Julian Marchesi's presentation slides from our previous Microbiome R&D and Business Collaboration Forum. For information about this years event please visit http://www.globalengage.co.uk/microbiota.html

1. Moving into the Post-
Metagenomic Era of Gut
Microbiome Research
Julian R. Marchesijr@cardiff.ac.uk
School of Biosciences
Department of Surgery and Cancer

2. Some definitions
• Microbiota (microflora) – the qualitative and
quantitative information about the different microbes
present in a system – so who is there and how abundant.
• Microbiome – the functions that these microbiota have,
e.g. bile metabolism – their gene catalogue.
• Metagenomics – either “gain-of-function” or DNA based
approach to create gene catalogues, used to define the
microbiome.
• Metaxonomics – creation of 16S rRNA gene inventories,
used to define the microbiota.
• Metabolomics – a catalogue of the metabolites in a
sample.

3. The main focus in human biology has been to
explain how it functions in terms of the genome
+
Disease Health
X
X
Human microbiome
• Microbiome is host
specific
• Can be changed by diet,
drugs and surgery
X=mutation

4. Ecological interactions between members
of different species
+ positive (win); - negative (loss); 0 neutral
 Species 1/Microbiota; Species 2 = host
e.g. H. pylori or C. difficile
e.g. FMT for CDAD
Amensalism
e.g. probiotic microbes which do
not reproduce in the host L. lactis.
This is the modern issue for
microbiome studies
Pathologies not associated
with a pathogen

5. The concept of amensalism involves one
organism growing and in doing so
inadvertently damages another, but it is
not evolved to do so – collateral damage.
The study of Inflammatory Bowel Disease has been grappling with this for the
last 20 years.
A disease that is multi-factorial and involves environmental factors, but for
which no pathogen has been detected.
A disease which does not develop in sterile rodent models
• Colorectal cancer
• Breast cancer
• Metabolic syndrome
• Diabetes
We can no longer use Koch’s
postulate for verifying pathogenic
traits

6. We have no idea of the consequences of
position on this continuum
The variability clouds the issue of a healthy microbiota
Bio geography
complicates
the story
Burkina Faso Italy

7. The absence of a microbiome impacts the whole host
We have a dearth of
mechanisms and only
a few have been
identified.

8. Diabetes
Inflammatory bowel
disease
Obesity
Asthma/Eczema
Depression
Heart disease
Colon Cancer
Non-alcoholic
fatty liver
disease
All these disease have evidence for a role of the
gut microbiota

9. Inventories of
16S rRNA genes
“Omic” approaches available to investigate the gut
Bottom up
Top down
f

10. Metabolic Roux-en-Y Gastric Bypass Surgery (RYGB) and gut microbiota/metabolites
Rat modelPCA of metabolite profiles PCA of bacterial profiles
RAT MODEL
Li et al., (2011) Gut
A marriage of metataxonomics and metabonomics

11. Time point post surgeryWeek 2 Week 8
2. Wistar Rat Study

12. Determination of the relative suspension growth as a measure of cytotoxicity
(A) and genotoxicity (B) of fecal water extracts following treatment of L5178Y
mouse lymphoblastoid cells for 24 h.

13. -60
-40
-20
0
20
40
60
-100 -50 0 50 100
PC1 (R X=24.9%)
PC2(RX=8.2%)relativeintensityofobservedunconjugatedbileacids
0
200
400
p=0.7
p=0.001 p=0.026
relativeintensityofobservedtaurine-conjugatedbileacids
A B
C D
Sham
RYGB
Sham
RYGB
Sham
RYGB
p=0.3
p=0.073 p=0.049
Week 6 Week 8 1 week pre-op Week 6 Week 81 week pre-op
1 week pre-op Week 6 Week 8
0
1
2
p=0.4
p=0.012 p=0.087
0
100
0
200
300
relativeintensityofobservedcholicacid
2
2
The Relative concentrations of observed unconjugated and taurine-conjugated bile acids
show a significant difference between sham and RYGB-operated rats at weeks 6 and 8.
Cholic acid
Unconjugated bile acids Taurine-conjugated bile acids
PCA scores plot
Altered faecal bile acid levels post RYBG
Conclusion: Our data hence suggest that BABR could be useful
for the management of the impaired glucose tolerance of the
metabolic syndrome, since they not only lower cholesterol
levels, but also reduce obesity and improve insulin resistance.

14. The “normal” vaginal microbiome (non-pregnant)
‣ Represents95%of healthy
women in these ethnic grou
- 5%of women are different t
these
• 5 Community State Types
(CST)
• Many lactic acid producers
• Maintain low pH
• Produce antimicrobials
Ravel et al. PNAS 2011; 108: 4680-7
• Lactobacillus spp.
characteristic of “normal”,
“healthy” reproductive age
women.

15. Vaginal microbiome CSTs in our UK population
• Hierarchical clustering analysis identified 5 major CSTs
• 60% of post-partum samples are high diversity CST IV
• CST * may be a new UK specific CST

16. Longitudinal assessment of vaginal CSTs throughout pregnancy and post
partum
Increaseddiversity
So far this is one of the
only scenarios where
more diversity is
deemed to be bad

17. The vaginal microbiome in Preterm Premature Rupture Of Membrane
at 28 weeks gestation
16S rRNA genes from the vaginal microbiome enables stratification of PPROM patients
into Lactobacillus- dominant (PPROM 9-15) and Lactobacillus-depleted heterogeneous
groups (PPROM 1-8).
Two separate aetiologies? Treat differently?

18. The dialogue between the host and
microbiome in a healthy context
Disease Health
X
X
Human microbiome
X=mutation
These arrows really depict the
movement of proteins and
metabolites - in other words the
communication is via the
proteomes and metabonomes

19. Exploring the gut microbiome via activity – emergent properties
Why?
Bacterial proteases are a potential virulence factor in colorectal
cancer and IBD. They have also been shown to compromise tight
junction integrity.
Steck N et al., Gastroenterology. 2011 141(3):959-71.
Metaproteome HostMicrobiome
The missing link is the proteome

20. IBD and the degradome
The degradome and degradomics is the study of enzymes which
degrade proteins i.e. proteases. So does it play a role in
inflammatory bowel disease?
Casein Collagen Keratin

21. Use of targeted proteases inhibitors to understand the degradome
in IBD and healthy samples
Bacterial
PlantFungal
Mammalian
IBD and the degradome cont.

22. The proteolytic activity varies in both cohorts

23. PROTEASE
INTESTINAL
ALKALINE
PHOSPHATASE
LPS
BACTERIAL
ALKALINE
PHOSPHATASE
INFLAMMED NASH
NOT INFLAMMED NASHX
The microbiome and the healthy person
Tight junction integrity
There are many more generic bacterial
enzymes which may be involved in health
and disease.

24. We are now moving into a post-metagenomics era of
gut microbiome research, which moves further away
from metagenomics and towards multiplatform
omics?
Emergent features and functions should be the focus
and not necessarily gene content e.g. protease levels.
What roles does a healthy microbiome play?
SCFA production, niche exclusion and unknown roles in
development.
Conclusions

25. Impact can be at a local level
Off tumour Dysplasia Cancer
On tumour mucosal
cancer microbiota
are specific to
pathology and
maybe prognostic

26. The gut microbiome also influences drug metabolism