1. Introducing the Microbiome
Dr Ailsa Hart
Director IBD Unit, Consultant Gastroenterologist, St Mark’s
Honorary Senior Clinical Lecturer, Imperial College

2. Introduction What is the gut microbiome?
How does the gut microbiome play a role in disease?
- role in inflammatory bowel disease
How can we modulate the gut microbiota as a therapy
- faecal transplant

3. The gut microbiota 1014 gut bacteria and 1013 cells in body
Most densely populated ecosystem on Earth
4 major phyla (Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria)
Provide traits we have not had to evolve on our own
“Virtual” organ
Genes in gut flora 100 times our own genome
Mostly anaerobic, steep stomach acid driven proximal distal gradient

4. Function of gut microbiota
Germ-free animals
Exist and survive
But abnormalities of:
Immune function (oral tolerance)
Metabolic function (altered enzymes)
Physiological function (altered motility)
Trophic function (altered cell turnover)
What is the function of the gut flora – well much can be deduced from studies in germ free animals – These animals exist and survive indicating that the gut flora is not a prerequisite for life but the animals do have profound abnormalities of immune function – reduced GALT, small PP, dec LP AND defective CMI and induction of oral tolerance.
In addition these animals have abnormalities of…

5. Host – microbiota interactions
Function of gut microbiota
Host – microbiota interactions
Hooper et al. Science 2001
A single commensal bacteria switches on genes involving
Mucosal barrier function
Nutrient absorption/ dietary energy extraction
Enteric nervous system
Intestinal maturation
Immune system development
One of the most illuminating studies assessing the function of the gut flora is this one by Hooper et al. reported in Science in This group introduced a single commensal bacteria into a germ free animal and assessed by gene array analysis the genes which were switched on. They found that a multitude of genes were switched on including …… Although clearly a reductionist model it does highlight the huge potential of the gut flora in its entirity to interact with and influence host physiology.

6. The Meta HIT project

7. The Human Microbiome Project

8. The human microbiome project
5 year project
242 healthy men/women - samples from different body sites
10,000 different types of organism found
Is there a core set of microbes that all humans share?
Diversity of microbes across human beings
Diet, host genetics, early microbial exposure
Unique communities of microbes at different body sites
At specific body sites, many microbes had similar genes/functions
Challenges thinking of one-microbe model of disease
More likely “function” of group of microbes changes

9. Gut microbiota “pathology”
e.g. inflammatory bowel diseases
functional bowel disease
gastrointestinal infections
non-GI diseases
- obesity, metabolic syndrome, atopy/allergy

10. Genetics of IBD >160 independent IBD susceptibility loci
2001
2007
2008
2011
2012
For many loci fine mapping is underway as part of the Immunochip experiment

11. What has genetics taught us about IBD?
> 75,000 cases & controls
Information about pathways involved in disease process
Over 2/3 of genes are shared between UC &CD
30 CD-specific and 23 UC-specific
Particular overlap between ankylosing spondylitis and psoriasis
1Jostins et al Nature 491 (7422): ; 2Lees et al Gut 2011;60: (diagram)

12. Experimental models of colitis
Germ-Free
No Colitis
Animal models
of colitis
What is the evidence that the gut flora drives the inflammation in IBD. The best evidence comes from animal models

13. Experimental models of colitis
Germ-Free
No Colitis
Animal models
of colitis
Bacterial
Colonisation
Colitis

14. In humans… Faecal stream diversion alleviates Crohn’s
Reanastomosis triggers recurrence
Infusion of luminal contents into excluded normal bowel induces inflammation

15. What part of the gut microbiota drives inflammation?
Single organism?
Expansion or relative contraction?
“Functional” changes?

16. Reduced diversity of faecal microbiota In CD

17. Reduced Firmicutes in Crohn’s disease

18. - Reduction of a major member of Firmicutes, F
- Reduction of a major member of Firmicutes, F. prausnitzii, associated with higher risk of postoperative recurrence of ileal CD.
Experimental replacement of F. prausnitzii had anti-inflammatory effects

19. But changes in “function” of microbiota….
Mucosa/stool samples from 231 IBD patients & controls
16S gene pyrosequencing/shotgun metagenomics
Major shifts in oxidative stress pathways
Decreased carbohydrate metabolism
Decreased amino acid synthesis
In ileal Crohn’s, increases in virulence and secretion pathways
Overall CD is not caused by diminished diversity alone – but requires a susceptible genotype – as confirmed by research in mice with human relevant susceptibility mutations
Mucin, which is rich in cysteine and glycosylated sugars, is abundant in the intestinal epithelium, and it is
upregulated during inflammation. The increases in cysteine metabolism and N-acetylgalactosamine
transporters may reflect a shift in the microbiome towards greater abundance of microbes that use
mucin as a primary energy source (Figure 6). This functionality suggests activity at the mucosa and this
may be problematic for a damaged IBD epithelium with compromised barrier function.
This
raises the interesting possibility that E. coli or related species in IBD may be highly represented because
they gain a competitive advantage from oxidative stress and are better able to compensate for it with
glutathione production.
Microbial function more consistently altered than microbial composition
Morgan et al. Genome Biology Sept 2012

20. Challenges Clinical phenotype confounders “healthy” controls Sampling
age, gender, smoking
ethnicity, diet, surgery
medications
“healthy” controls
Sampling
faeces v mucosa
axial and longitudinal variation
replication
multiple samples from same region
longitudinal sampling
Communication
clinicians
microbial ecologists
bioinformatics
statisticians
Technical
16S sequence
metagenomics
metatranscriptomics
metabonomics
… economics

21. Can the gut microbiota be modified? If so, how?

22. Faecal transplantation - history
1600s – “transfaunation” – gastric contents transplanted into animals unable to ruminate
Bedouin of northern Africa – “coprphagia” – ingestion of camel dung as treatment for dysentery
WWII – same practice in soldiers – led to Bacillus subtilis as probiotic
Faecal transplant (FT ) - treatment in humans for pseudomembranous colitis in1958
Landy et al. Aliment Pharmacol Ther 2011; 34:

23. Publications on faecal transplantation

24. Faecal transplantation in C. difficile
Over 376 cases of faecal transplant (FT) for C. diff infection
17/22 studies of FT are in refractory/fulminant C. diff
Enemas; via duodenal tubes; via colonoscopes
Overall success rates of >90%
Landy et al. Aliment Pharmacol Ther 2011; 34:

25. Faecal transplantation in C. difficile
First randomised controlled trial
Aimed to recruit 120 patients – stopped early
13/16 (81%) in FT group – resolution of diarrhoea
4/13 (31%) in vancomycin group
3/13 (23%) in vancomycin + bowel lavage
After FT, ↑diversity (similar to healthy donors)
Van Nood et al. NEJM January 2013

26. Faecal transplantation in IBD
41 patients with IBD (27 UC; 12 CD; 2 indeterminate)
Majority (19/25) had reduction in symptoms/ 15/24 remission
Resolution of C. diff infection in 15/15
Damman et al. Am J Gastro. 2012; 107: ; Anderson et al. APT Sept 2012

27. with faecal transplantation may be effective in refractory pouchitis
Faecal transplantation in pouchitis
Hypothesis – increasing diversity and altering selection of resistant bacteria
with faecal transplantation may be effective in refractory pouchitis
Pilot study of faecal transplantation in patients with chronic pouchitis
Microbiological; immunological; histological & clinical assessment
pre and post transplant

28. Faecal transplantation in pouchitis
PDAI CGQoL score pouch frequency
No major adverse events; 3 patients reported adverse effects nausea (n=3); bloating (n=2)
Landy et al. ECCO 2013

31. Unresolved issues… Can microbiota be altered in IBD with FT?
If so, does genetic pressure / indigenous bacteria affect ability to change?
Donor - mixture of phylogenetically diverse bacteria – which?
Frequency and route of administration?
Use of concomitant treatment?
Fresh versus frozen (banks)?
Safety?

32. Altering microbiota - beyond IBD …..
Other gastrointestinal disorders
Functional bowel disorders
Gastrointestinal infections e.g. Clostridium difficile, rotavirus
Non-intestinal disorders
Atopic diseases
Type I diabetes/ metabolic syndrome
Obesity ……

33. Obesity

34. Could manipulation of gut microbiota influence obesity?
Calorie extraction from food varies
depending on gut flora
“Obese flora” more effective at
extracting energy from food
Could manipulation of gut microbiota influence obesity?

35. Could manipulation of gut microbiota influence malnutrition?
Calorie extraction from food varies
depending on gut flora
“Obese flora” more effective at
extracting energy from food
Could manipulation of gut microbiota influence malnutrition?

36. Summary Gut microbiota – virtual organ; microbiome; metobonome
Role of microbiota in IBD
Modulation of gut flora as a therapeutic intervention
Mechanistic approach
Far reaching potential in GI (and non-GI) disease