Is the microbiome really an importantplayer in human disease?

Karen Madsen, PhDUniversity of Alberta

Microbiomania – not a new phenomena

Eclectic Medical Journal 1898

Publications 2000-2014

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Microbiome

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New technologies…

Culture based Whole genome sequencing

Internet 2015

All diseases and humanbehaviour are influenced

by gut microbes!

Therefore, fix yourmicrobes and you willcure your disease and

change your life!

GenesX

Jonathan Eisen blog

Attraction to partners and relationship success is determined byfinding a partner with similar microbiomes to oneself….

And that if the microbiome in a kissing partner is a match thisleads to bliss and addiction

if the bacteria don’t align with your microbes, you actuallyfeel disgust and revolt

Which has led to things like this interview onCBC Fresh Air….

Host microbiota and health

Jeremiah J. Faith et al. PNAS 2015;112:633-640

Genetic and Microbial Inheritance

• Microbial inheritance couldinvolve acquisition of one or anumber of effector strains thatindividually or collectivelymodulate risk of developingcomplex diseases

Model of Microbial Inheritance

Probability that the gut of any individual is colonized with aparticular strain

P(transmission) = depends on factors such as its ability to surviveenvironmental insults and competitiveness

P(access) – depends on microbes abundance in the community aswell as hygiene, frequency of contact with and healthy status

P(resistance) – depends on the current composition of thepotential recipients microbiota

Jeremiah J. Faith et al. PNAS 2015;112:633-640

Changing microbial colonization throughout life

PNAS 2011:108(Suppl 1):4578

Succession of microbes in an infant

Fever

Introductionof formulaand food

In 2011, we were divided into 3 enterotypes!

(Based onsequencingabout 300people…..)

Enterotype 1= High Bacteroides

Enterotype 2= High Prevotella

Enterotype 3= High Ruminococcus

Arumugam et al. Nature 473, 173-180. 2011

2014: Revision: Diet is the main determinant ofgut microbial composition

Voreades et al. Front. Microbiol. Sept 22, 2014

Jeremiah J. Faith et al. Science 2013;341:1237439

Stability of Microbiota over Time

• Followed 30 individuals up to 5 years• Analyzed microbiota at a strain level• Found ~30% of strains shared between unrelated individuals

Microbiome science needs a healthy dose ofskepticism….

• Do microbiome studies showcausation or just correlation?

• Are changes in microbial compositionbiologically relevant?

• Could anything else explain theresults?

Hanage WP (2014) Nature 512:247-8

CORRELATION CAUSATION

Nature 178:488. 2012

MJ Claesson et al. Nature 000, 1-7 (2012) doi:10.1038/nature11319

Living in long-term care resulted in a poorer diet,worse health, and altered microbiome

Diet Microbiome Poor Health

??

178 seniors

Community

Residential care

If gut microbial dysbiosis is the causeof the disease, then

Fixing the dysbiosis should cure the disease

IBD patients have dysbiosis

What about replacing gut microbes by fecalmicrobial transplantation (FMT)?

• Instillation of gut microbiota from ahealthy person into a sick person

• FMT delivered by colonoscopy,nasogastric tube, or pills

– Colonoscopy

• 300-500 ml

– NG/NJ tube• 60 ml

– Pills• 20-40/dose

FMT does cure C. difficile colitis - clearinfectious cause of disease

• Trial terminated earlybecause it wasdeemed unethical tocontinue antibiotictreatment

2 RCT using FMT to treat UC

GroupTotal N

(active/placebo)

Dutch48 (23/25)

McMaster75 (38/37)

Patient population Mild-mod UC Mild-mod UC

Active arm (realFMT)

500 cc nasoduodenal infusion(120 g donor stool) at wk 0 and3

50 cc enema (8g donor stool)weekly X6

Control Sham FMT (patient stool) Water

OutcomesPrimary Remission (SCCAI < 2 + and

> 1 point reduction in MayoRemission (Mayo score < 3and Mayo endo score=0) @wk 7

Primary outcome 7/23 (30%) vs 5/25 (20%)p=0.51

9/38 (24%) vs 2/37 (5%)p=0.03

*Both trials were stopped early by DSMB due to futility

Moayyedi et al Gastroenterology 2015 April 7Rossen et al. Gastroenterology 2015 March 30

Results are generally not maintained in IBDpatients

This suggests that the “dysbiosis” in IBDpatients is a consequence, not the cause

Intestinal inflammation enhances thegrowth of certain facultative

anaerobes while decreasing thegrowth of obligate anaerobes

Intestinal Inflammation InducesDysbiosis

R3-N+-O-

(TMAO)

R2-SO(Sulfoxide)

R2-S(Sulfide)

R3-NH(Trimethyl Amine)

O2-

H2O2

HOCl

NO3-

(nitrate)

SO4-2

(Sulfate)

Intestinal Inflammation

ONOO-

MucolyticBacteria NOCHO

MucolyticBacteria

Inflammation Provides a Selective GrowthAdvantage for Disease-Producing Pathobionts

Increased oxidative stress protection pathways● increased cysteine and GSH transport

● increased riboflavin and sulfur metabolism● increased pentose phosphate shunt pathway

Increased sulfate transport and metabolism

Increase in amino acid transport

Increase in auxotrophy

Decrease in short chain fatty acids and metabolism

Decreased in amino acid biosynthesis

from Morgan et. al., 2012; Kostic et. al. 2014

Alterations in Microbial Function in IBD

AnaerobicRespiration

ClostridiaBacteriodia

Enterobacteriaceae

CHO

Products of Inflammation Feed the Expansion ofColitogenic Pathobionts

Modified from Winter et. al. EMBO, 2013

Microbiome science needs a healthy dose ofskepticism….

• Do microbiome studies showcausation or just correlation?

• Are changes in microbialcomposition biologically relevant?

• Could anything else explain theresults?

Hanage WP (2014) Nature 512:247-8

What do changes in composition anddiversity really tell us?

There are many species of bacteria, but only somehave adapted to life within us

• Over 50 known bacterial phyla

• Generally only 6 phyla found in gut• Bacteroidetes*

• Firmicutes*• Actinobacteria• Proteobacteria

• Verrucomicrobria• Fusobacteria

Tree of life

• 10-100 trillion organisms• >1000 different species• Bacteria, fungi, viruses

The Phylum Level

Bacteria are very different genetically from oneanother

Bacteriodes fragilis Escherichia coli

Shared genes = 40%

Caenorhabditis elegans(nematode)

Human

Shared genes = 38%

What do changes in ratios at the phylumlevel really mean?

Carlotta De Filippo et al. PNAS 2010;107:14691-14696

Microbiome science needs a healthy dose ofskepticism….

• Do microbiome studies showcausation or just correlation?

• Are changes in microbial compositionbiologically relevant?

• Could anything else explain theresults?

Hanage WP (2014) Nature 512:247-8

Technical andcontamination issues

are rampant in themicrobiome literature

Dilution of starting material increases contaminationeffects

• Sequencing results from a pure cultured sample• Increasing dilution = increased contamination• 3 different sites = 3 different results

Salter et al. BMC Biology 2014:12:87

Contamination is kit dependent

Salter et al. BMC Biology 2014:12:87

Analysis of nasopharyngeal samples from children inThailand as a function of age (Birth-2 yrs of age)

Samples cluster byage

No, samples clusterby extraction kit

Re-extractedNo clustering

Salter et al. BMC Biology 2014:12:87 Turner et al. Plos One 2012:7:e38271

Challenges with microbiome studies

• Problems with human studies

– Confounded by age, geographic locations, lifestyles,diets, host genetics

– Lack of standardization (sampling, extraction,processing, sequencing, analyzing)

– Batch effects – technical challenges

Human Microbiome

GOINGFORWARD

Specificity

Ecosystem Effects

Olle Nature Biotechnology 31(4):309. 2013

BUGS IN THE NEWS

Review ArticleAssociation between Faecalibacterium prausnitziiReduction and Inflammatory Bowel Disease: A Meta-Analysisand Systematic Review of the LiteratureYuan Cao, Jun Shen, and Zhi Hua RanRenji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institution of Digestive Disease,Shanghai Inflammatory Bowel Disease Research Center, Shanghai 200127, China

Gastroenterol Res Pract. 2014;2014:872725.

Faecalibacterium prausnitzii

LETTER doi:10.1038/nature13828

Precision microbiome reconstitution restores bileacid mediated resistance to Clostridium difficileCharlie G. Buffie1,2, Vanni Bucci3,4, Richard R. Stein3, Peter T. McKenney1,2, Lilan Ling2, Asia Gobourne2, Daniel No2, Hui Liu5,Melissa Kinnebrew1,2, Agnes Viale6, Eric Littmann2, Marcel R. M. van den Brink7,8, Robert R. Jenq7, Ying Taur1,2, Chris Sander3,Justin R. Cross5, Nora C. Toussaint2,3, Joao B. Xavier2,3 & Eric G. Pamer1,2,8

• “Identify a “probiotic” candidate that cancorrect a clinically relevant microbiome

deficiency”

• Clostridium scindens, a bile acid 7α-dehydroxylatingintestinal microbe, increases resistance to infection

Nature 2015 Jan 8;517(7533):205-8

Clostridium sp and C. difficile colitis

Akkermansia muciniphila and obesity

• A. muciniphila is a normal commensal thatdigests mucus

• Obese people (and fat mice) and those withtype 2 diabetes have much lower levels

• Mice on a high fat diet had less A. muciniphilaand when the levels were restored the micelost weight, had reduced insulin resistance,and reduced metabolic disorders

• Could A. muciniphila be used as a “probiotic”to treat diabetes or help people lose weight?

Everard et al. Proc Natl Acad Sci USA 2013;;110;220:9066

BUGS AND THE BRAIN

My bacteria made me do it…

•Microbes in the gut canalter brain function andbehaviour– Norepinephrine

– Vagus nerve stimulation

– Release of activebiomolecules

Concepts and Questions

• How to identify effector strains within a microbiome thatare responsible for shaping human biology?

• Majority of resident strains are acquired during the first 3years of life

• Once acquired, majority of strains are retained in anindividual for decades

• Effect of a strains residency may take decades tomanifest

Study designs for identifying microbes that modulatecomplex disease risk – Case Control Studies

Jeremiah J. Faith et al. PNAS 2015;112:633-640

Study designs for identifying microbes that modulatecomplex disease risk – Familial Disease

Jeremiah J. Faith et al. PNAS 2015;112:633-640

Geographic Disease Cluster

Jeremiah J. Faith et al. PNAS 2015;112:633-640

Conclusions

• The gut microbiota is dominated by only a few bacterialphyla but great strain-diversity

• Difficulty in identifying causative strains for complexdiseases as most strains are also found in healthyindividuals

• Complex diseases are more likely to reflect thecontributions of multiple microbial strains that both mediateand mitigate pathogenesis in the context of a specific hostgenotype

The End

Multiple stable compositional states in the developing gutmicrobiota of the premature infant.

Morowitz M J et al. PNAS 2011;108:1128-1133

Back in 1917

Clark Stanley, the“Rattlesnake King” in

1917