Humans are Microbial Carrying Vessels

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

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

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

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

Studying the microbiome - series of eras

Studying Microbiome Era IMicroscopy

Human Commensals

• Skin• Conjunctiva• Oral cavity• Intestinal tract• Upper respiratory

tract• Urogenital tract

Table 14.1c

Studying Microbiome Era II - Culturing

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.

BACTERIA COMMONLY FOUND ON THE SURFACES OF THE HUMAN BODY

BACTERIUM Skin Conjunctiva Nos e Pharynx Mouth Lower Intestine

Anterior urethra

Vagina

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. +/- + + + + + +

BACTERIA COMMONLY FOUND ON THE SURFACES OF THE HUMAN BODY

BACTERIUM Skin Conjunctiva Nos e Pharynx Mouth Lower Intestine

Anterior urethra

Vagina

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 + + + +/- +

Once Cultured ….

• Experimental biology

• Place on a tree of life

• Genome sequencing

• Genetic engineering

alanine/glycine

Na+

alanine/glycine

Na+

alanine/glycine

Na+

proline/betaine

H+

proline/betaine

H+WD0168

WD0414

WD1046

WD1047

WD0330

Na+

glutamate/aspartate

Na+

WD0211

WD0229

glutamate/aspartate

ornithine

putrescineWD0957

H+ Na+H+ Na+

WD0316 WD0407

H+

WD1107WD1299WD1300WD1391WD0816WD0765

Mg2+

WD0375

H+ Zn2+/Cd2+

WD1042

ATPADP

Zn2+

WD0362WD0938WD0937

ATPADP

Fe3+

WD1136WD0153WD0897

glycerol-3-phosphate/hexose-6-phosphate

phosphateWD0619

H+

drugs

H+

drugs

WD0056

WD0248

H+

drugs

H+

drugs

WD1320

WD0384

H+

?

H+

?

WD0621

WD0099

H+

metabolite?

WD0470

H+

metabolite?

WD1033

H+WD0249

metabolite?

ATPADP

heme

WD0411WD1093WD0340

K+

WD1249

Na+H+

drugsATP

ADP WD0400

phosphate

ATPADP

ORF00100ORF00714ORF00927ORF00940

(2?)

H+

F-type ATPase

ATP ADP

WD1233WD0203WD0204WD0427WD0428WD0429WD0655WD0656

phosphoenolpyruvate

1,3-bisphosphoglycerate

3-phosphoglycerate

2-phosphoglycerate

pyruvate

acetyl-CoA

citrate

isocitrate

oxaloacetate

suc-CoAsuccinate

fumarate

malate

oxaloacetate

TCA CYCLE

glyceraldehyde-3P

fructose-1,6-P2

dihydroxyacetone-P

WD1238

WD0091

WD0451

WD1167

WD0868

WD0494

WD0690

WD0105

WD0791

WD1309WD0544WD0751

WD1209WD1210

WD0437WD0727WD1221WD1222

WD0492

WD1121

mannose-1P mannose-6PWD0695

MALATE WD0488 WD1177WD0416WD0473WD0751WD0325

Non-oxidative Pentose Phosphate Pathway

xylulose-5P

glyceraldehyde-3P

sedoheptulose-7P

fructose-6P

ribose-5P

ribulose-5P

glyceraldehyde-3P

WD0551WD0387

WD0387

WD0712

erythrose-4P

WD1151

glycerol-3P

WD0731

Amino Acid catabolism

GLUTAMATE glutamineWD1322

GLUTAMINE glutamateWD0535

CYSTEINE alanineWD0997

THREONINE glycineWD0617,WD0617

PROLINE glutamateWD0103

SERINE glycineWD1035

Fatty Acid Biosynthesis WD0985, WD0650, WD1083, WD1170, WD0085

PRPP

WD0036

Thiamine metabolismWD1109,WD0763,WD0029,WD0913,WD1018,WD1024

AMP,ADP,dAMP, dADP,ATP,dATP,ITP,dITP,IMP,XMP,GMP,GDP,dGDP,dGTP,dGMP

WD1142WD1305WD1023WD0786WD0867WD0337WD0786

WD0661WD1183WD0197WD0089WD0195WD0439WD0197

adenylosuccinate WD0786

Purine Metabolism

UMPUDP

WD0684WD1295WD0895WD0230WD1239WD0228WD0461

aspartate semialdehydeaspartateWD1029 WD0960 WD0954

Wu et al., PLoS Biology 2004

Limitations of Culturing

• Can’t perfectly mimic niche

• Can’t mimic communities

• Great plate count anomaly

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.

Great Plate Count Anomaly

Culturing Microscope

CountCount

Great Plate Count Anomaly

Culturing Microscope

CountCount <<<<

Great Plate Count Anomaly Solution

Culturing Microscope

CountCount <<<<

DNA

PCR Saves the Day

rRNA Phylotyping

• Extract DNA

• Run rDNA PCR

• Sequence products

• Infer evolutionary tree

rRNA and Uncultured Microbes

Eisen et al. 1992

Majority of Microbes are “Uncultured”Numbers and Diversity

Phylotyping Can Be Used to Count

Phylotyping Diversity Indices

Bohannan and Hughes 2003

Hugenholtz 2002

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.1365-2958.2006.05056.x

Rawls et al. 2006

Palmer et al 2007

Palmer et al. 2007

Mouth Diversity

GI Tract

Problems with rRNA PCR

• Doesn’t predict biology of organisms well

• Doesn’t work for viruses

• Not very quantitative

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

Environmental Shotgun Sequencing

shotgunshotgun

sequencesequenceWarner Brothers, Inc.Warner Brothers, Inc.

Using a rRNA anchor allowed the

identification of a new form of phototrophy:

Proteorhodopsin

Beja et al. 2000

rRNA Phylotypes

Venter et al., 2004

taxonomic content per SHOTGUN 16S

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

GS-02

GS-03

GS-04

GS-05

GS-06

GS-07

GS-08

GS-09

GS-10

GS-11

GS-12

GS-13

GS-14

GS-15

GS-16

GS-17

GS-18

GS-19

GS-20

GS-21

GS-22

GS-23

GS-25

GS-26

GS-27

GS-28

GS-29

GS-30

GS-31

GS-32

GS-33

GS-34

GS-35

GS-36

Station

Shotgun Sequencing Detects More Diversity than PCR-methods

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

ABCDEFG

TUVWXYZ

Binning in More Complex Systems?

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