1. Clostridium difficile Colitis or Dysbiosis

2. Symbiostasis/Dysbiosis

3. 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

4. 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

5. 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

6. 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

7. How many Bacterial Phyla?

8. How diverse are the Microbes?

9. 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)

10. Diversity and Evenness http://www.tiem.utk.edu/~gross/bioed/bealsmodules/simpsonDI.html 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

11. 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

12. 16S rRNA Gene Aids in Microbial Community Profiling V4 UCSC.edu Bioinformatics-toolkit.org V5 V3 V2 V1 V7 V8 V9 V6

13. 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

14. 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

15. 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

17. Residents vs Tourists

20. 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 Effect of Antibiotic Treatment on Fecal Microbiome

21. 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;108:4554-4561

22. ©2011 by National Academy of Sciences Dethlefsen L, Relman D A PNAS 2011;108:4554-4561 PCoA of unweighted UniFrac distances, a phylogenetically aware measure of intersample (β) diversity.

24. C. difficile toxins: Enterotoxin (toxin A): produces chemotaxis, hemorrhagic necrosis Cytotoxin (toxin B): depolymerization of actin

26. Culture-Based Study Microbes were cultured from fecal samples, identified and quantitated

27. Clostridium difficile associated diarrhea

30. Hamster Animal Model Disease is induced/treated Microbiome monitored

31. In hamsters

32. Toxin antibody treatment protects

33. After 40 days

34. qPCR of C. difficile 16S rRNA

36. Change in Bacteroidales composition

37. Human Fecal Transplant Trial Microbiome monitored before and after transplant

45. 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.