Transcript advancesinibd.com

Critical Design Issues Involved in Mouse
Studies of the Intestinal Microbiota
Ajay S. Gulati, MD
Associate Professor of Pediatrics and Pathology
Division of Pediatric Gastroenterology
University of North Carolina at Chapel Hill
Functions of the Intestinal Microbiota
 Modulating host innate and
adaptive immune function
 Regulating energy utilization and
metabolism
 Enhancing epithelial responses
to injury and repair
 Regulating brain development
and behavior
“The Microbial Cloud”
- Jonathan Eisen
Sekirov et al. (2010) Physiol Rev 90:859-904
Functions of the Intestinal Microbiota
 Modulating host innate and
adaptive immune function
 Regulating energy utilization and
metabolism
 Enhancing epithelial responses
to injury and repair
 Regulating brain development
and behavior
“The Microbial Cloud”
- Jonathan Eisen
Demonstrated using mouse models
Sekirov et al. (2010) Physiol Rev 90:859-904
… a healthy dose of scepticism?
“Microbiome science needs a
healthy dose of scepticism”
Questions
Mouse Studies

Do microbiome studies show
causation or just correlation?

Subject mice to defined
exposures and measure direct
effects on microbial communities

What is the mechanism
proposed?

Study mechanisms by which
exposures regulate bacterial
communities

Could anything else explain the
results?

Control confounders such as
breeding strategy and
environmental conditions
Hanage WP (2014) Nature 512:247-8
… a healthy dose of scepticism?
“Microbiome science needs a
healthy dose of scepticism”
Questions
Mouse Studies

Do microbiome studies show
causation or just correlation?

Subject mice to defined
exposures and measure direct
effects on microbial communities

What is the mechanism
proposed?

Study mechanisms by which
exposures regulate the gut
microbiota

Could anything else explain the
results?

Control confounders such as
breeding strategy and
environmental conditions
Hanage WP (2014) Nature 512:247-8
… a healthy dose of scepticism?
“Microbiome science needs a
healthy dose of scepticism”
Questions

Do microbiome studies show
causation or just correlation?

What is the mechanism
proposed?

Could anything else explain the
results?
Mouse Studies

Subject mice to defined
exposures and measure direct
effects on microbial communities
Hanage WP (2014) Nature 512:247-8
… a healthy dose of scepticism?
“Microbiome science needs a
healthy dose of scepticism”
Questions
Mouse Studies

Do microbiome studies show
causation or just correlation?

Subject mice to defined
exposures and measure direct
effects on microbial communities

What is the mechanism
proposed?

Study mechanisms by which
exposures regulate the gut
microbiota

Could anything else explain the
results?
Hanage WP (2014) Nature 512:247-8
… a healthy dose of scepticism?
“Microbiome science needs a
healthy dose of scepticism”
Questions
Mouse Studies

Do microbiome studies show
causation or just correlation?

Subject mice to defined
exposures and measure direct
effects on microbial communities

What is the mechanism
proposed?

Study mechanisms by which
exposures regulate the gut
microbiota

Could anything else explain the
results?

Control confounders such as
breeding strategy and
environmental conditions
Hanage WP (2014) Nature 512:247-8
… a healthy dose of scepticism?
“Microbiome science needs a
healthy dose of scepticism”
Questions
Mouse Studies

Do microbiome studies show
causation or just correlation?

Subject mice to defined
exposures and measure direct
effects on microbial communities

What is the mechanism
proposed?

Study mechanisms by which
exposures regulate the gut
microbiota

Could anything else explain the
results?

Control confounders such as
breeding strategy and
environmental conditions
Hanage WP (2014) Nature 512:247-8
Talk Outline

Intrinsic
− Mouse genetic background

Legacy effect
‾ Heritability of the intestinal
microbiota

Extrinsic
‾ Cage effects
‾ Housing conditions
RALPH STEADMAN
Impact of Mouse Genetic Background
on the Gut Microbiota

Profiled the cecal
microbiota of 10 distinct
mouse strains

All mice were purchased
from same vendor

Analyzed using 454
sequencing of the 16S
rRNA gene
Campbell JH et al (2012) ISME J 6:2033-2044
Impact of Mouse Genetic Background
on the Gut Microbiota
Campbell JH et al (2012) ISME J 6:2033-2044
Impact of Mouse Genetic Background
on the Gut Microbiota


Effect of strain was found
to be significant (P =
0.0001)
Accounted for 41.1% of
all variation
Campbell JH et al (2012) ISME J 6:2033-2044
Impact of Mouse Genetic Background
on the Gut Microbiota




Effect of strain was found
to be significant (P =
0.0001)
Accounted for 41.1% of
all variation
Effect of gender was also
significant
(P = 0.01)
Accounted for only 0.7%
of variation in the data
Campbell JH et al (2012) ISME J 6:2033-2044
Reciprocal Colonization of Germ-Free Mice
Gulati et al (2012) PLoS One 7:e32403
Reciprocal Colonization of Germ-Free Mice
 Stool was collected at 2, 4, and 12 weeks after colonization
 Mice were sacrificed at 12 weeks for tissue procurement
 Microbiota profiled using 454 sequencing of 16S rRNA gene
Gulati et al (2012) PLoS One 7:e32403
Reciprocal Colonization of Germ-Free Mice
1. Ensured that mice from different backgrounds are exposed
to the same initial bacteria at colonization
2. Allowed us to characterize the establishment of bacterial
communities over time
Gulati et al (2012) PLoS One 7:e32403
Mouse Background Drives Composition of the
Intestinal Microbiota
Stool From
SPF C57BL/6 Mice
ILEAL MICROBIOTA
(12 weeks post-colonization)
Stool From
SPF 129/SvEv Mice
0.3
GF
C57BL/6
Mice
GF
129/SvEv
Mice
GF
C57BL/6
Mice
GF
129/SvEv
Mice
(B6→B6)
(B6→129)
(129→B6)
(129→129)
0.2


If donor stool is driving microbial
composition, the solid squares will
cluster independently from the open
squares
If recipient background strain is
driving microbial composition, the red
squares will cluster independently
from the blue squares
PC2
0.1
0.0
-0.1
-0.2
-0.3
-0.4
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
PC1
Gulati et al (2012) PLoS One 7:e32403
Mouse Background Drives Composition of the
Intestinal Microbiota
Stool From
SPF C57BL/6 Mice
ILEAL MICROBIOTA
(12 weeks post-colonization)
Stool From
SPF 129/SvEv Mice
0.3
GF
C57BL/6
Mice
GF
129/SvEv
Mice
GF
C57BL/6
Mice
GF
129/SvEv
Mice
(B6→B6)
(B6→129)
(129→B6)
(129→129)
129 Recipients
0.2


If donor stool is driving microbial
composition, the solid squares will
cluster independently from the open
squares
If recipient background strain is
driving microbial composition, the red
squares will cluster independently
from the blue squares
PC2
0.1
P = 0.01
0.0
-0.1
-0.2
-0.3
-0.4
B6
Recipients
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
PC1
Gulati et al (2012) PLoS One 7:e32403
Mouse Background Directs Ecological
Succession of Gut Microbiota After Colonization
Fecal Microbiota
(2 weeks)
0.2
129
Recipients
PC2
0.1
0.0
-0.1
-0.2
-0.2
0.0
0.2
PC1
0.4
Donor
B6
129
B6
129
Recip
129
129
B6
B6
Mouse Background Directs Ecological
Succession of Gut Microbiota After Colonization
Fecal Microbiota
(2 weeks)
0.2
129
Recipients
0.1
PC2
B6 stool
0.0
B6
Recipients
-0.1
-0.2
129 stool
-0.2
0.0
0.2
PC1
0.4
Donor
B6
129
B6
129
Recip
129
129
B6
B6
Mouse Background Directs Ecological
Succession of Gut Microbiota After Colonization
Fecal Microbiota
(2 weeks)
Fecal Microbiota
(4 weeks)
0.3
0.2
129
Recipients
0.1
0.1
0.0
B6
Recipients
-0.1
PC2
PC2
B6 stool
-0.1
B6
-0.2
B6
129 stool
-0.2
0.0
0.2
PC1
0.4
-0.3
-0.1
0.1
PC1
0.3
0.5
Donor
B6
129
B6
129
Recip
129
129
B6
B6
Mouse Background Directs Ecological
Succession of Gut Microbiota After Colonization
Fecal Microbiota
(2 weeks)
Fecal Microbiota
(12 weeks)
Fecal Microbiota
(4 weeks)
0.3
0.3
0.2
129
Recipients
129
Recipients
0.1
PC2
B6
Recipients
PC2
PC2
0.0
-0.1
0.1
0.1
B6 stool
-0.1
B6
-0.2
B6
Recipients
-0.1
B6
129 stool
-0.2
0.0
0.2
PC1
0.4
-0.3
-0.1
0.1
PC1
0.3
0.5
-0.3
-0.3
-0.1
0.1
0.3
PC1
Donor
B6
129
B6
129
Recip
129
129
B6
B6
Mouse Background Directs Ecological
Succession of Gut Microbiota After Colonization
Fecal Microbiota
(2 weeks)
Fecal Microbiota
(12 weeks)
Fecal Microbiota
(4 weeks)
0.3
0.3
0.2
129
Recipients
129
Recipients
0.1
PC2
B6
Recipients
PC2
PC2
0.0
-0.1
0.1
0.1
B6 stool
-0.1
B6
-0.2
B6
129 stool
-0.2

B6
Recipients
-0.1
0.0
0.2
PC1
0.4
-0.3
-0.1
0.1
PC1
0.3
0.5
-0.3
Mouse background dictates the rate at which
microbial homeostasis is achieved after colonization
-0.3
-0.1
0.1
0.3
PC1
Donor
B6
129
B6
129
Recip
129
129
B6
B6
Mouse Background Directs Ecological
Succession of Gut Microbiota After Colonization
Fecal Microbiota
(2 weeks)
Fecal Microbiota
(12 weeks)
Fecal Microbiota
(4 weeks)
0.3
0.3
0.2
129
Recipients
129
Recipients
0.1
PC2
B6
Recipients
PC2
PC2
0.0
-0.1
0.1
0.1
B6 stool
-0.1
B6
-0.2
B6
Recipients
-0.1
B6
129 stool
-0.2
0.0
0.2
PC1
0.4
-0.3
-0.1
0.1
PC1
0.3
0.5
-0.3

Mouse background dictates the rate at which
microbial homeostasis is achieved after colonization

Mouse background influences the individual
variability within a given strain
-0.3
-0.1
0.1
0.3
PC1
Donor
B6
129
B6
129
Recip
129
129
B6
B6
Maternal Effects on the Gut Microbiota
BDF1
Female
Agouti
Embryos
C57BL/6
Embryos
Friswell MK et al (2010) PLoS One 5:e8584
Maternal Effects on the Gut Microbiota
BDF1
Female
C57BL/6
Embryos
Agouti
Embryos
Fecal bacterial communities of offspring were
indistinguishable from one another
Friswell MK et al (2010) PLoS One 5:e8584
Legacy Effect
Homozygous Breeding
KO × KO
WT × WT
WT
WT
WT
WT
WT
WT
KO
KO
KO
KO
KO
KO
Legacy Effect
Homozygous Breeding
KO × KO
WT × WT
 Differences between WT and
KO mice may be due to the
gene of interest
OR
WT
WT
WT
WT
WT
WT
KO
KO
KO
KO
KO
KO
 Random differences between
the founders that are
transmitted to their progeny
by the legacy effect
Legacy Effect
Homozygous Breeding
KO × KO
WT × WT
 Differences between WT and
KO mice may be due to the
gene of interest
OR
WT
WT
WT
WT
WT
WT
KO
KO
KO
KO
KO
KO
 Random differences between
the founders that are
transmitted to their progeny
by the legacy effect
Legacy Effect
Heterozygous Breeding
Het × Het
Het
Het
Het
Het
WT
WT
KO
KO
 Ensures that littermates are
exposed to the same
microbiota
 Differences between WT and
KO mice are likely due to the
gene of interest
The Role of Nod2 in Regulating the
Intestinal Microbiota
Homozygous Breeding
KO × KO
WT × WT
WT
WT
WT
WT
KO
WT
KO
KO
Nod2 KO
Wild-type
KO
WT
KO
KO
Nod2 genotype had a profound
influence on fecal microbial
composition
Rehman et al (2011) Gut 60:1354-62
The Role of Nod2 in Regulating the
Intestinal Microbiota
PCo1 versus PCo2
0.2
Heterozygous Breeding
Nod2 KO
Het × Het
Het
WT
WT
KO
PCo2
Het
Wild-type
0.1
0.0
KO
Het
Het
-0.1
-0.2
-0.1
0.0
0.1
0.2
PCo1
Nod2 genotype had no effect on fecal
microbial composition
Shanahan et al (2014) Gut 63:903-10
The Role of Nod2 in Regulating the
Intestinal Microbiota
PCo1 versus PCo2
0.2
Heterozygous Breeding
Nod2 KO
Het × Het
Het
WT
WT
KO
PCo2
Het
Wild-type
0.1
0.0
KO
Het
Het
Similar findings have been shown
for other bacterial ligand receptors
including TLR- 2,4,5,9 (Ubeda et
al, JEM, 2012)
-0.1
-0.2
-0.1
0.0
0.1
0.2
PCo1
Nod2 genotype had no effect on fecal
microbial composition
Shanahan et al (2014) Gut 63:903-10
Cage Effects Have a Stronger Impact on the
Intestinal Microbiota than Nod2 Genotype
PCo1 versus PCo2
0.2
Heterozygous Breeding
Nod2 KO
Het × Het
Het
WT
WT
KO
PCo2
Het
Wild-type
0.1
0.0
KO
Het
Het
-0.1
-0.2
-0.1
0.0
0.1
0.2
PCo1
Mice colored by the cage they were
housed in
Shanahan et al (2014) Gut 63:903-10
Cage Effects Have a Stronger Impact on the
Intestinal Microbiota than Nod2 Genotype
PCo1 versus PCo2
One-Way ANOVA
0.2
P
Value
1
0.22
2*
2.35x10-5
3*
1.87x10-4
4*
2.41x10-4
5
0.29
Nod2 KO
Wild-type
0.1
PCo2
Principal
Coordinate
0.0
-0.1
-0.2
* Cage was a significant
driver for 3 of the first 5
principal coordinates
-0.1
0.0
0.1
0.2
PCo1
Shanahan et al (2014) Gut 63:903-10
Cage Effects on Gut Microbial Composition
Germ-Free
Specific Pathogen
Free (SPF)
Gavaged with the fecal
microbiota pooled from 4 SPF
mice

Gavaged
(n = 12)



Acquired
(n = 12 )
At
least
4 cages
were used
for
Cage
effects
developed
in both
each
group
groups
by 4 weeks postcolonization
Stool samples were collected at
1,
2, 4,after
8 weeks
after
transfer
Even
being
gavaged
with
the same microbiota, cage
Processed
16S rRNA
effects stillfor
develop
over time
sequencing
Allowed to naturally acquire a
microbiota over time
McCafferty et al (2013) ISME J 7:2116-25
Impact of Housing Conditions on the
Intestinal Microbiota
 In addition to the micro-environment of an individual cage, the
macro-environment that mice are housed in can also profoundly
impact the intestinal microbiota
Hemoccult Index
Conventional Housing
Irgm1 KO
Wild-type
*
DSS Duration (days)
Liu et al (2013) Am J Physiol GI Liv 305:G573-G584
Impact of Housing Conditions on the
Intestinal Microbiota
 In addition to the micro-environment of an individual cage, the
macro-environment that mice are housed in can also profoundly
impact the intestinal microbiota
Irgm1 KO
Wild-type
DSS Duration (days)
Specific Pathogen Free Housing
*
Hemoccult Index
Hemoccult Index
Conventional Housing
Irgm1 KO
Wild-type
DSS Duration (days)
Liu et al (2013) Am J Physiol GI Liv 305:G573-G584
Impact of Housing Conditions on the
Intestinal Microbiota
Irgm1 KO vs. WT
Fecal Microbiota

No differences in fecal microbiota
between wild-type (WT) and
Irgm1 knock-out (KO) mice
Impact of Housing Conditions on the
Intestinal Microbiota
Irgm1 KO vs. WT
Fecal Microbiota

No differences in fecal microbiota
between wild-type (WT) and
Irgm1 knock-out (KO) mice

Clear separation between mice
raised in conventional (CV)
versus specific pathogen free
(SPF) conditions
Impact of Housing Conditions on the
Intestinal Microbiota
Irgm1 KO vs. WT
Fecal Microbiota
Helicobacter genus
* p = 2.61x10-9
Impact of Housing Conditions on the
Intestinal Microbiota
Irgm1 KO vs. WT
Fecal Microbiota
Helicobacter genus
* p = 2.61x10-9
H. hepaticus

Now in the process of inoculating
WT and KO SPF mice with H.
hepaticus to determine if this
induces DSS-susceptibility
Summary of Key Issues That Can Impact Mouse
Studies of the Intestinal Microbiota
1. Mouse genetic background profoundly influences the gut microbiota
 Bacterial composition
 Establishment of bacterial communities over time
 Individual microbial variability between mice
Summary of Key Issues That Can Impact Mouse
Studies of the Intestinal Microbiota
1. Mouse genetic background profoundly influences the gut microbiota
2. The intestinal microbiota is heritable
 Homozygous versus heterozygous breeding strategies can influence how
the microbiota develops within a given mouse colony
Summary of Key Issues That Can Impact Mouse
Studies of the Intestinal Microbiota
1. Mouse genetic background profoundly influences the gut microbiota
2. The intestinal microbiota is heritable
3. The local cage environment strongly impacts the gut microbiota
 Relevant even in experiments which expose mice to the same initial
microbial colonization
Summary of Key Issues That Can Impact Mouse
Studies of the Intestinal Microbiota
1. Mouse genetic background profoundly influences the gut microbiota
2. The intestinal microbiota is heritable
3. The local cage environment strongly impacts the gut microbiota
4. Housing conditions can significantly affect mouse phenotype and
intestinal microbial composition
 Offers the ability to identify specific organisms relevant to disease
pathogenesis
Considerations for Experimental Design
1. Control what is feasible to control
 Includes genetic background, housing conditions, age, sex, diet
 Many of these are difficult to control in human studies, but can be regulated
in mouse experiments
2. Record metadata for factors that are difficult to control
 Cage, litter, mother – these can be adjusted for statistically in downstream
analyses
 Initial power analyses should consider not only the numbers of mice per
group, but also the number of cages and litters per group
3. Attempt to control initial microbial exposure
 Heterozygous breeding
 Colonization of germ-free mice
 Co-housing/co-fostering
Considerations for Experimental Design
1. Control what is feasible to control
 Includes genetic background, housing conditions, age, sex, diet
 Many of these are difficult to control in human studies, but can be regulated
in mouse experiments
2. Record metadata for factors that are difficult to control
 Cage, litter, mother – these can be adjusted for statistically in downstream
analyses
 Initial power analyses should consider not only the numbers of mice per
group, but also the number of cages and litters per group
3. Attempt to control initial microbial exposure
 Heterozygous breeding
 Colonization of germ-free mice
 Co-housing/co-fostering
Considerations for Experimental Design
1. Control what is feasible to control
 Includes genetic background, housing conditions, age, sex, diet
 Many of these are difficult to control in human studies, but can be regulated
in mouse experiments
2. Record metadata for factors that are difficult to control
 Cage, litter, mother – these can be adjusted for statistically in downstream
analyses
 Initial power analyses should consider not only the numbers of mice per
group, but also the number of cages and litters per group
3. Attempt to control initial microbial exposure
 Heterozygous breeding
 Colonization of germ-free mice
 Co-housing/co-fostering
Conclusions
 Because we can address causation and study
mechanism, mouse models are a powerful tool to study
the intestinal microbiota
 However, we do have to consider careful experimental
design and control confounders to draw appropriate
conclusions from these studies
Conclusions
 Because we can address causation and study
mechanism, mouse models are a powerful tool to study
the intestinal microbiota
 However, we do have to consider careful experimental
design and control confounders to draw appropriate
conclusions from these studies
 Numerous thorough reviews have been published on
this topic:
− Spor et al. (2011) Nat Rev Microbiol, 9: 279-90
− Kostic et al. (2013) Genes Dev, 369: 517-28
Acknowledgements
Balfour Sartor
Alexi Schoenborn
Michael Shanahan
Greg Taylor
Raad Gharaibeh
Anthony Fodor
Ian Carroll
Allison Rogala
Bo Liu
Richard von Furstenberg
Susan Henning
Christian Jobin
Janelle Arthur
Maureen Bower
Lisa Holt
Funding
K08DK095917 (Gulati)
KL2RR025746 (Gulati)
CCFA/CDHNF Career Development
Award (Gulati)
Global Probiotics Council Young
Investigator Grant for Probiotic
Research (Gulati)
P30 DK34987 (CGIBD)
National Gnotobiotic Rodent Resource
Center P40R018603 (Sartor)
Cores
UNC Gnotobiotic Core (CGIBD)
Cell Service & Histology Core (CGIBD)
Microbiome Core (CGIBD)
The Role of Nod2 in Regulating the
Intestinal Microbiota
Homozygous Breeding
WT
WT
WT
WT
Nod2 KO
KO × KO
WT × WT
KO
WT
KO
Wild-type
KO
KO
WT
KO
KO

Nod2 genotype had a
profound influence on fecal
microbial composition
Rehman et al (2011) Gut 60:1354-62
The Role of Nod2 in Regulating the
Intestinal Microbiota
107
**
B. vulgatus cfu
B. vulgatus cfu
107
105
103
101
****
Nod2-/-
105
WT
103
N.D.
WT
Nod2-/-
0
20
30
10
Days post separation
Ramanan et al (2014) Immunity 41:311-24
The Microbial Cloud
The Microbial Cloud
Impact of Mouse Genetic Background
on the Gut Microbiota
 Variability of the
microbiota from
mouse-to-mouse also
depends on
background strain
 Circled strains have
tight clustering of
biological replicates
(BL6J, C3HRI, DBAJR,
PWK, WSB)
 Others have greater
individual variation
(129S1, AJ, CAST,
NOD, NZO)
Campbell JH et al (2012) ISME J 6:2033-2044
Reciprocal Colonization of Germ-Free Mice
Stool From
SPF C57BL/6 Mice


Stool From
SPF 129/SvEv Mice
GF
C57BL/6
Mice
GF
129/SvEv
Mice
GF
C57BL/6
Mice
GF
129/SvEv
Mice
(B6→B6)
(B6→129)
(129→B6)
(129→129)
If donor stool is driving microbial
composition, the solid squares will
cluster independently from the open
squares
If recipient background strain is
driving microbial composition, the red
squares will cluster independently
from the blue squares
CECAL CONTENTS
(12 weeks post-colonization)
 Samples segregate based on background
strain of recipient mice (p < 0.05)
Mouse Background Directs Ecological
Succession of Gut Microbiota After Colonization
2 weeks
Post-colonization
0.2
PC2
0.1
0.0
-0.1
-0.2
-0.2
0.0
0.2
PC1
Donor
B6
129
B6
129
Recip
129
129
B6
B6
0.4
Mouse Background Drives Composition of the
Intestinal Microbiota
Stool From
SPF C57BL/6 Mice
FECAL MICROBIOTA
(12 weeks post-colonization)
Stool From
SPF 129/SvEv Mice
0.3
GF
129/SvEv
Mice
GF
C57BL/6
Mice
GF
129/SvEv
Mice
(B6→B6)
(B6→129)
(129→B6)
(129→129)
129
Recipients
0.1
PC2
GF
C57BL/6
Mice

If donor stool is driving microbial
composition, the solid squares will
cluster independently from the open
squares

If recipient background strain is
driving microbial composition, the red
squares will cluster independently
from the blue squares
B6
Recipients
-0.1
-0.3
-0.3
-0.1
0.1
PC1
0.3
 Samples segregate based on background
strain of recipient mice (p < 0.0006)
Gulati et al (2012) PLoS One 7:e32403
Legacy Effect
Homozygous Breeding
KO × KO
WT × WT
WT
WT
WT
WT
WT
WT
Heterozygous Breeding
KO
KO
KO
KO
KO
KO
• Offspring can be re-bred to
generate additional mice for
downstream analysis
Het × Het
Het
Het
Het
Het
WT
WT
KO
KO
• Hets re-bred to generate
additional littermates
• WT and KO mice used for
analysis
Cage Effects on Gut Microbial Composition
Germ-Free
Specific Pathogen
Free (SPF)
Gavaged
(n = 12)

Cage effects developed in both
groups by 4 weeks postcolonization

Even after being gavaged with
the same microbiota, cage
effects still develop over time
Acquired
(n = 12 )
McCafferty et al (2013) ISME J 7:2116-25
Summary of Key Issues That Can Impact Mouse
Studies of the Intestinal Microbiota
1. Mouse genetic background profoundly influences the gut microbiota
 Includes composition, but also the development of bacterial communities
over time, and the individual microbial variability between mice
2. The intestinal microbiota is heritable
 Homozygous versus heterozygous breeding strategies can dictate how
the microbiota develops within a given mouse colony
3. The local cage environment strongly impacts the gut microbiota
 Relevant even in experiments which expose mice to the same initial
microbial colonization
4. Housing conditions can significantly affect phenotype and intestinal
microbial composition
 Offers the ability the identify specific organisms relevant to disease
pathogenesis
Conclusions
“When aware of the environmental and legacy effects
in mouse models and their implications for
microbiome experimental design, mice can still be a
stalwart tool for unraveling mechanisms of hostmicrobiota interactions relevant to humans.”
Impact of Mouse Genetic Background
on the Gut Microbiota

Analyzed the cecal microbiota of 10 distinct mouse strains using 454 sequencing
of the 16S rRNA gene

8 of these strains are currently being used to develop the Collaborative Cross

All are derived from these original 8 lines, which have their genomes sequenced

Will allow investigators to link specific genetic loci to phenotypic traits, and
patterns of microbial colonization
Campbell JH et al (2012) ISME J 6:2033-2044