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Using gut microbes as therapy

for C.difficile infection

and beyond Emma Allen-Vercoe

University of Guelph

PHO Rounds Microbiology

October 8th 2015

Presenter disclosure

• Faculty: Emma Allen-Vercoe

• Name of the commercial interest:

Nubiyota LLC

• Nature of the relationship: Co-founder

and scientific advisor

NuBiyota Better ecosystem, better health

Only very few microbes are pathogens In fact, human health depends on microbiota health

We are the vessels for our microbial passengers

https://www.broadinstitute.org/files/news/images/2012/gevers_cover_nature.png

There are more bacteria living in your gut

than there are people on the planet…

Gut: 200-500

bacterial

species

Also Archaea as

well as yeasts &

other

microscopic

eukaryotes

Everyone is different

Gut microbial ecosystems are highly variable in

composition and abundance profiles between people

http://farm9.staticflickr.com

How do we acquire

our microbes?

Toh & Allen-Vercoe, MEHD Feb 2015

How do we acquire

our microbes?

Toh & Allen-Vercoe, MEHD Feb 2015

What do our gut microbes do for us?

Metabolic

• Fermentation of non-digestible substrates

• Production of vitamins, SCFA

• Removal of toxins, carcinogens

• Differentiation of IECs

Structural

• Intestinal villi and crypts

• Tight junctions

• sIgA production

• Mucus secretion

Protective

• Colonization resistance

• Innate and adaptive

immunity

• Inflammatory cytokine

oversight

Competition

for sites and

nutrients

Immune

system and

barrier

function

Antimicrobial

secretion Energy

After Cryan et al.

We exist with our microbes in a delicate

state of equilibrium

http://eco-chick.com/2008/10/1621/still-rock-n-roll-the-balanced-stone-art-of-shane-hart/

Maintaining the equilibrium

High diversity of species:

•Healthy ecosystem

•Balance

•Functional redundancy

•Resistance to disease

Low diversity of species:

•Sick ecosystem

•Imbalance

•Functional disability

•Susceptibility to disease

‘Extinction events’ may impact health • Hygiene hypothesis

– We are preventing proper colonization by being too clean

• Missing microbiota hypothesis – Blaser & Falkow Nature Reviews Microbiology 2009

– We are disturbing proper colonization across generations through e.g. antibiotic use, poor diet, over-sanitary lifestyle

• Antimicrobial use (especially in early childhood) may be particularly problematic

• Highly refined Western-style diet exacerbates the problem further

http://www.medicaldrugeffects.com/save-environment/

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• Several studies have shown:

– Gut microbiota changes significantly with

antibiotic use

– Takes a long time afterwards to return to

baseline

– Sometimes does not return to baseline at all

– Repeated ‘hits’ cause vast changes from

which the ecosystem does not recover

Looft et al., PNAS 2012; Robinson & Young Gut Microbes. 2010; Jakobsson et al. PLoS One. 2010;

Antonopoulos et al., Infect Immun. 2009; Dethlefsen et al. PLoS Biol. 2008.

• Lack of microbial

diversity

• Loss of

‘keystone’

species

• Overgrowth of

opportunistic

pathogens

• Poor diet/lifestyle

• Drug interactions

DISEASE

“Dysbiosis”

• How do we look inside the black box?

• How do we study it?

Examples of diseases associated with

reduced gut microbiota diversity (published research)

Infant colic

Neonatal necrotizing enterocolitis

Obesity

Inflammatory bowel diseases

Clostridium difficile infection

Eczema Colorectal cancer

Allergic asthma Celiac disease

Autism

Irritable Bowel Syndrome

• The Good

• Lactic Acid Bacteria

(LAB)

– E.g. Bifidobacterium

and Lactobacillus spp.

• Butyrate-producing

bacteria

– E.g. Faecalibacterium

prausnitzii, Roseburia

spp.

• The Bad

• Opportunistic

pathogens

– E.g. E.coli,

Pseudomonas

aeruginosa,

Clostridium difficile,

Bacteroides fragilis

• Sulfate-reducing

bacteria

– E.g. Desulfovibrio spp.

The Ugly: it really is not that clear-cut

What’s inside the black box?

Some microbes are like bad teenagers in a

subway station…

In a crowded

environment they tend to

behave well

When the crowds are gone,

they tend to start behaving

in antisocial ways

Wikimedia commons images

C.difficile:

a ‘bad teenager’

Normal colon

Non-spore-forming anaerobes predominate;

C.difficile absent or numbers low

Antibiotics

-clindamycin

-cephalosporins

-ampicillin

Vancomycin or

metronidazole

Symptoms abate

Cessation of therapy

Return to normal

Reduction in major

genera of anaerobes:

C.difficile grows

to high numbers

C. Carlucci, A-V lab, 2012

Production of

exotoxins A and B

Diarrhea

Ulceration

of colon

Death Wikimedia commons

So, C.difficile is a pathogen that

needs to be destroyed!

• We need to throw our best chemical

weapons at it to rid ourselves of the

scourge!

• …NO, this is NOT the best strategy

http://forums.bistudio.com/showthread.php?98772-Grenade-Throwing-Animation

We simply need to

repopulate the subway

…but how do we do that?

The human gut microbiota is

a complex microbial

ecosystem

http://www.sciencephoto.com

Its function and behaviour

is best studied as a whole

Microbes in a microbiology lab… •Almost always exist on their own as part of a

pure culture

•Usually have to adapt to survive this way

•Are often grown logarithmically

•Are usually given access to rich nutrient

sources

Microbes in nature… •Almost always exist as part of microbial

communities

•Benefit from their microbial friends (& host)

•Rarely grow logarithmically

•Rarely have access to rich nutrient

sources

…thus, chemostats

can be used to

emulate the human

colonic environment

The human colon is a type of

chemostat…

How do we study the black box?

Our chemostats (‘Robogut’)

•Seeded with fresh feces or defined communities and set to model the

distal gut ecosystem

•Host-free system

•Can be used to ‘culture the unculturable’

•Can support whole gut microbial ecosystems for several weeks at a time

Microbes ‘talk’ each other using

small molecules

Wikimedia commons images

Many of these small molecules are also absorbed by the host

“Liquid gold”

Allows us to eavesdrop on microbial conversations

Patterns of perturbation differ between donors Different

people’s

microbiota

responds to

antibiotic

disturbance in

different ways

Kathleen Schroeter and Julie McDonald

‘Defined’ microbial ecosystems

• As well as studying fecal ecosystems, we can culture diversity from fecal samples

• Reconstitute into defined ‘experimental’ ecosystems

– Strain composition is known • Genomes, transcriptomes, metabolomes etc. known or

measureable

• Strain composition can be easily (and cleanly) altered

• We can look at deliberate changes in composition and how they affect ecosystem function

Defined communities from ulcerative colitis patients

UC1

UC3

Rifaximin treatment

effects vary

according to

community

Kaitlyn Oliphant

Yen et al., Journal of

Proteome Research,

2015

1H NMR profiling of

liquid gold

‘Liquid gold’ derived

from different donors

produces metabolic

profiles unique to the

respective hosts

Marc

Aucoin, U

Waterloo

Sandi Yen

•Results in cure of the

patient in >90% of cases

•Rapid resolution of CDI

•Only rare recurrence of

disease

•Fresh homogenate instilled

into patient within a few hrs

of preparation •Rectal enema

•Colonoscopy

•Nasoduodenal tube

•“Poop pills”

Back to CDI… FMT: one way to put the people back on the

subway platform…

But not without risk…

Known

• Potential to transmit

infection

• Risks of procedure

• Colonoscopy and

associated sedation

Unknown • Changes in

microbiome that increase risk of other diseases (e.g., IBD, cancer, obesity, etc)

• Risk of acquiring new pathogens or infection

• Development of autoimmune disorders

Long-term risks: UNKNOWN

Case report: sudden and

unexpected weight gain after FMT

Sciencedaily.com

Alang and

Kelly, 2014

What if we could create ‘synthetic stool’ to

treat CDI?

Good idea – why hasn’t anyone

done this before?

• They have! Early studies used small number

of gut microbial isolates to treat CDI

– Tvede & Rask Madsen, Lancet 1989

• Fell out of favour with increased antibiotic use

• Difficult to do – most gut anaerobes

(erroneously) thought to be ‘unculturable’

• Prior lack of understanding of the importance

of the gut microbiota to health

Steps to MET

1. Find a healthy donor

2. Use chemostat to isolate microbial diversity

3. Characterize, curate and preserve isolates

4. Test for, and remove, undesirable

components

Antibiotic resistance, presence of virulence genes,

toxicity, etc.

5. Assemble, test and optimize ecosystem Determine optimal growth conditions/scalability

“RePOOPulate” • Acidaminococcus intestinalis

• Bacteroides ovatus

• Bifidobacterium adolescentis (x2)

• Bifidobacterium longum (x2)

• Collinsella aerofasciens

• Dorea longicatena (x2)

• Escherichia coli

• Eubacterium eligens

• Eubacterium limosum

• Eubacterium rectale (x4)

• Eubacterium ventriosum

• Faecalibacterium prausnitzii

• Lactobacillus casei

• Lactobacillus paracasei

• Parabacteroides distasonis

• Raoultella sp.

• Roseburia faecalis

• Roseburia intestinalis

• Ruminococcus torques (x2)

• Streptococcus mitis

• Likely novel species (x5)

• Likely novel genus & species (x1)

(Closest species by full-length 16S alignment)

“RePOOPulate” • Acidaminococcus intestinalis

• Bacteroides ovatus

• Bifidobacterium adolescentis (x2)

• Bifidobacterium longum (x2)

• Collinsella aerofasciens

• Dorea longicatena (x2)

• Escherichia coli

• Eubacterium eligens

• Eubacterium limosum

• Eubacterium rectale (x4)

• Eubacterium ventriosum

• Faecalibacterium prausnitzii

• Lactobacillus casei

• Lactobacillus paracasei

• Parabacteroides distasonis

• Raoultella sp.

• Roseburia faecalis

• Roseburia intestinalis

• Ruminococcus torques (x2)

• Streptococcus mitis

• Likely novel species (x5)

• Likely novel genus & species (x1)

Lachnospiraceae and Ruminococcaceae family species (Generally lacking in CDI, IBD)

RePOOPulate proof-of-principle trial

• 2 elderly ladies with severe, recurrent C.diff infections were treated (April and June 2011)

• RePOOPulate made fresh at Guelph, driven to KGH, and administered via colonoscopy

– 1 dose, 100mLs

• Both patients recovered within 2 days and have remained C.diff-free ever since (despite numerous subsequent antibiotic exposures)

2013: 1 (1): 6

Petrof et al. Microbiome, 2013 In both patients, RePOOPulate

signatures could be seen 6

months following administration:

colonization had taken place

Why only n=2? • Trial temporarily stopped by Health

Canada

– Is RePOOPulate/MET1 a probiotic or a biologic drug?

• Health Canada (& FDA): should be considered as a biologic

– Potentially easier to regulate than fecal bacteriotherapy

– But so far, no guidelines for us!

From Health Canada Guidelines for FMT

Where we currently are with MET

• Developing MET1+ as well as other METs

from a range of healthy donors

• Creating a QC pipeline

– Based on metagenomics,

metabolomics/metabonomics and other

proprietary measures

• Scale up and stabilization of product

– For encapsulation and simple, oral delivery

– Sounds easy, but it isn’t!

• Gearing up for new clinical trial

The challenges

• What’s the definition of a healthy gut microbiota? – Defining species content is likely not enough

• What determines success or failure of engraftment? – And how can we modulate this?

• What are the long-term risks? – (Much easier to measure with a defined MET)

• Can the MET approach be applied for other disease indications?

Do we need to match patient to

ecosystem?

Consider different people’s bodies as different

models of car Consider the gut microbiota as an engine

Wikimedia commons images

Maybe we should just replace the faulty part of the engine

Birchall et al. in prep

…Ushering in the era of personalized medicine

I foresee a time when… • People will regularly have a

microbiome check-up

• Antibiotic use will be minimal and personalized to reduce risk of microbiota damage

• Microbial Ecosystem Therapeutics will enter mainstream medicine

• “Symbiontology” will become a new medical specialty

– A merger of Gastroenterology, microbial ecology, nutrition science, and many other disciplines

Acknowledgements EA-V lab members

Christian Ambrose

Erin Bolte

Christian Carlucci

Kyla Cochrane

Michelle Daigneault

Kaitlyn Oliphant

Rafael Peixoto

Kathleen Schroeter

Mariia Taguer

Mike Toh

Sandi Yen

And alumni

Eric Brown

Ian Brown

Julie McDonald

Queen’s U

Elaine Petrof

Adriana Breen

Curtis Noordhof

KGH clinical staff

Western U

Greg Gloor

Jean Macklaim

U Waterloo

Marc Aucoin

NuBiyota Better ecosystem, better health

I found the problem, Mr. Smith. Instead of

probiotics, you have been taking amateur biotics.