"The Quest for A field Guide to the Microbes" talk by Jonathan Eisen February 2, 2014.

!
!

The Quest for a Field Guide to the Microbes
!

Jonathan A. Eisen
@phylogenomics
University of California, Davis
!

Talk for Science in the River City
January 28, 2014

The Quest for A Field Guide to the Microbes

Part I:
!

My Obsessions


Part II:
!

The Story of a Bird

!14
from http://www.birdlist.co.uk/americanrobin.shtml

!30
http://www.birdsofbritain.co.uk/bird-guide/

!31
from Google Maps w/ some addition

Field Guides
• What should be included
•
•
•
•
•

Catalog of types of organism
Functional diversity
Biogeography (space and time)
Niche information
Means for identification

• Provides a guide to interpret normal
states and abnormalities


Part III:
!

A Micro Bit  
about Microbes

The Microbe Challenge

• Microbes are small
• But diversity and numbers

are very high
• Appearance not a good
indicator of type or function
• Field observations of limited
value

Diversity of Function
The Bad

The Good

The Bad

The Good

The Unusual

The Bad

The Consumable

The Good

The Unusual

The Bad

The Consumable

The Good

The Burnable

The Unusual

The Bad

The Consumable

The Good

The Burnable

The Unusual

The Planet


Part IV:  
 
CSI Microbiology

Great Plate Count Anomaly

Culturing

Microscopy


Culturing

Count

Microscopy

Count


Culturing

Count

Microscopy

<<<<

Count


Solution???

Culturing

Count

Microscopy

<<<<

Count


DNA

Culturing

Count

Microscopy

<<<<

Count

DNA Use Case I: Who is Out There?

Who is Out There?
DNA
extraction

PCR
Makes lots of
copies of the
rRNA genes
in sample

PCR

Phylogenetic tree
rRNA1

Sequence alignment = Data matrix

Yeast

C

A

C

A

C

T

A

C

A

G

T

E. coli
Humans

A

Yeast

E. coli

rRNA1

A

G

A

C

A

G

Humans

T

A

T

A

G

T

Sequence
rRNA genes

rRNA1
5’ ...TACAGTATAGGTG
GAGCTAGCGATCGAT
CGA... 3’

Sequences vs. Bones

Carl Woese

The Tree of Life 
2006

adapted from Baldauf, et al., in Assembling the Tree of Life, 2004

!60

The Tree of Life 
2006

adapted from Baldauf, et al., in Assembling the Tree of Life, 2004

Who is Out There?
DNA
extraction

PCR
Makes lots of
copies of the
rRNA genes
in sample

PCR

Phylogenetic tree
rRNA1


rRNA2

Yeast

C

A

C

A

T

A

C

A

G

T

A

G

A

C

A

G

Humans

T

A

T

A

G

T

Yeast

T

A

C

A

G

T

rRNA1
5’ ...ACACACATAGGTG
GAGCTAGCGATCGAT
CGA... 3’

C

E. coli
Humans

A

rRNA2

E. coli

rRNA1

Sequence
rRNA genes

rRNA2
5’ ...TACAGTATAGGTG
GAGCTAGCGATCGAT
CGA... 3’

Who is Out There?
DNA
extraction

PCR
Makes lots of
copies of the
rRNA genes
in sample

PCR

rRNA1
5’...ACACACATAGGTGGAGCTAG
CGATCGATCGA... 3’

Phylogenetic tree
rRNA1


rRNA2

rRNA1

Humans

E. coli
Yeast

A

C

A

C

A

C

rRNA2

T

A

C

A

G

C

A

C

T

G

T

rRNA4

C

A

C

A

G

T

E. coli

A

G

A

C

A

G

Humans

T

A

T

A

G

T

Yeast

T

A

C

A

G

T

rRNA2
5’..TACAGTATAGGTGGAGCTAGC
GACGATCGA... 3’

T

rRNA3

rRNA4

rRNA3

Sequence
rRNA genes

rRNA3
5’...ACGGCAAAATAGGTGGATTC
TAGCGATATAGA... 3’
rRNA4
5’...ACGGCCCGATAGGTGGATTC
TAGCGCCATAGA... 3’

DNA Sequencing Has Gone Crazy

1977

2010

Sanger sequencing method by F. Sanger
(PNAS ,1977, 74: 560-564)

1983
1953

2000

1990

1980

Approaching to NGS

AAATCGCTAGCGC
CGGCGAGCTAGC
CGAGCGATCGAGC
CGAGCATCGAGTA

PCR by K. Mullis
(Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73)

Discovery of DNA structure
(Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31)

Human Genome Project
(Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351)

1993

Development of pyrosequencing

(Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365)

Single molecule emulsion PCR

1998

Founded Solexa

1998

Founded 454 Life Science

2000

454 GS20 sequencer
(First NGS sequencer)

2005

Solexa Genome Analyzer
(First short-read NGS sequencer)

Illumina acquires Solexa
(Illumina enters the NGS business)

2006
2006

ABI SOLiD
(Short-read sequencer based upon ligation)

Roche acquires 454 Life Sciences
(Roche enters the NGS business)

2007
2007

GS FLX sequencer
(NGS with 400-500 bp read lenght)

NGS Human Genome sequencing
(First Human Genome sequencing based upon NGS technology)

2008
2008

Hi-Seq2000
(200Gbp per Flow Cell)

From Slideshare presentation of Cosentino Cristian
http://www.slideshare.net/cosentia/high-throughput-equencing

2010

Miseq
Roche Jr
Ion Torrent
PacBio
Oxford

A Thumb Drive DNA Sequencer?

From Oxford Nanopores Web Site

DNA Use II: What Are They Doing?

DNA Use Case IV: Human Microbiome

Microbes Can Make Mice Fat

Turnbaugh et al Nature. 2006 444(7122):1027-31.

The Human Microbiome

Censored

Censored

The Human Microbiome
Hair

External nose

Naris (L)

Lat. pinna (R)

Lat. pinna (L)

Ext. auditory
canal (L)

Axilla (R)

Dorsal tongue

Oral cavity

Axilla (L)

Volar
forearm (R)

Palm (R)

Palm (L)

Volar
forearm (L)

Palmar index
finger (R)

Gut

Umbilicus

Palmar index
finger (L)

Popliteal
fossa (R)

Plantar
foot (R)

Plantar
foot (L)

Popliteal
fossa (L)

Acinetobacter

Actinomycetales

Actinomycineae

Alistipes

Anaerococcus

Bacteroidales

Bacteroides

Biﬁdobacteriales

Branhamella

Campylobacter

Capnocytophaga

Carnobacteriaceae1

Carnobacteriaceae2

Clostridiales

Coriobacterineae

Corynebacterineae

Faecalibacterium

Finegoldia

Fusobacterium

Gemella

Lachnospiraceae

Lachnospiraceae (inc. sed.)

Lactobacillus

Leptotrichia

Micrococcineae

Neisseria

Oribacterium

Parabacteroides

Pasteurella

Pasteurellaceae

Peptoniphilus

Prevotella

Prevotellaceae

Propionibacterineae

Ruminococcaceae

Staphylococcus

Streptococcus

Veillonella

Other

Naris (R)

Forehead

Ext. auditory
canal (R)

Glans
penis

Labia
minora

Variation May Affect Health
• Microbial community different in many disease
states compared to healthy individuals
• Unclear if this is cause or effect in most cases

Colonization Gone Wrong

Necrotizing
enterocolitis

C-sections

The Built Environment
Microbial Biogeography of Public Restroom Surfaces
Gilberto E. Flores1, Scott T. Bates1, Dan Knights2, Christian L. Lauber1, Jesse Stombaugh3, Rob Knight3,4,
Noah Fierer1,5*
Bacteria of Public Restrooms

1 Cooperative Institute for Research in Environmental Science, University of Colorado, Boulder, Colorado, United States of America, 2 Department of Computer Science,
University of Colorado, Boulder, Colorado, United States of America, 3 Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, United
States of America, 4 Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, United States of America, 5 Department of Ecology and Evolutionary
Biology, University of Colorado, Boulder, Colorado, United States of America

Abstract

We spend the majority of our lives indoors where we are constantly exposed to bacteria residing on surfaces. However, the
diversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibited
by bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing of
The ISME Journal (2012), 1–11
the 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla:
& 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12
Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria. The communities clustered into three general categories: those
www.nature.com/ismej
found on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched with
hands. On illustrations of the relative abundance of discriminating suggesting fecal contamination of these surfaces. Floor
Figure 3. Cartoon toilet surfaces, gut-associated taxa were more prevalent, taxa on public restroom surfaces. Light blue indicates low
surfaces dark blue indicates high abundance of taxa. (A) contained several taxa taxa (Propionibacteriaceae, Corynebacteriaceae,
abundance while were the most diverse of all communities and Although skin-associated commonly found in soils. Skin-associated
Staphylococcaceae especially the Propionibacteriaceae, on all surfaces, they were relatively more abundant on surfaces routinely touched with
bacteria, and Streptococcaceae) were abundant dominated surfaces routinely touched with our hands. Certain taxa were more
hands. (B) Gut-associated taxa (Clostridiales, Clostridiales group XI, vagina-associated Lactobacillaceae were widelyBacteroidaceae)in female
common in female than in male restrooms as Ruminococcaceae, Lachnospiraceae, Prevotellaceae and distributed were most
abundant on toilet surfaces. from urine contamination. Use of the SourceTracker algorithm confirmed Nocardioidaceae) taxonomic
restrooms, likely (C) Although soil-associated taxa (Rhodobacteraceae, Rhizobiales, Microbacteriaceae and many of our were in low
abundance on all restroom surfaces, they were relatively more abundant on the floor of the surfaces. Overall, theseFigure not drawn to scale.
observations as human skin was the primary source of bacteria on restroom restrooms we surveyed. results demonstrate that
doi:10.1371/journal.pone.0028132.g003
restroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with clear
Bacteria
linkages between communities on or in different body sites and those communities found on restroom surfaces. More of P
show that SourceTracker analysis support the taxonomic
the stallgenerally,were likely dispersed manuallypublicwomen used as we Results of human-associated microbes are commonly found
in), they this work is relevant to the after health field
1
1
1,2
1,2
1,2
Steven W Kembel , Evan Jones , Jeff Kline , Dale Northcutt , Jason Stenson ,
on Coupling these observations with those of the
patterns highlighted above, indicating that human skin was the
the toilet. restroom surfaces suggesting that bacterial pathogens could readily be transmitted between individuals by the touching
1
Bohannan1, G Z Brown1,2 and Jessica L Green1,3
Ann
time, the M Womack , Brendan JM 100
of surfaces.
SOURCES
source of bacteria on all public restroom surfaces
bacteria indicate that routine can
1
Bathroomdistribution of gut-associatedon indoor surfaces, an approach of high-throughput analyses ofpathogen communities to determine
biogeography.Furthermore, we demonstrate that we use use primary be used to track bacterial transmission and test the
By
Biology and the Built Environment Center, Institute of Ecology and Evolution, Department of
sources the bacteria of urine- and fecal-associated bacteria
of dispersal
whichexamined, while the human gut was an important source on or
could
toilets results in
Soil
un to take
swabbing throughout surfaces in While these results are not unexpected,
different the restroom. practices.
Biology, University of Oregon, Eugene, OR, USA; 2Energy Studies in Buildings Laboratory,
efficacy of hygiene
around the toilet, and urine was an important source in women’s
Water
80
of outside
Department of Architecture, University of Oregon, Eugene, OR, USA and 3Santa Fe Institute,
public restrooms,highlight the importance of hand-hygiene when using
restrooms (Figure 4, Table S4). Contrary to expectations (see
they do researchers
Mouth
Santa Fe, NM, USA
om plants
Microbial Biogeography of Public by the SourceTracker 6(11): e28132.
public microbes vary in ST, surfaces could also be potential
restrooms GE, Bates
determined thatCitation: Floressince these Knights D, Lauber CL, Stombaugh J, et al. (2011)above), soil was not identifiedRestroom Surfaces. PLoS ONEalgorithm as
Urine
doi:10.1371/journal.pone.0028132
60
being a major source of bacteria on any of the surfaces, including
ours after
where theyvehicles from dependcome for the transmission of human pathogens. Unfortunately,
Gut
Editor: Mark R. Liles, Auburn University, college students
floors (Figure 4). Although the floor samples contained family-level
previous studies have documented that United States of America are
ing on the surface (chart).frequent users of the studied restrooms)(who not
ere shut
taxa 23, 2011
likely Received September 12, 2011; Accepted November 1, 2011; Published November that are common in soil, the SourceTracker algorithm
the most
are
Buildings are complex ecosystems that house trillions of microorganisms interactingSkin each
with
40
ortion of
other, with humans and with their environment. Understanding the ecological and evolutionary
probably underestimates the relative importance of which permits
always the most ß 2011 Flores et al. This is an[42,43].
Copyright: diligent of hand-washers open-access article distributed under the terms of the Creative Commons Attribution License, sources, like

ORIGINAL ARTICLE

Average contribution (%)

Architectural design influences the diversity and
structure of the built environment microbiome

February 9, 2012

Do
or
Do in
or
ou
t
St
all
in
Fa Sta
uc
et ll ou
So han t
ap
d
dis les
pe
ns
To
T
e
ile oile r
tf
lus t sea
hh t
a
To ndle
ile
tf
lo
Si or
nk
flo
or

processes that determine the diversity and composition of the built environment microbiome—the
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
pant in indoor microbial
community of microorganisms that live indoors—is important for understanding the relationship
20
Funding: This work was supported
Foundation
their Indoor Environment program, and
ecology research,ofPeccia the Howard with funding from the Alfred P. Sloan had no role andstudy design, data collection and analysis, in part bytothe National
between building design, biodiversity and human health. In this study, we used high-throughput
Institutes
Health and
Hughes Medical Institute. The funders
in
decision
publish, or
sequencing of the bacterial 16S rRNA gene to quantify relationships between building attributes and
preparation of has
thinks that the fieldthe manuscript.
airborne bacterial communities at 0 health-care facility. We quantified airborne bacterial community
a
Competing Interests:
structure and environmental conditions in patient rooms exposed to mechanical or window
wh i c h
yet to gel. And the Sloan The authors have declared that no competing interests exist.
* E-mail: noah.fierer@colorado.edu
ventilation and in outdoor air. The phylogenetic diversity of airborne bacterial communities was
26 JanuFoundation’s Olsiewski
lower indoors than outdoors, and mechanically ventilated rooms contained less diverse microbial
communities than did window-ventilated rooms. Bacterial communities in indoor environments
Journal,
shares some of his concontained many taxa that are absent or rare outdoors, including taxa closely related to potential
communities and revealed a greater diversity of bacteria on
Introduction
hanically
cern. “Everybody’s genhuman pathogens. Building attributes, specifically the source of ventilation air, airflow rates, relative
indoor surfaces than captured using cultivation-based techniques
humidity and temperature, were correlated with the diversity and composition of indoor bacterial
had lower
erating vastMore than ever, individuals across the globe spend a large [10–13]. Most of the organisms identified in these studies are
amounts of
communities. The relative abundance of bacteria closely related to human pathogens was higher
portion of their lives indoors, yet relatively little is known about the
related to human
are
y than ones with openthan outdoors, and higher in rooms withquantify those con- lower relative humidity. looking across data sets 2. Relationship between bacterial communities associated with commensals suggesting that the organisms were
they move around. But to lower airflow rates and data,” she says, but
indoors winFigure
ten public restroom surfaces. Communities
microbial diversity of indoor environments. Of the studies that
not actively
The observed relationship between building design and airborne bacterial can be difficult because groups choose dif- of the unweighted UniFrac distance matrix. Each point represents agrowing on the surfaces but rather were deposited
ility of fresh air translated tributions, Peccia’s team has had to develop diversity suggests that
PCoA
single sample. Note that the floor (triangles) and toilet (as
have examined microorganisms associated with indoor environdirectly (i.e. touching) or indirectly (e.g. shedding of skin cells) by
we can manage indoor environments, altering through building design and operation the community
form
tions of microbes associ- new methods to collect airbornemicrobiomeand our timeanalytical tools. With ments, most have relied upon cultivation-based techniques hands. humans. Despite these efforts, we still have an incomplete
Sloan support, clusters distinct from surfaces touched with to
of microbial species that potentially colonize the human bacteria during ferent indoors.
doi:10.1371/journal.pone.0028132.g002
detect organisms residing on a variety of household surfaces [1–5].
The ISME Journal extract their DNA, 26 January 2012; are much
understanding of bacterial communities associated with indoor
an body, and consequently, advance online publication,as the microbesdoi:10.1038/ismej.2011.211 a data archive and integrated analytthough,
Subject Category: microbial population and community ecology
Not surprisingly, these studies have identified surfaces in kitchens
environments because limitations of traditional 16 S rRNA gene
high diversity
communities is likely due to
of
related
relative abundances of s
pathogens. Although this less abundant in air than on surfaces.
ical tools dispersal;
Keywords: aeromicrobiology; bacteria; built environment microbiome; community ecology;are in the works. and restrooms as being hot of floorof bacterial contamination.the frequencyand sequencingdifferences in the made replicate sampling
spots
cloning
techniques have

e human
ck to pre-

contact
shoes, which would track a diversity
some surfaces (Figure 1B, Table
notably
environmental filtering In one recent study, they used air filters
Because several pathogenic bacteria are known
hat having natural airflow
To foster collaborations between micro- with the bottom aofvariety of to survive on inand in-depth characterizations of abundant onS2). Most surfaces
of microorganisms from
sources including soil, which is
were clearly more the communities prohibitive.
certain
surfaces for extended periods of time [6–8], these studies are of
With the
Green says answering that to sample airborne particles and microbes biologists, architects, and building scientists,in preventing the spread microbial habitat [27,39]. Indeed,advent of high-throughputrestrooms (Figure 1B). Some
known to be a highly-diverse
restrooms than male sequencing techniques, we
obvious importance
of human disease.
can now
bacteria commonly associated with soil (e.g.
family indoor microbial communities at abun
often most an
Introduction
microbiome—includes the foundation and comclinical data; she’s hoping in a classroom during 4 days during which human pathogensalso sponsored a symposium widely recognized that the majority of Rhodobacteraceae, investigate are the most common, andthe relationship
However, it is now
unprecedented depth and begin to understand

mensals interacting with each other and with their

Rhizobiales, Microbacteriaceae and Nocardioidaceae) were, on average,

found in the vagina of healthy reproductive age w

The Microbe Era

MICROBES
RUN THE
PLANET

Acknowledgements
• GEBA:
• $$: DOE-JGI, DSMZ
• Eddy Rubin, Phil Hugenholtz, Hans-Peter Klenk, Nikos Kyrpides, Tanya Woyke, Dongying Wu, Aaron Darling,
Jenna Lang

• GEBA Cyanobacteria
• $$: DOE-JGI
• Cheryl Kerfeld, Dongying Wu, Patrick Shih

• Haloarchaea
• $$$ NSF
• Marc Facciotti, Aaron Darling, Erin Lynch,

• Phylosift
• $$$ DHS
• Aaron Darling, Erik Matsen, Holly Bik, Guillaume Jospin

• iSEEM:
• $$: GBMF
• Katie Pollard, Jessica Green, Martin Wu, Steven Kembel, Tom Sharpton, Morgan Langille, Guillaume Jospin,
Dongying Wu,

• aTOL
• $$: NSF
• Naomi Ward, Jonathan Badger, Frank Robb, Martin Wu, Dongying Wu

• Others (not mentioned in detail)
• $$: NSF, NIH, DOE, GBMF, DARPA, Sloan
• Frank Robb, Craig Venter, Doug Rusch, Shibu Yooseph, Nancy Moran, Colleen Cavanaugh, Josh Weitz
• EisenLab: Srijak Bhatnagar, Russell Neches, Lizzy Wilbanks, Holly Bik

"The Quest for A field Guide to the Microbes" talk by Jonathan Eisen February 2, 2014.

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"The Quest for A field Guide to the Microbes" talk by Jonathan Eisen February 2, 2014.