Presentation by Jonathan Eisen on the human microbiome for SciFoo 2007

1. Humans are Microbial Carrying
Vessels

2. Some Microbial Trivia
• Microbes rule the planet
• There are 100x more microbial cells in the
human body than human cells
• Microbes are really small
• Human health may be determined more by
microbes than environment or genes
• Most microbes in/on humans are beneficial
- known as commensals

4. Top Known Benefits of Human
Commensals
• Preventing infection by pathogens
• Development of the immune system
• Digestion of food
• Vitamin production
• Toxin degradation
• Appearance and odor

5. Why Study Commensals?
• Diagnostic for health status
• Disturbed by antibiotics
• Colonization disrupted by C-sections
• Imbalances may cause disease (autoimmune, IBS,
obesity)
• Probiotics and prebiotics could improve health
• Likely have many as of yet unknown functions

6. Microbes are Very Small
• Makes them hard to study
• Even when examined in microscopes -
appearance is not a reliable indicator of
microbial type or biology
• Culturing allows biology to be studied in
detail in the lab
• Genome sequencing of cultured species
very informative

7. Studying the microbiome - series
of eras

8. Studying Microbiome Era I
Microscopy

9. Human Commensals
• Skin
• Conjunctiva
• Oral cavity
• Intestinal tract
• Upper respiratory
tract
• Urogenital tract
Table 14.1c

10. Studying Microbiome Era II -
Culturing

11. Guide to the Normal Bacterial Flora of Humans
Clostridia
• Members of the genus
Clostridium
• Found in the the
intestinal tract
Clostridium difficile. Clostridia are
anaerobic endospore-forming bacteria,
found mainly in the large intestine.

13. BACTERIA COMMONLY FOUND ON THE SURFACES OF THE
HUMAN BODY
Lower Anterior
BACTERIUM Skin Conjunctiva Nos e Pharynx Mouth Vagina
Intestine urethra
Staphylococcus
++ + ++ ++ ++ + ++ ++
epidermidis (1)
Staphylococcus
+ +/- + + + ++ +/- +
aureus* (2)
Streptococcus
+ ++ +/- + +
miti s
Streptococcus
++ ++
salivarius
Streptococcus
+ ++
mutans* (3)
Enterococcus
+/- + ++ + +
faecalis * (4)
Streptococcus
+/- +/- + + +/-
pneumoniae* (5)
Streptococcus
+/- +/- + + +/- +/-
pyogenes* (6)
Neisseria sp. (7) + + ++ + + +
Neisseria
+ ++ + +
meningitidis * (8)
Veillonella e sp. + +/-
Enterobacteriaceae*
(Escherichia col i) +/- +/- +/- + ++ + +
(9)
Proteus sp. +/- + + + + + +

14. BACTERIA COMMONLY FOUND ON THE SURFACES OF THE
HUMAN BODY
Lower Anterior
BACTERIUM Skin Conjunctiva Nos e Pharynx Mouth Vagina
Intestine urethra
Pseudomon as
aeruginosa* +/- +/- + +/-
(10)
Haemophilus
+/- + + +
influenzae* (11)
Bacteroides
++ + +/-
sp.*
Bifidobacterium
++
bifidum (12)
Lactobacillus
+ ++ ++ ++
sp. (13)
Clostridium
+/- ++
sp.* (14 )
Clostridium
+/-
tetani (15)
Corynebacteria
++ + ++ + + + + +
(16)
Mycobacteria + +/- +/- + +
Actinomycetes + +
Spirochetes + ++ ++
Mycoplasmas + + + +/- +

16. Once Cultured ….
• Experimental biology
• Place on a tree of life
• Genome sequencing
• Genetic engineering

17. ORF00100
ORF00714
ORF00927
ORF00940
phosphate
AMP,ADP,dAMP, dADP,ATP,dATP,ITP,
glycerol-3P dITP,IMP,XMP,GMP,GDP,dGDP,dGTP,dGMP
ADP ATP fructose-1,6-P2 WD1142 WD0661
(2?) WD1238 WD1305 WD1183
WD0731
WD1023 WD0197
Purine Metabolism WD0786 WD0089
WD0867 WD0195
dihydroxyacetone-P WD0091
glyceraldehyde-3P WD0337 WD0439
WD0786 WD0197
WD0451 PRPP WD1109,WD0763,WD0029,WD0913,WD1018,WD1024
Thiamine metabolism
glycerol-3-phosphate/ WD0036
hexose-6-phosphate 1,3-bisphosphoglycerate
phosphate
WD0619 WD1167 ribose-5P sedoheptulose-7P
metabolite?
WD0470 H +
3-phosphoglycerate glyceraldehyde-3P
ATP
metabolite? WD0868 WD0712 drugs
ribulose-5P xylulose-5P
WD1033 H +
ADP WD0400
WD0551
2-phosphoglycerate WD0387 drugs
metabolite? erythrose-4P
WD0249 H+ WD0494 WD0387 H+
WD0248
glyceraldehyde-3P drugs
phosphoenolpyruvate fructose-6P
H+
Non-oxidative Pentose Phosphate Pathway WD0056
WD0690 drugs
WD0695
putrescine pyruvate mannose-1P mannose-6P H+
WD0957 MALATE WD0488 WD1177 WD0384
ornithine WD0416 WD0684 drugs
WD1295
WD0473 Amino Acid catabolism WD0895
glutamate/aspartate
WD0751
WD0325 WD0230 H+
WD0997 WD1239 WD1320
WD0211
Na+ WD0985, WD0650, WD1083, WD1170, WD0085 CYSTEINE alanine WD0228 ?
Fatty Acid Biosynthesis acetyl-CoA WD1322 WD0461
glutamate/aspartate
GLUTAMATE glutamine UMP H+
WD0099
WD0229 Na+ GLUTAMINE WD0535 glutamate UDP ?
WD0103
proline/betaine
WD1151
citrate PROLINE glutamate H+
WD0621
WD1035
WD0168 H + SERINE glycine
oxaloacetate WD0105 WD0617,WD0617
proline/betaine THREONINE glycine
WD0414 WD1121
H+
isocitrate
alanine/glycine malate TCA
WD1046 Na+ CYCLE
WD0791
WD0492
alanine/glycine
WD0786
WD1047
Na+ adenylosuccinate fumarate oxaloacetate WD1029 aspartate
WD0960 WD0954
aspartate semialdehyde
WD0437
alanine/glycine WD0727 WD1309 F-type
WD1221 WD0544
WD0330 Na+ WD1222 WD0751 ATPase
succinate WD1209 suc-CoA
WD1210
ADP ATP ADP ATP ADP ATP ATP ADP
H+
H+ K + Mg2+ H+ Na+ H+ Na+ H+ Na+ H+ Zn2+/Cd2+ Zn2+ Fe3+ heme WD1233
WD1107 WD0203
WD1249 WD0375 WD0316 WD0407 WD1042 WD0362 WD1136 WD0411
WD0204
Wu et al., PLoS Biology 2004
WD1299 WD0938 WD0153 WD1093
WD1300 WD0427
WD0937 WD0897 WD0340
WD1391 WD0428
WD0816 WD0429
WD0765 WD0655
WD0656

19. Limitations of Culturing
• Can’t perfectly mimic niche
• Can’t mimic communities
• Great plate count anomaly

20. The Uncultured Majority
• Vast majority of microbes have never been
cultured (total numbers and phylogenetic
diversity)
• Particularly true for endosymbionts and
extremophiles
• Main questions
– Who is out there?
– What are they doing?
– Connect who and what.

21. Great Plate Count Anomaly
Culturing Microscope
Count Count

22. Great Plate Count Anomaly
Culturing Microscope
Count <<<< Count

23. Great Plate Count Anomaly
Solution
DNA
Culturing Microscope
Count <<<< Count

24. PCR Saves the Day

25. rRNA Phylotyping
• Extract DNA
• Run rDNA PCR
• Sequence products
• Infer evolutionary tree

26. rRNA and Uncultured Microbes
Eisen et al. 1992

27. Majority of Microbes are “Uncultured”
Numbers and Diversity

28. Phylotyping Can Be Used to Count
Phylotyping Diversity Indices
Hugenholtz 2002 Bohannan and
Hughes 2003

29. Zoetendal, Erwin G., Vaughan, Elaine E. & de Vos, Willem M.A microbial world within
us.Molecular Microbiology ﾊ 59 ﾊ (6), ﾊ 1639-1650.doi: 10.1111/j.1365-

30. Rawls et al. 2006

31. Palmer et al 2007

33. Palmer et al. 2007

34. Mouth Diversity

35. GI Tract

36. Problems with rRNA PCR
• Doesn’t predict biology of organisms well
• Doesn’t work for viruses
• Not very quantitative

37. Genome-Scale Methods and
Uncultured Species
• High throughput rDNA PCR (e.g., Sogin, Eisen)
• rDNA “phylochips” (e.g., Brown, Anderson)
• Virus chip (e.g., Derisi)
• Metagenomics
– Large inserts (e.g., Delong)
– Environmental shotgun sequencing (Venter, Banfield, everyone
doing because of power of random sampling)
• Single cell genomics

38. Environmental Shotgun Sequencing
shotgun
Warner Brothers, Inc.
sequence

39. Using a rRNA anchor
allowed the
identification of a new
form of phototrophy:
Proteorhodopsin
Beja et al. 2000

40. rRNA Phylotypes
Venter et al., 2004

41. taxonomic content per SHOTGUN 16S
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G
S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S-
02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25 26 27 28 29 30 31 32 33 34 35 36
Station

42. Shotgun Sequencing Detects More
Diversity than PCR-methods

44. What Next?
• Selected as new NIH Roadmap Initiative
• Current state of knowledge incredibly
limited
• 100s of body locations, and likely variation
across people, places, diets, ages, etc need
to be surveyed
• New molecular and informatics methods
needed

45. Binning in More Complex
Systems?
A T
B U
C V
D W
E X
F Y
G Z

46. Metagenomic Challenges
(Examples)
• Fragmentary data
• Sparse sampling
• Parasitizing methods from standard genome
analysis
• Structure of communities unknown
• Analyses frequently cover multiple levels
and multiple fields of methods