Complex Gnotobiology: an Emerging 

Paradigm in the Era of Next Generation 
Sequencing
Craig Franklin, DVM, PhD, DACLAM
MMRRC, RRRC, MUMC
University of Missouri

– Archives and distribution repositories of genetically 
engineered mouse and rat models of human disease
– OVERARCHING QUESTION: How are models distributed 
influenced by differing gut microbiota (GM)

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

Validation

Observation generating

– 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
Defined communities

Questions
• Do complex GMs vary in contemporary rodent 
colonies?
– What factors cause (correlate with) such variation?
– Does variation influence model phenotypes?

• How can we exploit complex GM variability in 
animal modeling?
– Refine models?
– Assess reproducibility?
– Improve translatability?

Does GM vary 
in 
contemporary 
rodent 
colonies?
Bacterial family

PLoS One. 2015 Feb 12;10(2):e0116704

Producer

PLoS One. 2015 Feb 12;10(2):e0116704

Diet? Bedding? Housing?

Cecal microbiota

Cecal microbiota

Ventilated Rack / Aspen
Ventilated Rack / Paper
Static MI / Aspen
Static MI / Paper

What factors cause (correlate with) variation?
• Host



Genetics
Age
Sex? ongoing
Producer

• Manipulation
– Antibiotics
– Disease
– Shipping?

• Sampling
– Location
– Time of day
– Extraction technique

• Environment







Cage type
Bedding
Diet (acute         chronic     )
Housing density
Water treatment?
Social interaction?
Enrichment? ongoing
Light cycle?

• Production
– Natural vs. caesarian 
rederivation or cryorecovery

• Many, Many more

Does variation alter model phenotypes?

M1 = GMCRL
M2 = GMJAX
M3 = GMTAC

Does variation alter model phenotypes?

C3H/HeJ IL‐10‐/‐

Differing microbiota associated with 
differing colonic tumor counts

Host/microbiota interaction

Can we exploit existing GM 
variability?
• Options
Axenic /  germ‐free
Mono‐colonized
Defined flora
“Humanized”

– Complex microbiota
• Barrier/specific 
pathogen‐free (SPF)
• Conventional 
• “Wild‐type”?

Richness & Diversity of GM




Complex 
Microbiota 
Targeted 
Rederivation
(CMTR)

IL‐10 KO, Smad‐3 KO, and Apc‐Min mice; Apc‐PIRC rat

Outbred colonies with differing microbiota for CMTR
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)

Outbred colonies with differing microbiota for CMTR
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

0

PC1 22.4% Variability

0.2

0.4

0.6

Comparison of CMTR surrogate source
Original surrogates (CD‐1, B6, B6)

B6 IL‐10‐/‐

CD‐1 surrogates

Can we use banked feces to 
reconstitute GM?
Low diversity donor  →  High diversity recipient       High diversity donor  →  Low diversity recipient     

Reciprocal post‐antibiotic GM transfers
Ericsson, et al., Reciprocal fecal microbiota transfer between mice with low and high richness microbiota using fresh or 
frozen feces or fresh cecal contents. Front Microbiol (accepted pending revisions) 

Reconstitution 
is incomplete

Ericsson, et al., Reciprocal fecal microbiota transfer between mice with low and high richness microbiota using fresh or 
frozen feces or fresh cecal contents. Front Microbiol (accepted pending revisions) 

Are murine GM translatable?

Embrace complexity

Impact on Animal 
Welfare and Ethics
• Improve reproducibility by defining 
GM and minimizing variability
• Improve study design
• Optimize models
• Biomarkers for non‐terminal 
endpoints?
• Ultimately decrease animal 
numbers

Many future challenges
• Reporting microbiota
– Consistent methods, databases etc.
– $$$
– Standardize?







Bioinformatics
Translatability
Correlation vs. causation
Metabolomics / function
Unculturable agents
Genome + Metagenome surveys
Viruses, fungi, protozoa, etc.

Metagenomics                  Genomics

Acknowledgements
• Aaron Ericsson 
& MUMC
• Elizabeth Bryda
& RRRC
• MU DNA Core
• MU Informatics 
Research Core 
Facility
• J. Amos‐Landgraf
& lab
• Yuksel Agca & lab
• Catherine Hagan 
& lab
• J. Wade Davis 
& lab

U42 OD010918‐17

P40 OD011062‐16

Comparative Metagenomics Laboratory
(aka poop group)

T32 OD011126‐37

Mizzou Advantage – One Health

Questions?

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