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Defined Microbiomes in
LaboratoryMice
Agenda 3Rsofethicalanimalexperimentation
Phenotypicvariance(differencesrevealedinexperimentalmeasurements)
hasahostgeneticandanenvironmentalcomponent
Theenvironmentalcomponentoftheassociatedmicrobiotaishardestto
controlorstandardise thebiologyisnotfullyunderstood.
Thisimpactsourabilitytomakerobustmeasurements,investigatethe
underlyingbiology,designsuitablecontrolsandreducenumbersof
animalsineachgroup.
Vphenotypic =Vgenetic +Venvironmental +Vexperimental
IfVgenetic 0(throughinbreeding)Vphenotypic = Venvironmental +Vexperimental
HowfarcanVenvironmental 0(throughgnotobiology)?
Nonpathogenic
environmental
microorganisms
=
Eukaryotic cells
Microbiome genes
~2000000
>
Eukaryotic genes
~20000
1010
Pharynxandupper
respiratorytract
10810/mlcontents
Distalsmall intestine
(ileum)
1011
1039/cm2
Skin
10910
Femalelower
genitaltract
Biomedicalimportance of
the underlying biology:
diseases shaped by our
microbiota
Liver disease
(NASHcirrhosis)
Asthma
Colonisation
resistance to GIand
respiratory infections
Inflammatory bowel
disease
Obesity
Detoxification
Type1diabetes
Tumours (e.g.colon
cancer)
Autoimmunearthritis
Skindisease
(eczema/psoriasis)
2012Science336,12681273
Skin A
Fecal B
Skin
Skin B
Fecal C
Skin C
Fecal D
Skin D
Fecal
Fecal E
Skin E
Strongeffects onbody
systems inthe presence of
nonpathogenic microbes
Germfree
isolators
Germfree
mice
Exgermfree mice
(d21)with non
pathogenic
microbes (SPF)
Effects of
microbiota
extend to
essentially
ALLbody
systems
Effects of
microbes
extend far
beyond their
livebiomasses
Promiscuous
metabolite
penetration
Compartmentalised
local immune
induction for benign
mutualists
Differenttypes ofexperimentalmodels
Definition
1. Limited scope
2. May omit microbial
metabolic pathways and
metabolite exchanges
between bacteria in
complex microbiotas.
3. Mostly mouse models
Advantages
Disadvantages
Bottomupmodels
Convergence
Topdownstudies
1. Imprecise definitions of
microbial consortia
2. Ambiguity in assigning
effects to species
3. Reproducibility issues
4. Ethical issues limit
human experimentation
Representation of a
natural situation that
models or directly shows
the human condition
Principlesofusinginbredanimals
Vgenetic 0(throughinbreeding)
Lessinherentphenotypicvariability(truefor
mostwholeanimalbiologyincludingbehavioural
andpharmacologicstudies).
Experimentseasiertopowerwithsmalleranimal
numbers.
Lossof(hostgenetic)traitscanbecompensated
byusingdifferentstrainswithafactorialdesign
(allofwhichareproperlypowered).
Isogenicmouse
timeline
Standardisation achievablewithgenetically
uniformanimals:exampleofkidneyweightin
inbredoroutbredrats
Animal type
Standard deviation of
kidney weights
13.5
30
18.3
54
Outbred
79
65
Mycoplasma free
18.6
56
Mycoplasma infected
43.3
296
Littermatebreedingapproachtoclarifying
microbiomeorhostgeneticcontributionto
phenotypictraits
Determinethe
mechanismsfor
genetic
differences
Characterise the
geneandits
underlying
mechanism
Determinethe
mechanismsfor
microbial
differencesinthe
originalbreeding
colonies
Metagenomics
Conbiotic mice
Stappenbeck &Virgin
2016,Nature534191199
BUT:excludingpathogensleavesanimmature
immunesystemin(colonised)laboratorymice
Cervicaltissue
CD8Tcells
Homing
markerfor
lymphoid
tissues
Peripheral
bloodCD8T
cells
Antigenstimulation
Beura etal.,2016,Nature532
5126
(alsoseeReeceetal.2016,Cell
HostMicrobe,1971319)
InbreedingofBalb/cmicetoattemptto
homogenise themicrobiota
Line1
Generation
Line2
Line 1
/ similarity in
breeding pair
(%)
Litter
size
Microbiota
similarity in
litter (%)
78
13
66
F1
78
73
F1
79
67
F2
79
66
F2
66
62
67
F3
67
67
55
F3
Generation / similarity in
breeding pair
Line 2
(%)
Litter
size
Microbiota
similarity in
litter (%)
Experimentalmanipulationofthemicrobiotaindifferent
mousestrains
1.
Birth
Maternal(orpaternal!)microbiota
2.
Germfree
Gavagewithorganismsfrompureculture,
fecesoraddcolonised animaltocage
3.
Germfree
Gavagewith
livebacteria
Colonised
?Phenotype
Colonised
?Immunity
Germfreeagain
?Immunity
Embryotransfer
newstrain
acquiresmaternal
microbiota
Germfreestrain
acquiresthe
desiredconsortia
subjectto
compatibilityand
extinctions
Uncouples
microbial
exposureand
permanent
colonisation
Gnotobiotic microbiotas
reducebetweenanimalandbetweencagediversityasfaras
possible
Initiallydevisedtoavoidfatalopportunistic
infectionsingermfreeanimals
Comparedwithisogenicmodels,relativelylittle
efforthasbeenmadetohavestandardised
stablemicrobiotasthatarerepresentativeof
diverselycolonised animalsforatleastsome
traits.
Isobiotic mouse
timeline
Embryotechniquesofconverting
isogenicandisobiotic mousestrain
Fecal culture on
supplemented medium
Hapfelmeier etal.,Science32817059
Functionalmicrobialshapingofthe
immunesystemstartswiththeMATERNAL
microbiota
Bacteria
Antimicrobial
peptides
Mucus
Antibodies
<<<<
Epithelial
cell
PIGR
NKp46+
ILC3
Goblet
cell
F4/80+
CD11c+
iMNC
Bcell
Mothertransientlycolonised
Controlgermfreemother
Bacteria
Epithelial
cell
Globet
cell
NKp46+
ILC3
Gomez,Ganal,etal.,Science35112961302
DC
F4/80+
CD11c+
iMNC
Bcell
2.Gnotobiotics withmoderatelydiverseconsortia
MetadataofalteredSchaedler flora(ASF)
ASF356
ASF360
ASF361
ASF457
ASF492
ASF500
ASF502
ASF519
From
mouse
strain
NelsonCollins
Swiss(NCS)
NCS
NCS
NCS
CD1
CD1
CD1
NCS
Stomach
Int./Cecum
Int./Cecum
Int./Cecum
Int./Cecum
Int./Cecum
Int./Cecum
Isolated
by
Russell
Schaedler
Rockefeller
Russell
Schaedler
Rockefeller
Russell
Schaedler
Rockefeller
Russell
Schaedler
Rockefeller
RogerOrcutt
CharlesRiver
RogerOrcutt
CharlesRiver
RogerOrcutt
CharlesRiver
Russell
Schaedler
Rockefeller
Taxonomy
Clostridiumsp.
Lactobacillus
intestinalis
Lactobacillus
murinus
Mucispirillum Eubacterium
schaedleri
plexi
caudatum
Pseudo
Clostridium
flavoni
sp.
bacteriumsp.
Parabacter
oides
goldseinii
Genome
size(Mb)
2.91
2.01
2.17
2.33
3.70
6.87
Intestine/Cecum
6.51
6.48
WymoreBrandetal.,2015,ILAR56,169
(alsoseeWannemuehler etal.2014,Genome
Announcementse00287fordraftgenomesequences)
IntestinalregulatoryTcellresponseon
colonisation withASFmicrobiota
Germfreemice
i)Maintainedgermfree
ii)CohousedwithsentinelswithAltered
SchaedlerFlora(ASF)microbiota
t=28days
IntestinalleucocyteFACS
Generationofcoreisobiotic mouselines
Stabledefined
moderatelydiverse
microbiotas (sDMDM)
Adddefinedmouseor
humancommensals
n=1030
Allon
standardised
(sterile)diets
Conbiotic microbiota
1ormoreorganisms
Interbreed
isobiotic line
Proposedcriteriaforgnotobiotic (isobiotic)strains
ASF
sDMDM
Microbial stabilityovergenerationsonopensourcediets
()
Transferabilityto differentgeneticbackgroundsandtodifferentinstitutionalvivaria
Canberegeneratedfrompureculture
()
Noabnormalitiesinclinicalchemistry,hematology,organhistology, bodycomposition,
developmentorfecundity
Representativemetabolic andimmunologicalprofilescomparedwithdiversemicrobiota
Normalpathogenresistance,inflammatoryandautoimmunemodeldiseasesusceptibility
()
Completenessofmicrobialmetabolicpathways
Relativestability underaseptichusbandryinisolatedventilatedcages
3.Gnotobiotics withmoderatelydiverseconsortia
MetadataofsDMDM
YL32
KB18
I48
YL27
Isolation
YL58
YL31
YL2
I49
YL44
KB1
2.75
Enterococcusfaecalis
2.04
Akkermansia muciniphila
1.80
Lactobacillusreuteri
3.82
Bifidobacteriumanimalis
subsp.animalis
5.13
Flavonifractor plautii
4.47
Blautia sp.
2.92
Erysipelotrichaceae
3.31
Burkholderiales
4.84
Parabacteroidessp.
3.80
Bacteroides sp.
7.22
Ruminiclostridium sp.
Genome
size(Mb)
I46
Lachnoclostridium sp.
Taxonomy
YL45
3.03
Improvedcolonisation
resistancetopathogens
Microbiotatransplant
Salmonellatyphimuravir
d1 d2
ASF+
Control
or
OligoMM(sDMDM)
or
SPFdiversemicrobiota
Beschluss andChallenges
Itispossibletocreateavarietyofisogenicmicrobiotaswithreasonable
stabilityovertimewiththatapproach:theycanbetransferredbetween
institutions.
Thesearepowerfultoolsformetatranscriptomic andmetagenomic
experimentatation withsmallgroupnumbers.
Thereisinevitablegeneticdriftanddietmustbecontrolled.
Theyarecapableofdiseasemodelling.
Wearguethattheseshouldbeasfreelyavailableacrossdifferent
institutionsasisogenicanimalmodels reproducibilityindifferentvivaria.
Nooneisobiotic modelshouldbeconsideredexclusive ratherafactorial
approachshouldbetakenacrossdifferentstandardised microbiotas.
Acknowledgements
UNIVERSITY
UNIVERSITY
ETHZRICH
of BERN
of MUNICH
UweSauer
BrbelStecher
TobiasFuhrer
SandrineBrugiroux
WolfHardt
KathyMcCoy*
SigiHapfelmeier
JulienLimenitakis
CatherineMooser
MercedesGomez
MarkusGeuking*
HaiLi
Yasuhiro Uchimura
StephanieGanal
IreneKeller
*Now atUniversityof Calgary