Clostridium difficile Colitis
or Dysbiosis
Symbiostasis/Dysbiosis
Overview
• The human microbiome and how one studies
it
• Effect of antibiotic on microbiome
• Clostridium difficile colitis
• Changes in the microbiome
• Fecal transplants as a treatment option
Aspects of the Microbiome to
Consider:
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Amount
Richness (how many taxa)
Diversity (how many taxa and how even)
Evenness (how even the distribution of taxa)
Specific organisms presence / abundance
Specific genes presence / abundance
Expression profiles
Complexity of Human Gut Microbiota
• Eckburg et al, 2005. Science.
• At least 500 species are
present in human intestine.
• Each subject had a “distinct”
microbiota.
• Red = Gram-positives
• Yellow = Gram-negative
anaerobes
• Blue = “Enteric” Bacteria
Complexity of Human Gut Microbiota
• Eckburg et al, 2005. Science.
• At least 500 species are
present in human intestine.
• Each subject had a “distinct”
microbiota.
• Red = Gram-positives
• Yellow = Gram-negative
anaerobes
• Blue = “Enteric” Bacteria
How many Bacterial Phyla?
How diverse are the Microbes?
Alpha-Diversity
Diversity within a sample
• Shannon’s diversity index
– H proportion of individual species to all species
– S total number of species (richness)
– EH equitability (H'/ln(S), evenness, 0 to 1, 1 = even)
• ACE: richness based on estimation of sample coverage
• Simpson’s
– D diversity index
– S total number of species (richness)
– EH equitability (D/S, evenness, 0 to 1, 1 = even)
Diversity and Evenness
1 species accounts for
90%, the other 10%
are evenly distributed
Shannon’s diversity index
H proportion of individual
species to all species
EH equitability (evenness, 0 to
1, 1 = even)
Simpson’s
D diversity index
EH equitability (evenness, 0 to
1, 1 = even)
http://www.tiem.utk.edu/~gross/bioed/bealsmodules/simpsonDI.html
Characterization of Microbiomes
DNA Isolation from
Microbial Community
within Mouse Small
Intestine
PCR Amplification of the V4 Hypervariable Region of the 16S rRNA Gene
Illumina MiSeq Amplicon Sequencing
Illumina.com
Illumina.com
Microbial Composition
Analyses
16S rRNA Gene
Aids in Microbial
Community
Profiling
V4
V6
V7
V5
V3
V8
V1
V2
V9
UCSC.edu
Bioinformatics-toolkit.org
Complexity of Human Gut Microbiota
• Eckburg et al, 2005. Science.
• At least 500 species are
present in human intestine.
• Each subject had a “distinct”
microbiota.
• Red = Gram-positives
• Yellow = Gram-negative
anaerobes
• Blue = “Enteric” Bacteria
Beta-Diversity of Samples
The diversity between samples
• Bray Curtis, an OTU abundance-based metric
• Ochiai, an OTU presence/absence-based
metric
• Unweighted Unifrac, phylogenetic-based
metric
• Weighted Unifrac, abundance and
phylogenetic-based metric
Another way to Depict the Data:
PCoA
• Principle Coordinates Analysis (different from
a PCA or Principle Component Analysis)
• The distance matrix data from UniFrac is used
• Look for the % on each axis to see how much
of the total distance is explained by each axis
Residents vs Tourists
Effect of Antibiotic Treatment on Fecal Microbiome
Heat map displaying the relative abundance of refOTUs in three prominent clades of bacteria.
Dethlefsen L , Relman D A PNAS 2011;108:4554-4561
©2011 by National Academy of Sciences
PCoA of unweighted UniFrac distances, a phylogenetically aware measure of intersample (β)
diversity.
Dethlefsen L , Relman D A PNAS 2011;108:4554-4561
©2011 by National Academy of Sciences
PCoA of unweighted UniFrac distances, a phylogenetically aware measure of intersample (β)
diversity.
Dethlefsen L , Relman D A PNAS 2011;108:4554-4561
©2011 by National Academy of Sciences
C. difficile toxins:
Enterotoxin (toxin A):
produces chemotaxis,
hemorrhagic necrosis
Cytotoxin (toxin B):
depolymerization of actin
Culture-Based Study
• Microbes were cultured from fecal samples,
identified and quantitated
Clostridium difficile
associated diarrhea
Hamster Animal Model
• Disease is induced/treated
• Microbiome monitored
In hamsters
Toxin antibody treatment protects
After 40 days
qPCR of C. difficile 16S rRNA
Change in Bacteroidales composition
Human Fecal Transplant Trial
• Microbiome monitored before and after
transplant
Conclusions
• The microbiome is very complex and when
disturbed lead to dysbiosis.
• C. difficile infected patients have a large scale
change in the microbiome, some microbes
that are thought to be antagonistic to C.
difficile disappeared.
• Fecal transplants benefit patients
immediately, even before the microbiome
rebounds to a normal state.