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: 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

Eckburg et al, 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, 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

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) – E H 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) – E H equitability (D/S, evenness, 0 to 1, 1 = even)

Diversity and Evenness Shannon’s diversity index H proportion of individual species to all species E H equitability (evenness, 0 to 1, 1 = even) Simpson’s D diversity index E H equitability (evenness, 0 to 1, 1 = even) 1 species accounts for 90%, the other 10% are evenly distributed

Illumina.com Illumina MiSeq Amplicon Sequencing DNA Isolation from Microbial Community within Mouse Small Intestine Illumina.com PCR Amplification of the V4 Hypervariable Region of the 16S rRNA Gene Characterization of Microbiomes Microbial Composition Analyses

16S rRNA Gene Aids in Microbial Community Profiling V4 UCSC.edu Bioinformatics-toolkit.org V5 V3 V2 V1 V7 V8 V9 V6

Eckburg et al, 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

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

Heat map displaying the relative abundance of refOTUs in three prominent clades of bacteria. Dethlefsen L, Relman D A PNAS 2011;108: ©2011 by National Academy of Sciences Effect of Antibiotic Treatment on Fecal Microbiome

PCoA of unweighted UniFrac distances, a phylogenetically aware measure of intersample (β) diversity. ©2011 by National Academy of Sciences Dethlefsen L, Relman D A PNAS 2011;108:

©2011 by National Academy of Sciences Dethlefsen L, Relman D A PNAS 2011;108: PCoA of unweighted UniFrac distances, a phylogenetically aware measure of intersample (β) diversity.

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.