RPMI, under-layed with 80% percoll, and centrifuged at 2,200 rpm for 20 min at room temperature. LPMCs were then collected at the interface and used for subsequent flow cytometric analyses.
Flow Cytometry
Cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 500 ng/ml Ionomycin for 4 hr at 37 C in the presence
of brefeldin A (GolgiPlug, Becton Dickinson, Franklin Lakes NJ). Following this in vitro stimulation, cells were stained with LIVE/DEAD
Fixable Aqua (LifeTechnologies), anti-CD3, and anti-CD4 and fixed in 4% paraformaldehyde in PBS. Cells were then permeabilized in
Perm/Wash buffer (Becton Dickinson) and stained with anti-IL-17A and anti-IL-22 (eBioscience, San Diego CA). Cells were acquired
on an LSRII (Becton Dickinson) and analyzed with FlowJo (Tree Star, Ashland OR) software.
Hepatic and Ileal Gene Expression Profiling
Ileal and liver tissues were collected at sacrifice and stored in RNA later for 24 hr, then transferred to a new tube and stored at 80 C.
Total RNA was extracted from the mouse ileum and liver by the RNeasy Mini Kit (QIAGEN, Valencia CA), according to the manufacturers instructions.
Quantitative PCR
Total RNA was reverse transcribed to cDNA using the VersoTM cDNA Synthesis Kit (Thermo Scientific). To generate standards for
expression analysis of each targeted gene, the DNA or cDNA region of interest was PCR-amplified and the PCR product was cloned
using the pGEM-Teasy vector system (Promega). qPCR was performed with a LightCycler 480 SYBR Green _ Master (Roche, Indianapolis IN) and run in a LightCycler 480 system (Roche, Indianapolis IN). Target mRNA was normalized to GAPDH mRNA as an internal
control in each sample.
Microarray
Expression profiling of the liver from 30-week old LDP and control male mice fed NC and HFD (n = 3 each) and the ileum of 8-week old
LDP and control mice (n = 3 and 4, respectively) fed HFD was performed using the Affymetrix Genechip Chip Mouse 430_2 system
(Affymetrix, Santa Clara CA). Total RNA quality and quantity were determined using the Agilent 2100 Bioanalyzer and Nanodrop ND1000. Total RNA (100ng) was used to prepare cDNA following the Affymetrix 30 IVT Express Kit labeling protocol. Standardized array
processing procedures recommended by Affymetrix were performed, including hybridization, fluidics processing and scanning of the
Affymetrix MG-430 2.0 arrays. The raw microarray data were normalized using the Robust Multi-Array (RMA) method (Irizarry et al.,
2003). Affymetrix QC metrics were within acceptable limits, including average background, scale factor, number of genes present,
and 30 to 50 ratios for B-actin and GAPDH (Gautier et al., 2004). The Limma package in R was used to fit a linear model to the expression data using empirical Bayes moderated t-statistics (Smyth, 2004). Multiple testing adjustments were done by FDR correction.
Probes with FDR < 0.05 were called to be differentially expressed.
RNA-Seq
RNA-seq libraries were generated using the TruSeq v2 RNA sample prep (Illumina, San Diego, CA) following manufacturers recommendations. Libraries were sequenced with 2x50bp paired-end reads in an Illumina HiSeq 2500 in either high-output mode (batch 1)
or rapid mode (batch 2) to a mean read depth of 30.6 M (batch 1) or 57.8 M (batch 2). Fastq files were processed as follows: alignment
was done with STAR (version 2.3.0e for the first batch, version 2.3.1x for the second batch) (Dobin et al., 2013), with an index build
from the mm10 reference sequence. The genes annotated in UCSC mm10 were quantified with HTSeq-count v0.5.3p9
(featureCounts v1.4.3-p1 for the second batch of files) (Anders et al., 2014; Liao et al., 2014). Differential expression analysis was
performed with the Bioconductor package DESeq (Anders and Huber, 2010).
Cell 158, 705721, August 14, 2014 2014 Elsevier Inc. S3
Nanostring
Ileal gene expression was measured in 8-week old male and female mice exposed to LDP or not (control) from the Dura-LDP experiment and in 8-week old germ-free female mice colonized with LDP- or control-microbiota (n = 4 each) using the nCounter GX Mouse
Immunology Kit (NanoString Technologies, Seattle, WA). Counts were normalized to a panel of housekeeping genes, and significant
differences between LDP and control were detected by t test.
Microbial Community Analysis
DNA was extracted using the PowerSoil DNA Extraction Kit (MoBio, Carlsbad CA). Total bacterial populations were assessed by
quantitative PCR on a Rotor Gene 3000 quantitative PCR cycler using the LightCycler FastStart DNA Master PLUS SYBR Green I
kit (Roche) using universal 16S rRNA primers (Cho et al., 2012). To assess microbial composition, the microbial 16S rRNA gene
was amplified with barcoded fusion primers, targeting either the V1-2 region or the V4 region (Table S7). For Dyna-LDP, fusion
primers targeted the V1-2 region (Fierer et al., 2008), and 24 unique barcoded primers were used in 4 sequencing regions for a total
of 94 samples. Amplification was confirmed by visualizing a band on gel electrophoresis, primers were removed using the Ampure
Bead kit, then quantified using Quant-iT PicoGreen dsDNA regent (LifeTechnologies), and pooled at equal DNA quantity. The
sequencing library was amplified by emulsion PCR and pyrosequenced on the Roche 454 platform according to standard protocols.
For the remainder of the experiments (Fat-LDP, Trans-LDP1, and Trans-LDP2), the fusion primers amplified the V4 region as
described (Caporaso et al., 2012). DNA concentrations from each amplicon was measured using Quant-iT PicoGreen dsDNA regent
(LifeTechnologies), and pooled at equal DNA quantity. After samples were combined in sub-pools in groups of up to 96 samples,
excess primers were removed with the Qiaquick PCR purification kit (QIAGEN). DNA concentration in these sub-pools were quantified with the Qubit high sensitivity dsDNA Assay (LifeTechnologies), and combined at equal concentration. The amplicons were
sequenced on the Illumina MiSeq I platform, with 30%50% PhiX spiked into the amplicon pool to improve the signal from a low
diversity library, in 3 separate sequencing runs at Argonne National Laboratory and NYU, see Figure S4. The sequencing was carried
out in three separate reads, (i) 151-base pair forward read, (ii) 12-base pair barcode read, and (iii) 151-base pair reverse read. Only
reads that passed the Illumina quality filter were used for downstream analysis.
Bioinformatics Analysis
Paired end reads were joined with FastqJoin from EA-utils (Aronesty, 2013), and only reads that perfectly matched in the overlapping
region were kept. QIIME (Caporaso et al., 2010) was used to demultiplex and quality filter the samples. Reads were truncated at a
stretch of 4 or more consecutive low quality bases (phred q score < 20), and only reads at R 75% of the original length were retained.
Taxonomy was assigned using the open reference method in QIIME, with an RDP confidence interval of 50%, and the Green Genes
2013 May database release was used as a reference (McDonald et al., 2012). QIIME was used to generate relative abundance plots,
to calculate a-diversity metrics (richness, phylogenetic diversity, and Shannon index), and to calculate UniFrac distances and
generate PCoA plots. To test for significance of the inter- and intra-group differences in all beta diversity analyses, we permuted
the labels of the distance matrix 10,000 times and calculate the p-value as the proportion of the times that the distance between
the compared groups is greater than the one computed. Differences in beta-diversity visualized on the PCoA plots were calculated
with the ADONIS test at 1,000 permutations (Anderson, 2001) using the R vegan package (Oksanen et al., 2013). Spearman correlations were calculated using the R statistical framework, and ellipse plots generated with the R package Ellipse. Metagenomic content of the microbiota samples was predicted from the 16S rRNA profiles and KEGG pathway functions were categorized at level 3
using the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) tool (Langille et al., 2013).
Linear discriminant analysis effect size (LEfSe) (Segata et al., 2011) was used via the Galaxy Browser (Blankenberg et al., 2010; Giardine et al., 2005; Goecks et al., 2010) to detect significant changes in relative abundance of microbial taxa between control and LDP
mice. To reduce the number of features, the 16S analysis was limited to taxa with relative abundance R 1% in any sample. When
applicable, the classes were defined as treatment groups (control or LDP), the subclasses were fecal time points. Pairwise comparisons among classes were restricted to within the same subclass. Significance thresholds were performed at the default settings:
alpha % 0.05 for the Kruskal-Wallis test among classes, alpha % 0.05 for the pairwise Wilcoxon tests between subclasses,
and R 2.0 for the logarithmic linear discriminant analysis (LDA) score.
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Lactobacillus
***
60
**
10
5
**
10
0
8
16
18
30
30
10
Control
LDP
20
16
18
30
Allobaculum
***
**
***
Not detected in
microbioata samples.
30
20 0.15
Candidatus
Arthromitus
CTL-NC
LDP-NC
CTL-HFD
LDP-HFD
40
Lactobacillus
16
18
30
Rikenellaceae
Candidatus Arhtromitus
8
***
*
6
**
*
*
**
3.5
20
50
Rikenellaceae
15
**
20
Candidatus
Arthromitus
Not detected in
inocula or recipient
samples.
10
30
3.5
Age(week)
60
40
10
Age(week)
***
***
Allobaculum
70
Control Recipients
LDP Recipients
***
**
20
3.5
Age(week)
Lactobacillus
30
0
3
**
3.5
Age(week)
***
**
10
Trans-LDP
Females
High fat diet
MiSeq
V4 region
**
**
10
Rikenellaceae
15
***
**
15
20
***
50
Dura-LDP
Females
pre-weaning
MiSeq
V4 region
Allobaculum
70
0
4F 16F 26F 30C 30I
60
0
4F 16F 26F 30C 30I
20
***
10
Lactobacillus
3
2
25
20
Candidatus Arhtromitus
30
40
20
Rikenellaceae
40
Fat-LDP
Males
NC and HFD
MiSeq
V4 region
50
Allobaculum
12
Control
LDP
80
**
Time-LDP
Males
Normal Chow
454 seqeuencing
V1-2 region
10
0
0
D T0
10
20
30
C I
0
D T0
Days post-transfer
10
20
Days post-transfer
30
C I
D T0
10
20
30
C I
Days post-transfer
Figure S1. Relative Abundance of Taxa Consistently Reduced in Early Life by LDP, Related to Figure 2
(AD) Mice were treated with LDP from birth, or did not receive antibiotics (control) (A-C), or germ-free mice were conventionalized with microbiota from control or
LDP mice (D). The intestinal microbiota was surveyed by sequencing the V4 region of the 16S rRNA gene. 4 taxa were significantly reduced in early life in multiple
experiments. Plots show mean SEM of the relative abundance of the 4 taxa in fecal samples over time and in cecal (C) or ileal samples (I). *p < 0.05, **p < 0.01,
***p < 0.001, Mann-Whitney U test.
Figure S2. Effect of HFD and LDP on Liver Histology and Gene Expression, Related to Figure 3
Male and female control (CTL) mice did not receive antibiotics; LDP mice received low-dose penicillin from birth. All mice were started on a normal chow (NC) diet,
and then half were maintained on normal chow and half switched to a high fat diet (HFD) at week 17; the number of mice is listed in Figure 3A.
(A and B) (A) Hepatic lobular inflammation scored using hematoxylin and eosin stained and (B) fibrosis scored in trichrome-stained liver sections.
(CN) Hepatic gene expression measured by qPCR, n = 5 per group. *p < 0.05 for penicillin-mediated effect, p < 0.05 for diet-mediated effect by 1-way ANOVA
with Bonferronis post-test correction.
(O) Hepatic gene expression was measured by microarray in male mice at 30 weeks of age (n = 3 in each of the four exposure groups). The Venn diagram indicates
the number of overlapping genes in three separate pairwise comparisons: penicillin effect in HFD mice (Control versus LDP), diet effect in control mice (NC versus
HFD) and diet effect in LDP mice (NC versus HFD). Heatmaps depict gene expression values of Venn regions IV-VI.
Control NC
a
b
c
d: c__Actinobacteria
e: o_Bifidobacteriales
f
h
i
j
k
l
LDP NC
g__Akkermansia;s__muciniphila
c__Mollicutes;o__RF39
g__Anaeroplasma
g__Acinetobacter;s__johnsonii
f__Enterobacteriaceae
f__Enterobacteriaceae;Other
g__Desulfovibrio;s__C21_c20
g__Sutterella
p__Proteobacteria
g__[Eubacterium];s__dolichum
g__Coprobacillus
g__Allobaculum
f__Erysipelotrichaceae
g__Oscillospira
f__Peptostreptococcaceae
g__[Ruminococcus];s__gnavus
g__Dorea
g__Coprococcus
g__Blautia;s__producta
f__Lachnospiraceae
f__Lachnospiraceae;Other
f__Clostridiaceae;g__SMB53
f__Clostridiaceae
o__Clostridiales
g__Turicibacter
g__Streptococcus
g__Lactococcus
g__Lactobacillus;s__reuteri
g__Lactobacillus
g__Lactobacillus;Other
g__Enterococcus
g__Staphylococcus
o__Bacteroidales;f__S24-7
f__Rikenellaceae
g__Prevotella
g__Parabacteroides
g__Bacteroides;s__acidifaciens
o__Bacteroidales
f__Coriobacteriaceae
g__Bifidobacterium
Low_Threshold
Age (week): 4
Control HFD
Control
LDP
LDP HFD
16
18
30 C I
Start HFD
Figure S3. Effect of HFD and LDP on the Intestinal Microbiota, Related to Figure 4
(A) Relative abundance of intestinal microbiota in LDP and control mice in fecal samples at weeks 4-30, and cecal (C) and ileal (I) samples at week 30.
(B and C) Cladogram representing taxa enriched in week 30 fecal samples according to (B) diet (class: diet, subclass: treatment), or by (C) treatment (class:
treatment, subclass: diet), detected using the LEfSe tool.
LDP n = 9
**
10
25
*
**
30
Control, n = 20
4-LDP, n = 15
8-LDP, n = 14
32-LDP, n = 13
Total
Lean
Fat
*
**
20
*
* *
15
10
10
20
0
0
30
15
10
200
20
20
**
25
40
10
C
Fat
30
50
20
30
Food intake
180
160
140
120
100
10
20
CTL
30
LDP
Feed efficiency
8
6
4
2
0
CTL
Male
Age (week)
**
Lean
Total
Control n = 9
Mass (g)
Mass (g)
4 week males
15
LDP
CTL
Female
LDP
Male
Biological functions
Transfer-Females
LDP-Females
differentiation of cells
differentiation of blood cells
differentiation of lymphocytes
H2Eb1
differentiation of connective tissue cells
Hypoplasia
Cd74
quantity of Ca2+
release of Ca2+
H2Aa
*activation of lymphocytes
*adhesion of immune cells
Ceacam10
homing of cells
cell movement
Cd177
quantity of antigen presenting cells
H2Ab1
quantity of T lymphocytes
cell-mediated response
Ccl5
cytotoxicity of cells
recruitment of neutrophils
Itgae
eosinophilia
immune response of phagocytes
Slc9a3
quantity of B lymphocytes
quantity
of
immunoglobulin
Pcdh19
cell viability
C1 C2 C3 P1 P2 P3 P4
*organismal death
infection of mammalia
LDP
Control
1
-6 -4 -2 0 2 4 6 8
10
7 VII
0
I
116
10
49
3
VI
LDP-exposed
males
3
2
IV
III
53
52
LDP-microbiota
recipients
(transfer-females)
-4
-6 -4 -2 0 2 4 6
-2
z-score
Region II
-2
Region IV
LDP-Males
LDP-Females
Acot1
Gja4
Cyp4a10
Gbp1
Vnn1
Cyp26b1
Cyp4a31
Aqp8
Hamp
Aldh1b1
Lyve1
Igfbp3
Igfbp5
Cib3
Msln
H19
Esm1
Region VI
LDP-Females
Trans-Females
Csad
Cdkn1a
Socs2
A1bg
Gsta2
-2
0
2
Log2 Fold Change
Saa2
Tacc2
Map3k5
Sult1c2
Ugt1a5
Pcsk9
4
Sc4mol
Nsdhl
Fdps
Cyp51
Idi1
1810064F22Rik
Sqle
-2
0
2
Log2 Fold Change
I
Biological functions
cell movement of hepatoma cell lines
birthweight
conversion of acyl-coenzyme A
metabolism of triacylglycerol
synthesis of lipid
concentration of lipid
injury of mice
organismal death
homing of cells
cell movement of tumor cell lines
quantity of IgG
cell movement of phagocytes
Bacterial Infection
mass of organism
metabolism of terpenoid
apoptosis of epithelial cell lines
LDP-Male
Trans-Females
0
2
Log2 Fold Change
LDP-Females
Transfer-Females
-3 -2 -1 0 1 2 3
-3 -2 -1 0 1 2 3
z-score
-3 -2 -1 0 1 2 3
Figure S4. The Effect of Limited LDP on Body Composition, Food Intake, and Host Gene Expression, Related to Figure 5
Male and female did not receive antibiotics (control) or received 4-weeks, 8-weeks, or continuous LDP (28-weeks for females, 32-weeks for males).
(A and B) Body composition in male mice at (A) week 4 and (B) from 6 through 32 weeks of age measured by DEXA scans, *p < 0.05, **p < 0.01 t test.
(C) Cumulative 12-day food intake in male mice from week 6-8, n = 4 per group.
(D) Feed efficiency (%), calculated by weight gained (g)/ weight food consumed (g) during a 12 day period from 6-8 weeks of age. No significant differences
detected (p > 0.05).
(E) Genes with significantly (FDR-adjusted p-value < 0.05) altered ileal gene expression at week 8 in male control and LDP mice, determined by microarray.
(F) Ingenuity Pathway Analysis predicted biological functions increased or decreased in 8-week old male mice given LDP, female mice given LDP, or germ-free
female mice colonized with LDP microbiota compared to their respective controls. Bold = functions that are significant and have a z-score > j2j in two or more
groups, * = three groups.
(G) Number of genes with significantly (FDR-adjusted p-value < 0.05, DESeq) increased or decreased expression in LDP-exposed male and female mice or LDPmicrobiota recipients (transfer-females) compared to their respective controls, determined by RNaseq.
(H) Relative gene expression (Log2 fold change of LDP/control) hepatic genes significantly different between LDP and control in two independent experiments.
(I) Predicted biological functions that are significant (p < 0.05) with z-score > j2j in at least one group, determined by Ingenuity Pathway Analysis.
PC2 (11%)
Intergroup
b vs. c
a vs. c
c vs. c
0.2
0.0
Intragroup
Intergroup
Sample
Comparison
Run Comparison
a vs. b
***
0.4
Intragroup
0.2
Intergroup
Intragroup
b vs. c
a vs. b
a vs. c
c vs. c
a vs. a
b vs. b
***
0.6
b vs. b
UniFrac Distance
UniFrac Distance
0.4
Weighted
0.8
0.6
0.0
Unweighted
0.8
a vs. a
Intragroup
Intergroup
Sample
Comparison
Run Comparison
PC2 (22%)
PC1 (31%)
PC1 (17%)
PC3 (13%)
PC3 (8.3%)
g__Akkermansia
g__Desulfovibrio
g__Bilophila
g__Sutterella
g__Allobaculum
f__Erysipelotrichaceae
g__Ruminococcus
g__Oscillospira
f__Ruminococcaceae
f__Lachnospiraceae;g__[Ruminococcus]
10
11
12
13
g__Dorea
g__Coprococcus
g__Blautia
f__Lachnospiraceae
f__Lachnospiraceae;Other
f__Clostridiaceae;g__SMB53
f__Clostridiaceae
o__Clostridiales
g__Turicibacter
g__Lactococcus
14
15
16
17
18
19
20
g__Lactobacillus
g__Enterococcus
g__Odoribacter
o__Bacteroidales;f__S24-7
f__Rikenellaceae
g__Prevotella
g__Bacteroides
o__Bacteroidales
f__Coriobacteriaceae
g__Bifidobacterium
Figure S5. Comparison of Taxonomic Results between Sequencing Facilities, Related to Experimental Procedures
Amplicon libraries of the 16S rRNA V4 region were prepared for 718 samples, each with a unique molecular ID (barcode) on the reverse primer. Samples were
pooled and sequenced on the MiSeq Illumina instrument in four separate runs, including two at NYU with all specimens including either 50% or 40% PhiX, and two
at Argonne National Laboratory (ANL) including 30% PhiX and a different portion of the samples included in each run, so that each sample had 3 replicate
sequencing runs. The first ANL run had 123 samples and the second the remaining 595 samples. A random set of 10 samples was selected from the first and
second ANL runs for comparative analysis.
(A and B) Average pairwise unweighted (A) and weighted (B) UniFrac distances within (intragroup) or between (intergroup) runs (a = first NYU run, b = second NYU
run, c = ANL runs), and among the same samples (intergroup) or between different samples (intergroup). *** sample-intragroup distances are significantly lower
than all other distances (p < 0.001 for 1-way ANOVA with Bonferronis post-test correction). Similar intragroup and intergroup UniFrac distances in the run
comparison indicate that there is no significant bias between the different sequencing runs.
(C and D) Samples cluster together on principal coordinate analysis of unweighted (C) and weighted (D) UniFrac distances. The same colored symbols, connected
by line, are used for each of the individual samples in each of the three runs.
(E) Relative taxonomic abundance at the genus level. In each of the 10 triplets, the first two bars are from individual NYU sequencing runs, and the third is from the
ANL run. Patterns are nearly identical within the triplets; these analyses indicate that inter-site and inter-run variation is minimal.
S10 Cell 158, 705721, August 14, 2014 2014 Elsevier Inc.
Control
KEGG Pathway
Low-dose penicillin
Control-microbiota recipients
# of
# of
time
points
2
Transferred microbiota
KEGG Pathway
time
points
2
4
2
3
4
2
2
Glycolysis/Gluconeogenesis
2
2
2
Glycolysis/Gluconeogenesis
Peptidoglycan biosynthesis
Terpenoid backbone biosynthesis
2
2
2
Lysine biosynthesis
Peptidoglycan biosynthesis
Terpenoid backbone biosynthesis
4
4
2
Galactose metabolism
2
2
Protein kinases
Sphingolipid metabolism
2
3
3
3
2
2
Two-component system
Photosynthesis proteins
Restriction enzyme
2
2
Geraniol degradation
Glycerophospholipid metabolism
Glycosyltransferases
Ion channels
2
2
2
Metabolismofcofactorsandvitamins
Non - homologous end - joining
Penicillin and cephalosporin
biosynthesis
2
2
Prenyltransferases
Nitrogen metabolism
Other glycan degradation
3
3
KEGG Pathway
time
points
3
3
2
3
Lysine biosynthesis
Pantothenate and CoA biosynthesis
Phenylalanine, tyrosine and tryptophan
biosynthesis
2
3
Protein kinases
3
3
Sphingolipid metabolism
Two-component system
2
2
Benzoate degradation
Bladder cancer
2
2
Chromosome
2
2
Cytoskeleton proteins
D-Alanine metabolism
D-Glutamine and D-glutamate
metabolism
DNA replication
DNA replication proteins
Drug metabolism-other enzymes
3
3
3
Glycerophospholipid metabolism
Glycosyltransferases
3
3
Non-homologous end-joining
Sulfur metabolism
3
3
Tetracycline biosynthesis
2
2
2
Transcription machinery
Bacterial secretion system
3
2
2
2
2
Geraniol degradation
Inorganic ion transport and metabolism
2
2
2
2
2
2
2
interconversions
Energy metabolism
Peroxisome
Phenylpropanoid biosynthesis
2
2
Selenocompound metabolism
Signaltransductionmechanisms
2
2
2
2
2
2
2
Mismatch repair
Steroid biosynthesis
Steroid hormone biosynthesis
2
2
Glycerolipid metabolism
Homologous recombination
Methane metabolism
Nucleotide metabolism
Other ion-coupled transporters
2
2
Sulfur metabolism
Phenylpropanoid biosynthesis
2
2
2
2
2
2
Ribosome
Ribosome biogenesis
Starch and sucrose metabolism
Transcription factors
Translation factors
Transporters
6 shared pathways
biosynthesis
Aminobenzoate degradation
Atrazine degradation
Biotinmetabolism
Cyanoamino acid metabolism
Flagellar assembly
Pentose and glucuronate
# of
# of
time
points
KEGG Pathway
LDP-microbiota recipients
Lipopolysaccharide biosynthesis
proteins
Metabolism of cofactors and vitamins
2
2
Naphthalene degradation
Oxidativephosphorylation
2
2
2
2
2
Phenylalanine metabolism
PPAR signaling pathway
Prenyltransferases
2
2
2
2
2
Pyruvate metabolism
Replication, recombination and repair
proteins
Riboflavin metabolism
RNA degradation
Secretion system
2
2
2
Steroid biosynthesis
Steroid hormone biosynthesis
Toluene degradation
2
2
2
12 shared pathways
Figure S6. Microbial KEGG Pathways Significantly and Consistently Different in LDP and Control Mice in Independent Experiments, Related
to Figure 6
(A and B) Microbiota samples were obtained from female mice at 5 time points from (A) mice either exposed to low-dose penicillin (LDP, n = 8), or not (Control,
n = 13) between 3 and 8 weeks of life, and (B) in germ-free mice conventionalized with control (n = 7) and LDP (n = 8) microbiota between 2 and 21 days posttransfer. Predicted metagenomes were generated using PICRUST and differences between treatment groups were determined using LeFSe. All KEGG pathways
shown were significantly differentially present in R 2 time points. Pink-shading indicates pathways enriched in the same direction in multiple time points in both
experiments.
Cell 158, 705721, August 14, 2014 2014 Elsevier Inc. S11
CR1
CR2
PD
PR1
PR2
Control-microbiota recipient
14
14
CR1 PR1
CR2 PR2
1st Transfer
2nd Transfer
23 days post transfer
2nd Transfer
21
28
34
35
LDP-microbiota recipient
60
1st Transfer
40
21
28
34
35
C
g__Akkermansia;s__muciniphila
g__Proteus
g__Klebsiella
g__Desulfovibrio;s__C21_c20
g__Bilophila
g__Sutterella
c__Alphaproteobacteria;o__RF32
g__[Eubacterium];s__dolichum
g__Allobaculum
f__Erysipelotrichaceae
g__Ruminococcus
g__Oscillospira
g__Faecalibacterium;Other
f__Peptostreptococcaceae
g__Dorea
g__Coprococcus
g__Blautia;s__producta
1
23
69 70C g__Blautia
f__Lachnospiraceae
f__Lachnospiraceae;Other
f__Clostridiaceae;g__SMB53
f__Clostridiaceae
o__Clostridiales
o__Clostridiales;Other
g__Turicibacter
g__Lactococcus
g__Lactobacillus;s__reuteri
g__Lactobacillus
g__Lactobacillus;Other
g__Enterococcus
o__Lactobacillales;Other
g__Odoribacter
o__Bacteroidales;f__S24-7
f__Rikenellaceae
g__Bacteroides;s__ovatus
o__Bacteroidales
1
23
69 70C
g__Bifidobacterium
Recipient fecal microbiota
Low_Threshold
days post-transfer
J
Group Divergence
0.9
CR1
PR1
CR1 vs PR1
0.8
0.7
0.6
0.5
0
10
20
Cecal
Ileal
14 21 28 35
20
30
K
Group Divergence
0.9
CR2
PR2
CR2 vs PR2
b a
0.8
0.7
b,c
b,c
b,c
0.6
0.5
0
20
40
Cecal
ns
4
2
Donor
30
CR2 n = 6
PR2 n = 6
Age (week)
CR1 n = 7
PR1 n = 8
20
6
2
0
10
10
10
teu
s
Control
LDP
pro
10
10
Second Transfer
12
g__
D
First transfer
12
les
Donors
15
LDP-Recipient 2
ida
g__B
s__pro lautia
ducta
CD
tero
Bac
LDP
Second Transfer
Control
First Transfer
60
L
Unweighted UniFrac Distance
Donors
Intergroup Divergence
0.9
CR1 vs PR1
CR2 vs PR2
0.8
0.7
0.6
0.5
0
10
20
30
40
50
60
70
H 2nd Transfer
PC2 (7.1%)
PC2 (7.1%)
CR2 inoculum
CR2 recipient
PR2 inoculum
PR2 recipient
PC1 (14%)
PC3 (4.4%)
1 dpt
p = 0.007
PC2 (7.1%)
PC2 (7.1%)
CR1
CR2
PR1
PR2
PC1 (14%)
PC3 (4.4%)
69 dpt
p = 0.004
PC2 (7.1%)
PC1 (14%)
(1
PC1 (14%)
PC3 (4.4%)
23 dpt
p = 0.007
PC1 (14%)
PC3 (4.4%)
70 dpt
p = 0.002
PC3 (4.4%)
%)
S12 Cell 158, 705721, August 14, 2014 2014 Elsevier Inc.
Figure S7. Comparison of Adiposity and Bacterial Composition in Mice Subjected to Sequential Microbiota Transfers, Related to Figure 7
(A) Study Design: Control C57BL/6 mice did not receive antibiotics, LDP mice received low dose penicillin from birth until week 18 (see Figure 5). Then control and
LDP mice (n = 3 each) were sacrificed, cecal contents pooled, and transferred to germ-free Swiss-Webster mice designated as control or LDP recipients in TransLDP1 (first transfer, CR1, n = 7; and PR1, n = 8). From those first-transfer-recipients, cecal contents from three mice in each group were then transferred (TransLDP 2, second transfer) to new germ-free Swiss-Webster recipients (CR2, n = 6; and PR2, n = 6).
(BD) Fat mass, determined by DEXA scanning, of the (B) cecal microbiota donors for Trans-LDP1, (C) Trans-LDP1 microbiota recipients, and (D) Trans-LDP2
microbiota recipients.
(E) Fat mass 23 days after transfer in Trans-LDP1 and Trans-LDP2 microbiota recipients. *p < 0.05 t test. (F) Composition of transferred microbial communities in
two sequential transfers. Relative abundances in the donor (D), the transferred inoculum (T), and the recipient mice in serial fecal samples and the terminal cecal
(C) and ileal (I) specimens. Taxa displayed have relative abundance > 1% in at least one sample and are reported at the lowest possible level of identification
c = class, o = order, f = family, g = genus, s = species.
(G) Taxa associated with control or LDP recipients following a second sequential transfer. LEfSe cladogram represents taxa enriched in CR2 (green) or PR2 (red) in
fecal samples from 1 and 23 days post-transfer, as described in Figure 2.
(H) Community structure of transferred microbiota in a sequential transfer assessed by unweighted UniFrac distances of microbiota samples. The PCoA
plots show the transferred inoculum and microbiota in the Trans-LDP2 recipient mouse fecal samples 1, 23, and 69 days post-transfer (dpt), and cecal samples at
70 dpt.
(I) PCoA of all microbiota samples from the 1st (Trans1) and 2nd (Trans2) round of microbiota recipients. The colored ellipses indicate the clustering of the
microbiota samples from each of the groups.
(JL) Intra- and inter group divergence: mean UniFrac distances of pairwise comparisons between each of the control (CR) and LDP (PR) microbiota samples in
Trans-LDP1, for statistics, see Table S6 (J), Trans-LDP2 (K), and both transfers (L). Significant differences, p < 0.05 based on 10,000 permutations of the UniFrac
distance matrix, noted by letters, a = CR-intragroup versus PR intragroup, b = CR-intragroup versus intergroup (CR versus PR), c = PR-intragroup versus
intergroup (CR versus PR) (L).
Cell 158, 705721, August 14, 2014 2014 Elsevier Inc. S13