ComplexGnotobiology:anEmerging

ParadigmintheEraofNextGeneration
Sequencing
CraigFranklin,DVM,PhD,DACLAM
MMRRC,RRRC,MUMC
UniversityofMissouri

 Archivesanddistributionrepositoriesofgenetically
engineeredmouseandratmodelsofhumandisease
OVERARCHINGQUESTION:Howaremodelsdistributed
influencedbydifferinggutmicrobiota(GM)

Complex gnotobiology
Gnotobiosis = known life
With next generation sequencing, we can better define
microbial OTUs present in a variety of sites
Complexcommunities

Validation

Observationgenerating

Caveats - depth of coverage,

expanding databases,
consistency of methods,
bacteria-centric
Emerging statistical tools for
analysis of complex communities
Functional predictors or adjuncts

Complement classical
gnotobiotic studies
Definedcommunities

Questions
DocomplexGMsvaryincontemporaryrodent
colonies?
Whatfactorscause(correlatewith)suchvariation?
Doesvariationinfluencemodelphenotypes?

HowcanweexploitcomplexGMvariabilityin
animalmodeling?
Refinemodels?
Assessreproducibility?
Improvetranslatability?

DoesGMvary
in
contemporary
rodent
colonies?
Bacterialfamily

PLoS One.2015Feb12;10(2):e0116704

Producer

PLoS One.2015Feb12;10(2):e0116704

Diet?Bedding?Housing?

Cecal microbiota

Cecal microbiota

VentilatedRack/Aspen
VentilatedRack/Paper
StaticMI/Aspen
StaticMI/Paper

Whatfactorscause(correlatewith)variation?
Host

Genetics
Age
Sex? ongoing
Producer

Manipulation
Antibiotics
Disease
Shipping?

Sampling
Location
Timeofday
Extractiontechnique

Environment

Cagetype
Bedding
Diet(acutechronic)
Housingdensity
Watertreatment?
Socialinteraction?
Enrichment? ongoing
Lightcycle?

Production
Naturalvs.caesarian
rederivationorcryorecovery

Many,Manymore

Doesvariationaltermodelphenotypes?

M1=GMCRL
M2=GMJAX
M3=GMTAC

Doesvariationaltermodelphenotypes?

C3H/HeJ IL10/

Differingmicrobiotaassociatedwith
differingcolonictumorcounts

Host/microbiota interaction

CanweexploitexistingGM
variability?
Options
Axenic/germfree
Monocolonized
Definedflora
Humanized

Complexmicrobiota
Barrier/specific
pathogenfree(SPF)
Conventional
Wildtype?

Richness&DiversityofGM

Complex
Microbiota
Targeted
Rederivation
(CMTR)

IL10KO,Smad3KO,andApcMinmice;ApcPIRCrat

OutbredcolonieswithdifferingmicrobiotaforCMTR
CD1
Embryos

Embryo
Transfer

Surrogate Dams
(Different Vendors)
Crl:CD1

GMTAC

GMJAX

GMCRL
C57BL/6J

C57BL/6NTAC

CD1

Generation 1
CD1

Generation 2-6

16S rRNA sequencing (fecal pellets)

OutbredcolonieswithdifferingmicrobiotaforCMTR
0.5
0.4

PC2 14.8% Variability

0.3

CD1GMCRL
CD1GMJAX

CMTR Surrogate Dam

0.2

First generation

0.1
0

Second generation

-0.1
-0.2

CD1 GMTAC

-0.3
-0.4
-0.5
-0.8

-0.6

-0.4

-0.2

PC1 22.4% Variability

0.2

0.4

0.6

ComparisonofCMTRsurrogatesource
Originalsurrogates(CD1,B6,B6)

B6IL10/

CD1surrogates

Canweusebankedfecesto
reconstituteGM?
LowdiversitydonorHighdiversityrecipient HighdiversitydonorLowdiversityrecipient

ReciprocalpostantibioticGMtransfers
Ericsson,etal.,Reciprocalfecalmicrobiotatransferbetweenmicewithlowandhighrichnessmicrobiotausingfreshor
frozenfecesorfreshcecal contents.FrontMicrobiol (acceptedpendingrevisions)

Reconstitution
isincomplete

Ericsson,etal.,Reciprocalfecalmicrobiotatransferbetweenmicewithlowandhighrichnessmicrobiotausingfreshor
frozenfecesorfreshcecal contents.FrontMicrobiol (acceptedpendingrevisions)

AremurineGMtranslatable?

Embracecomplexity

ImpactonAnimal
WelfareandEthics
Improvereproducibilitybydefining
GMandminimizingvariability
Improvestudydesign
Optimizemodels
Biomarkersfornonterminal
endpoints?
Ultimatelydecreaseanimal
numbers

Manyfuturechallenges
Reportingmicrobiota
Consistentmethods,databasesetc.
$$$
Standardize?

Bioinformatics
Translatability
Correlationvs.causation
Metabolomics/function
Unculturable agents
Genome+Metagenomesurveys
Viruses,fungi,protozoa,etc.

MetagenomicsGenomics

Acknowledgements
AaronEricsson
&MUMC
ElizabethBryda
&RRRC
MUDNACore
MUInformatics
ResearchCore
Facility
J.AmosLandgraf
&lab
Yuksel Agca &lab
CatherineHagan
&lab
J.WadeDavis
&lab

U42OD01091817

P40OD01106216

ComparativeMetagenomicsLaboratory
(akapoopgroup)

T32OD01112637

Mizzou Advantage OneHealth

Questions?