Creating Stabilised and

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)?

Bodysurfaces and mucous

membranes are heavily
colonised with
microorganisms
Infections xn
1014
1012/mlcontents
Largeintestine
(colon)

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

Variability of the natural microbiota:

5differenthumanindividuals sequentially sampled over 6weeks
Genus Level
Fecal A

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

Studying axenic models or

those with very simple
microbiotas, e.g. germ-free or
monocolonisations

Disadvantages

Bottomupmodels

Convergence

Topdownstudies

Studying defined components

of complex microbiotas (e.g.
Studies of complex and
IgA-bound bacteria) in a
natural microbiotas (e.g.
gnotobiotic system defined
human samples, SPF)
system

Defined and reproducible

Molecular mechanisms and
system with a microbiota that
interactions between microbes
aims to be representative of a
or their metabolites and the
natural situation and is
immune system can be defined
amenable to experimentation

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

No of animals to detect 10%

change in mean with two-tailed
test and =0.05

F1 hybrid (cross of two inbred

strains [AB])

13.5

30

F2 hybrid (AA, AB, BB)

18.3

54

Outbred

79

65

Mycoplasma free

18.6

56

Mycoplasma infected

43.3

296

Garnter K, Lab. Animal 1990; 24: 71

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 (%)

Second breeding pair selected from the offspring of P

Pang et al., Laboratory Animals 2012; 46: 335337

Experimentalmanipulationofthemicrobiotaindifferent
mousestrains
1.

Birth

Maternal(orpaternal!)microbiota

2.

Germfree

Gavagewithorganismsfrompureculture,
fecesoraddcolonised animaltocage

3.

Germfree

Gavagewith
livebacteria

Colonised
?Phenotype

Colonised
?Immunity

Germfreeagain

?Immunity

Comparing host strains with different targeted

deletions dissects mechanisms in vivo

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

1.Reversiblecolonisation (HA107)uncouples livemicrobial

exposure and permanentcolonisation
1010 cfu E.Coli
HA107 or K12 by
aseptic gavage
into germ-free mice

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

Geuking etal.,Immunity 34794806

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

()

Membersculturable with publishedgenomicsequencesandopensourcestocks

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

Prof. Brbel Stecher Munich(reconstitutionProfKathyMcCoy,Bern)

Lachnoclostridium sp.

Taxonomy

YL45

3.03

Hai et al., 2014, Nature Comm.8, 6292

Brugiroux..Stecher 2016 Nature Microbiol
Uchimura et al. 2016, Genome Announcements for
circular genome sequences e00951

Improvedcolonisation
resistancetopathogens
Microbiotatransplant

Salmonellatyphimuravir
d1 d2

ASF+

Control
or
OligoMM(sDMDM)
or
SPFdiversemicrobiota

Brugiroux..Stecher 2016 Nature Microbiol. 21,16215

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

Staff of the CleanMouseFacilityin

Bernand the vivaria inMunich and
Zrich