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Diet Driven Evolution of Epidemic

Strains of an Enteric Pathogen


RobertBritton,Ph.D
CenterforMetagenomics andMicrobiome Research
DepartmentofMolecularVirologyandMicrobiology
BaylorCollegeofMedicine
Robert.Britton@bcm.edu

Projects in the Britton laboratory


Therapeutic microbiology
Role of the intestinal microbiota in resisting pathogen
invasion.
C. difficile
MBRAs

Traditional probiotics and host interactions


Probiotics and gut motility/IBS (J. Galligan, MSU)
Probiotics and bone health (L. McCabe, N. Parameswaran,
MSU)

Next-generation probiotics
Obesity and type 2 diabetes

Therapeutic delivery systems for the treatment of human


disease.
Intestinal inflammation (J. Tabor, Rice U.)
Secretion of human therapeutic proteins (IL-22 for GVHD, J.
Ferrara, Mount Sinai)
Recombineering and CRISPR technology available for
engineering Lactobacillus reuteri.

Ribosome Assembly

Projects in the Britton laboratory


Therapeutic microbiology
Role of the intestinal microbiota in resisting pathogen
invasion.
C. difficile
MBRAs

Traditional probiotics and host interactions


Probiotics and gut motility/IBS (J. Galligan, MSU)
Probiotics and bone health (L. McCabe, N. Parameswaran,
MSU)

Next-generation probiotics
Obesity and type 2 diabetes

Therapeutic delivery systems for the treatment of human


disease.
Intestinal inflammation (J. Tabor, Rice U.)
Secretion of human therapeutic proteins (IL-22 for GVHD, J.
Ferrara, Mount Sinai)
Recombineering and CRISPR technology available for
engineering Lactobacillus reuteri.

Ribosome Assembly

Minibioreactor arrays to study the functions


of complex microbial communities
C. difficile:microbiota interactions
Future directions and challenges

How do microbial communities resist


invasion by pathogens?
Supported by MSU ERIN NIH NIAID
U19AI0908972 and matching funds by
Michigan State University

Cathy Robinson

Jennifer Auchtung

Two Model Systems for studying C. difficile


interaction with human-derived microbiota
In vitro culturing in
bioreactors

- Relatively Simple
- Higher throughput
- Reduces the numbers of
animals needed for
experiments

In vivo studies in
humanized microbiota mice

- Complex host-microbiotapathogen interactions

An in vitro model to study interactions


between human microbiota and C. difficile
3 vessel continuous culture
model to study gut microbiota
developed by McFarlane et al1
Used by Freeman et al2 to study
C. difficile proliferation and toxin
production in the presence of
human microbiota
1McFarlane,

G.T., McFarlane, S., and Gibson, G.R.


(1998). Microb. Ecol. 35:180-187
2Freeman, J., ONeill, F.J., and Wilcox, M.H. (2003). J.
Antimicrob. Chemother. 52: 96-102.

SHIME

ROBOGUT

Development of in vitro model to study


Interactions between C. difficile and the
human intestinal microbiota
Pump

Pump

Fresh
Media

15 ml
bioreactor

Waste
Media

Environmental parameters:
-

Anaerobic
37C
pH 7-7.5
Continuously stirred
~8 hr retention time

Minibioreactor array (MBRA) setup allows


operation of up to 96 bioreactors

How does MBRA cultivation impact community


composition and dynamics?

- 3 different fecal donors (A,B,C) or pool of 3 donors (A+B+C)


- Cultivate triplicate reactors/donor
- Monitor community composition daily (days 0-21) through 16s
rRNA gene sequencing

Auchtung J.M., Robinson, C.D., and Britton, R.A. (2015). Microbiome. 3:42

MBRA cultivation leads to distinct,


stable communities
A.
NMDS Axis 2

0.0

Bray-Curtis Dissimilarity
stress=0.167

Donor B

Donor C
Pool
(A+B+C)

Donor A

0.0
NMDS Axis 1

Community composition
assessed by sequencing V4
region of 16S rRNA gene
(Illumina)
Dissimilarity measure based
upon shared OTUs (97% ANI)
Similar results observed with
Sorenson Dissimilarity

Auchtung J.M., Robinson, C.D., and Britton, R.A. (2015). Microbiome. 3:42

MBRA cultivation leads to distinct,


stable communities
Calculate average community
similarity of each day from
other days in culture

Avg BC Similarity

1.0
0.8
0.6

Plot as function of day in


culture

0.4
0.2
0.0

Average day of stabilization =


12 15 18 21 Day 8 (7 days of culture)

6 9
Time in Culture

Auchtung J.M., Robinson, C.D., and Britton, R.A. (2015). Microbiome. 3:42

Mean Bray-Curtis Similarity

MBRA cultivation leads to distinct,


stable communities
1.0
Days 8-13

0.8
0.6
Days 2-7

0.4

Day-to-day variation
~10% higher than
background

0.2
0.0
1

2
3
4
5
Days between Sampling
Auchtung J.M., Robinson, C.D., and Britton, R.A. (2015). Microbiome. 3:42

MBRA community composition


A

Relative Abundance

1.0

Actinobacteria
Bacteroidetes
Firmicutes
Proteobacteria
Unclassified Bacteria
Verrucomicrobia

Relative proportions of
dominant phyla similar to
fecal samples

0.5

0.0
01230123 01230123456
A
B
C
Pool
Auchtung J.M., Robinson, C.D., and Britton, R.A. (2015). Microbiome. 3:42

Overview of bioreactor growth and


C. difficile challenge
Start
flow
Inoculate
with fecal
slurry

Add
C. difficile
Treat with
clindamycin or
mock-treat

Monitor C. difficile abundance

10

11 12

Time in Culture (Days)

Compare responses between mock-treated


(water)/untreated and clindamycin-treated bioreactors

C. difficile Abundance (CFU/ml)

C. difficile invades clindamycin-treated


MBRA communities
107
clinda-treated
n=9

106
105
104
103

mock/untreated
n=10

102
101

3
4
5
Days in Culture

C. difficile Abundance (CFU/ml)

C. difficile can invade at low


infective doses
107

clinda-treated
n=7

106
105

clinda-treated
n=3

104

clinda-treated
n=9

103
mock/untreated
n=10

102
101

3
4
5
Days in Culture

Total bacterial load in the MBRAs does


not change with clindamycin treatment

Reduced diversity associated with


clindamycin treatment

Other uses of mini-bioreactor array


communities
Drug metabolism by the microbiota
Drug activation, inactivation

Production of beneficial and detrimental


metabolites
Trimethylamine production in CVD

Formation of defined microbial consortia from


purified strains
Preclinical testing models?

Establish microbial communities from other body


sites
Cultivation of hard to culture microbes

Future directions and challenges


Interface MBRAs with other host models:
enteroids and organoids.
Build in additional spatial niches into MBRAs.
Begin to model other intestinal sites small
intestinal microbial communities.
Media composition?
Functional assessment?
Genetics?

Funding in the Britton laboratory: NIH, NSF, Procter


and Gamble, Christian Hansen, and Biogaia.

Members of the Britton lab


Jennifer Auchtung
Laura Schaefer
James Collins
Darin Quach
Laura Ortiz-Velez
Kylie Farrell
Nikhil Jain
Stephanie LeValley
Catherine Tomaro-Duchesneau
Helene Velly
Jeff Galley
Emmanuel Ngall
MSU ERIN
Linda Mansfield
Shannon Manning
Kate Eaton
Bob Stedtfeld
MSU ERIN team

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