the era of the microbiome - talk by jonathan eisen

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DNA based Studies of Microbial Diversity !

Jonathan A. Eisen !

University of California, Davis !

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The Era of the Microbiome !

Jonathan A. Eisen University of California, Davis

!December 6, 2013

!Cleveland Clinic 11th Annual Dr. Roizen's

Preventive and Integrative Medicine Conference !

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Gratuitous Genomics Plot

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Controls?

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Microbiome Elvis

Pubmed Hits for “Microbiome” vs. “Elvis”

The Microbiome

“The Nobel laureate Joshua Lederberg has suggested using the term "microbiome" to describe the collective genome of our indigenous microbes (microflora), the idea being that a comprehensive genetic view of Homo sapiens as a life-form should include the genes in our microbiome”

Lora Hooper and Jeff Gordon (Commensal Host-Bacterial Relationships in the Gut Science 11 May 2001: Vol. 292. no. 5519, pp. 1115 - 1118

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The Rise of the Microbiome

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• We are covered in a cloud of microbes

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• We are covered in a cloud of microbes !

• This “microbiome” likely is involved in many important human phenotypes

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• This “microbiome” LIKELY is involved in many important human phenotypes

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• This “microbiome” LIKELY is INVOLVED in many important human phenotypes

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Why Now?

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Why Now I: Appreciation of Diversity

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Microbial Diversity

• Microscope picture

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• 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 Form

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Diversity of Function

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The Bad The Good The Unusual

The Consumable The Burnable The Planet

Phylogenetic Diversity

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Why Now II: Post Genome Blues

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Overselling the Human Genome

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Epigenetics

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Gene Regulation / Expression

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Other Genomic Variability

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Why NOT The Microbiome?

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Why Now III: Science of Communities

Culturing Microbes

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Great Plate Count Anomaly

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Culturing Microscopy

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CountCount!30


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CountCount


Culturing Microbes

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CountCount

Solution?


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Solution?

DNA


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CountCount

Solution?

rRNA PCR


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DNA extraction

PCRSequence

rRNA genes

Sequence alignment = Data matrixPhylogenetic tree

PCR

rRNA1

Yeast

Makes lots of copies of the

rRNA genes in sample

E. coli

Humans

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rRNA1 5’ ...TACAGTATAGGTGGAGCTAGCGATCGATCG

A... 3’

rRNA Gene PCR

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DNA extraction

PCRSequence

rRNA genes


PCR

rRNA1

Yeast



E. coli

Humans

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PRIMERS

rRNA Gene PCR

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DNA extraction

PCRSequence

rRNA genes


PCR

rRNA1

rRNA2



rRNA1 5’ ...ACACACATAGGTGGAGCTAGCGATCGATC

GA... 3’

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DNA extraction

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rRNA genes


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rRNA2 rRNA2 5’..TACAGTATAGGTGGAGCTAGC

GACGATCGA... 3’

rRNA3 5’...ACGGCAAAATAGGTGGATTCT

AGCGATATAGA... 3’

rRNA4 5’...ACGGCCCGATAGGTGGATTCT

AGCGCCATAGA... 3’

rRNA3 C A C T G T

rRNA4 C A C A G T

Yeast T A C A G T

Yeast

rRNA3 rRNA4

rRNA Gene PCR

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rRNA typing

• OTUs ! Taxonomic lists ! Relative abundance of taxa ! Ecological metrics (alpha / beta diversity)

• Phylogenetic metrics ! Binning ! Identification of novel groups ! Clades ! Rates of change ! LGT ! Convergence ! PD ! Phylogenetic ecology (e.g., Unifrac)

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<<<<


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Solution?

Not Just rRNA


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Solution?

metagenomics


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DNA extraction

PCRSequence all genes

Shotgun

Shotgun Metagenomics

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DNA extraction


Shotgun


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DNA extraction


Phylogenetic tree

Shotgun

rRNA1

E. coli Humans

rRNA2

Yeast

rRNA3 rRNA4

Phylotyping


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DNA extraction


Phylogenetic tree

Shotgun


inputs of fixed carbon or nitrogen from external sources. As withLeptospirillum group I, both Leptospirillum group II and III have thegenes needed to fix carbon by means of the Calvin–Benson–Bassham cycle (using type II ribulose 1,5-bisphosphate carboxy-lase–oxygenase). All genomes recovered from the AMD system

contain formate hydrogenlyase complexes. These, in combinationwith carbon monoxide dehydrogenase, may be used for carbonfixation via the reductive acetyl coenzyme A (acetyl-CoA) pathwayby some, or all, organisms. Given the large number of ABC-typesugar and amino acid transporters encoded in the Ferroplasma type

Figure 4 Cell metabolic cartoons constructed from the annotation of 2,180 ORFs

identified in the Leptospirillum group II genome (63% with putative assigned function) and

1,931 ORFs in the Ferroplasma type II genome (58% with assigned function). The cell

cartoons are shown within a biofilm that is attached to the surface of an acid mine

drainage stream (viewed in cross-section). Tight coupling between ferrous iron oxidation,

pyrite dissolution and acid generation is indicated. Rubisco, ribulose 1,5-bisphosphate

carboxylase–oxygenase. THF, tetrahydrofolate.

articles

NATURE | doi:10.1038/nature02340 | www.nature.com/nature 5© 2004 Nature Publishing Group

Metagenomics

Community structure and metabolismthrough reconstruction of microbialgenomes from the environmentGene W. Tyson1, Jarrod Chapman3,4, Philip Hugenholtz1, Eric E. Allen1, Rachna J. Ram1, Paul M. Richardson4, Victor V. Solovyev4,Edward M. Rubin4, Daniel S. Rokhsar3,4 & Jillian F. Banfield1,2

1Department of Environmental Science, Policy and Management, 2Department of Earth and Planetary Sciences, and 3Department of Physics, University of California,Berkeley, California 94720, USA4Joint Genome Institute, Walnut Creek, California 94598, USA

...........................................................................................................................................................................................................................

Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and theirroles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we reportreconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three othergenomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency ofgenomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a differentindividual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level.The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundancevariants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologousrecombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed thepathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extremeenvironment.

The study of microbial evolution and ecology has been revolutio-nized by DNA sequencing and analysis1–3. However, isolates havebeen the main source of sequence data, and only a small fraction ofmicroorganisms have been cultivated4–6. Consequently, focus hasshifted towards the analysis of uncultivated microorganisms viacloning of conserved genes5 and genome fragments directly fromthe environment7–9. To date, only a small fraction of genes have beenrecovered from individual environments, limiting the analysis ofmicrobial communities as networks characterized by symbioses,competition and partitioning of community-essential roles.Comprehensive genomic data would resolve organism-specificpathways and provide insights into population structure, speciationand evolution. So far, sequencing of whole communities has notbeen practical because most communities comprise hundreds tothousands of species10.

Acid mine drainage (AMD) is a worldwide environmentalproblem that arises largely from microbial activity11. Here, wefocused on a low-complexity AMD microbial biofilm growinghundreds of feet underground within a pyrite (FeS2) ore body

12–15.This represents a self-contained biogeochemical system character-ized by tight coupling between microbial iron oxidation andacidification due to pyrite dissolution11,16,17. Random shotgunsequencing of DNA from entire microbial communities is oneapproach for the recovery of the gene complement of uncultivatedorganisms, and for determining the degree of variability withinpopulations at the genome level. We used random shotgun sequen-cing of the biofilm to obtain the first reconstruction of multiplegenomes directly from a natural sample. The results provide novelinsights into community structure, and reveal the strategies thatunderpin microbial activity in this environment.

Initial characterization of the biofilmBiofilms growing on the surface of flowing AMD in the five-way region of the Richmond mine at Iron Mountain, California12,were sampled in March 2000. Screening using group-specific18

fluorescence in situ hybridization (FISH) revealed that all biofilmscontained mixtures of bacteria (Leptospirillum, Sulfobacillus and, ina few cases, Acidimicrobium) and archaea (Ferroplasma and othermembers of the Thermoplasmatales). The genome of one of thesearchaea, Ferroplasma acidarmanus fer1, isolated from the Richmondmine, has been sequenced previously (http://www.jgi.doe.gov/JGI_microbial/html/ferroplasma/ferro_homepage.html).A pink biofilm (Fig. 1a) typical of AMD communities was

selected for detailed genomic characterization (see SupplementaryInformation). The biofilm was dominated by Leptospirillum speciesand contained F. acidarmanus at a relatively low abundance (Fig. 1b,c). This biofilm was growing in pH 0.83, 42 8C, 317mM Fe, 14mMZn, 4mM Cu and 2mM As solution, and was collected from asurface area of approximately 0.05m2.A 16S ribosomal RNA gene clone library was constructed from

DNA extracted from the pink biofilm, and 384 clones were end-sequenced (see Supplementary Information). Results indicated thepresence of three bacterial and three archaeal lineages. The mostabundant clones are close relatives of L. ferriphilum19 and belongto Leptospirillum group II (ref. 13). Although 94% of the Lepto-spirillum group II clones were identical, 17 minor variants weredetected with up to 1.2% 16S rRNA gene-sequence divergence fromthe dominant type. Tightly defined groups (up to 1% sequencedivergence) related to Leptospirillum group III (ref. 13), Sulfobacillus,Ferroplasma (some identical to fer1), ‘A-plasma’15 and ‘G-plasma’15

were also detected. Leptospirillum group III, G-plasma andA-plasma have only recently been detected in culture-independentmolecular surveys. FISH-based quantification (Fig. 1c; seealso Supplementary Information) confirmed the dominance ofLeptospirillum group II in the biofilm.

Community genome sequencing and assemblyIn conventional shotgun sequencing projects of microbial isolates,all shotgun fragments are derived from clones of the same genome.When using the shotgun sequencing approach on genomes from an

articles

NATURE | doi:10.1038/nature02340 | www.nature.com/nature 1© 2004 Nature Publishing Group

!47

Environmental Genome ShotgunSequencing of the Sargasso SeaJ. Craig Venter,1* Karin Remington,1 John F. Heidelberg,3

Aaron L. Halpern,2 Doug Rusch,2 Jonathan A. Eisen,3

Dongying Wu,3 Ian Paulsen,3 Karen E. Nelson,3 William Nelson,3

Derrick E. Fouts,3 Samuel Levy,2 Anthony H. Knap,6

Michael W. Lomas,6 Ken Nealson,5 Owen White,3

Jeremy Peterson,3 Jeff Hoffman,1 Rachel Parsons,6

Holly Baden-Tillson,1 Cynthia Pfannkoch,1 Yu-Hui Rogers,4

Hamilton O. Smith1

Wehave applied “whole-genome shotgun sequencing” tomicrobial populationscollected enmasse on tangential flow and impact filters from seawater samplescollected from the Sargasso Sea near Bermuda. A total of 1.045 billion base pairsof nonredundant sequencewas generated, annotated, and analyzed to elucidatethe gene content, diversity, and relative abundance of the organisms withinthese environmental samples. These data are estimated to derive from at least1800 genomic species based on sequence relatedness, including 148 previouslyunknown bacterial phylotypes. We have identified over 1.2 million previouslyunknown genes represented in these samples, including more than 782 newrhodopsin-like photoreceptors. Variation in species present and stoichiometrysuggests substantial oceanic microbial diversity.

Microorganisms are responsible for most of thebiogeochemical cycles that shape the environ-ment of Earth and its oceans. Yet, these organ-isms are the least well understood on Earth, asthe ability to study and understand the metabol-ic potential of microorganisms has been ham-pered by the inability to generate pure cultures.Recent studies have begun to explore environ-mental bacteria in a culture-independent man-ner by isolating DNA from environmental sam-ples and transforming it into large insert clones.For example, a previously unknown light-drivenproton pump, proteorhodopsin, was discoveredwithin a bacterial artificial chromosome (BAC)from the genome of a SAR86 ribotype (1), andsoil microbial DNA libraries have been construct-ed and screened for specific activities (2).

Here we have applied whole-genome shot-gun sequencing to environmental-pooled DNAsamples to test whether new genomic approach-es can be effectively applied to gene and spe-cies discovery and to overall environmental

characterization. To help ensure a tractable pilotstudy, we sampled in the Sargasso Sea, a nutrient-limited, open ocean environment. Further, weconcentrated on the genetic material captured onfilters sized to isolate primarily microbial inhabit-ants of the environment, leaving detailed analysisof dissolved DNA and viral particles on one endof the size spectrum and eukaryotic inhabitants onthe other, for subsequent studies.The Sargasso Sea. The northwest Sar-

gasso Sea, at the Bermuda Atlantic Time-seriesStudy site (BATS), is one of the best-studiedand arguably most well-characterized regionsof the global ocean. The Gulf Stream representsthe western and northern boundaries of thisregion and provides a strong physical boundary,separating the low nutrient, oligotrophic openocean from the more nutrient-rich waters of theU.S. continental shelf. The Sargasso Sea hasbeen intensively studied as part of the 50-yeartime series of ocean physics and biogeochem-istry (3, 4) and provides an opportunity forinterpretation of environmental genomic data inan oceanographic context. In this region, for-mation of subtropical mode water occurs eachwinter as the passage of cold fronts across theregion erodes the seasonal thermocline andcauses convective mixing, resulting in mixedlayers of 150 to 300 m depth. The introductionof nutrient-rich deep water, following thebreakdown of seasonal thermoclines into thebrightly lit surface waters, leads to the bloom-ing of single cell phytoplankton, including twocyanobacteria species, Synechococcus and Pro-

chlorococcus, that numerically dominate thephotosynthetic biomass in the Sargasso Sea.

Surface water samples (170 to 200 liters)were collected aboard the RV Weatherbird IIfrom three sites off the coast of Bermuda inFebruary 2003. Additional samples were col-lected aboard the SV Sorcerer II from “Hydro-station S” in May 2003. Sample site locationsare indicated on Fig. 1 and described in tableS1; sampling protocols were fine-tuned fromone expedition to the next (5). Genomic DNAwas extracted from filters of 0.1 to 3.0 !m, andgenomic libraries with insert sizes ranging from2 to 6 kb were made as described (5). Theprepared plasmid clones were sequenced fromboth ends to provide paired-end reads at the J.Craig Venter Science Foundation Joint Tech-nology Center on ABI 3730XL DNA sequenc-ers (Applied Biosystems, Foster City, CA).Whole-genome random shotgun sequencing ofthe Weatherbird II samples (table S1, samples 1 to4) produced 1.66 million reads averaging 818 bpin length, for a total of approximately 1.36 Gbp ofmicrobial DNA sequence. An additional 325,561sequences were generated from the Sorcerer IIsamples (table S1, samples 5 to 7), yielding ap-proximately 265 Mbp of DNA sequence.Environmental genome shotgun as-

sembly. Whole-genome shotgun sequencingprojects have traditionally been applied to iden-tify the genome sequence(s) from one particularorganism, whereas the approach taken here isintended to capture representative sequencefrom many diverse organisms simultaneously.Variation in genome size and relative abun-dance determines the depth of coverage of anyparticular organism in the sample at a givenlevel of sequencing and has strong implicationsfor both the application of assembly algorithmsand for the metrics used in evaluating the re-sulting assembly. Although we would expectabundant species to be deeply covered and wellassembled, species of lower abundance may berepresented by only a few sequences. For asingle genome analysis, assembly coveragedepth in unique regions should approximate aPoisson distribution. The mean of this distribu-tion can be estimated from the observed data,looking at the depth of coverage of contigsgenerated before any scaffolding. The assem-bler used in this study, the Celera Assembler(6), uses this value to heuristically identifyclearly unique regions to form the backbone ofthe final assembly within the scaffolding phase.However, when the starting material consists ofa mixture of genomes of varying abundance, athreshold estimated in this way would classifysamples from the most abundant organism(s) asrepetitive, due to their greater-than-averagedepth of coverage, paradoxically leaving themost abundant organisms poorly assembled.We therefore used manual curation of an initial

1The Institute for Biological Energy Alternatives, 2TheCenter for the Advancement of Genomics, 1901 Re-search Boulevard, Rockville, MD 20850, USA. 3TheInstitute for Genomic Research, 9712 Medical CenterDrive, Rockville, MD 20850, USA. 4The J. Craig VenterScience Foundation Joint Technology Center, 5 Re-search Place, Rockville, MD 20850, USA. 5University ofSouthern California, 223 Science Hall, Los Angeles, CA90089–0740, USA. 6Bermuda Biological Station forResearch, Inc., 17 Biological Lane, St George GE 01,Bermuda.

*To whom correspondence should be addressed. E-mail: [email protected]

RESEARCH ARTICLE

2 APRIL 2004 VOL 304 SCIENCE www.sciencemag.org66

!48

Why Now IV: Sequencing’s Gone Crazy

Surpassing Moore Law

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Sequencing Revolution

•Metagenomics more feasible !

•Deeper sequencing • The rare biosphere • Relative abundance estimates !

•More samples (with barcoding) • Times series • Spatially diverse sampling • Fine scale sampling

Why Now V: Growing Appreciation of Microbiome Functions

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

http://www.ncbi.nlm.nih.gov/pubmed/17183312#

Drosophila microbiome

Both natural surveys and laboratory experiments indicate that host diet plays a major role in shaping the Drosophila bacterial microbiome.!!Laboratory strains provide only a limited model of natural host–microbe interactions!

The Human Microbiome as an Ecosystem

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Lesson 1: !

Think Like and Ecologist

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!!

Ecology of the Microbiome 1: !

Biogeography

Biogeography

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Censored

Censored

Human biogeography

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Cho and Blaser. Nature Reviews Genetics 13, 260-270 (April 2012)

Human biogeography

Fig. S13

Glanspenis

Hair

Labiaminora

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

Axilla (L)

Ext. auditorycanal (L)

Volarforearm (L)

Palmar indexfinger (L)

Poplitealfossa (L)

Naris (L)

Plantarfoot (L)

Oral cavity

Umbilicus

External nose

Lat. pinna (L)

Palm (L)

Gut

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Forehead

Dorsal tongue

Lat. pinna (R)

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Volarforearm (R)

Palmar indexfinger (R)

Poplitealfossa (R)

Naris (R)

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Human biogeography

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Human biogeography

Slide from Rob Knight

ARTICLES

A human gut microbial gene catalogueestablished by metagenomic sequencingJunjie Qin1*, Ruiqiang Li1*, Jeroen Raes2,3, Manimozhiyan Arumugam2, Kristoffer Solvsten Burgdorf4,Chaysavanh Manichanh5, Trine Nielsen4, Nicolas Pons6, Florence Levenez6, Takuji Yamada2, Daniel R. Mende2,Junhua Li1,7, Junming Xu1, Shaochuan Li1, Dongfang Li1,8, Jianjun Cao1, Bo Wang1, Huiqing Liang1, Huisong Zheng1,Yinlong Xie1,7, Julien Tap6, Patricia Lepage6, Marcelo Bertalan9, Jean-Michel Batto6, Torben Hansen4, Denis LePaslier10, Allan Linneberg11, H. Bjørn Nielsen9, Eric Pelletier10, Pierre Renault6, Thomas Sicheritz-Ponten9,Keith Turner12, Hongmei Zhu1, Chang Yu1, Shengting Li1, Min Jian1, Yan Zhou1, Yingrui Li1, Xiuqing Zhang1,Songgang Li1, Nan Qin1, Huanming Yang1, Jian Wang1, Søren Brunak9, Joel Dore6, Francisco Guarner5,Karsten Kristiansen13, Oluf Pedersen4,14, Julian Parkhill12, Jean Weissenbach10, MetaHIT Consortium{, Peer Bork2,S. Dusko Ehrlich6 & Jun Wang1,13

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Herewe describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundantmicrobial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set,,150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent)microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. Thegenes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entirecohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which arealso largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms offunctions present in all individuals and most bacteria, respectively.

It has been estimated that the microbes in our bodies collectivelymake up to 100 trillion cells, tenfold the number of human cells,and suggested that they encode 100-fold more unique genes thanour own genome1. The majority of microbes reside in the gut, havea profound influence on human physiology and nutrition, and arecrucial for human life2,3. Furthermore, the gut microbes contribute toenergy harvest from food, and changes of gut microbiome may beassociated with bowel diseases or obesity4–8.

To understand and exploit the impact of the gut microbes onhuman health and well-being it is necessary to decipher the content,diversity and functioning of the microbial gut community. 16S ribo-somal RNA gene (rRNA) sequence-based methods9 revealed that twobacterial divisions, the Bacteroidetes and the Firmicutes, constituteover 90% of the known phylogenetic categories and dominate thedistal gut microbiota10. Studies also showed substantial diversity ofthe gut microbiome between healthy individuals4,8,10,11. Although thisdifference is especially marked among infants12, later in life the gutmicrobiome converges to more similar phyla.

Metagenomic sequencing represents a powerful alternative torRNA sequencing for analysing complex microbial communities13–15.Applied to the human gut, such studies have already generated some3 gigabases (Gb) of microbial sequence from faecal samples of 33

individuals from the United States or Japan8,16,17. To get a broaderoverview of the human gut microbial genes we used the IlluminaGenome Analyser (GA) technology to carry out deep sequencing oftotal DNA from faecal samples of 124 European adults. We generated576.7 Gb of sequence, almost 200 times more than in all previousstudies, assembled it into contigs and predicted 3.3 million uniqueopen reading frames (ORFs). This gene catalogue contains virtuallyall of the prevalent gut microbial genes in our cohort, provides abroad view of the functions important for bacterial life in the gutand indicates that many bacterial species are shared by differentindividuals. Our results also show that short-read metagenomicsequencing can be used for global characterization of the geneticpotential of ecologically complex environments.

Metagenomic sequencing of gut microbiomes

As part of the MetaHIT (Metagenomics of the Human IntestinalTract) project, we collected faecal specimens from 124 healthy, over-weight and obese individual human adults, as well as inflammatorybowel disease (IBD) patients, from Denmark and Spain (Supplemen-tary Table 1). Total DNA was extracted from the faecal specimens18

and an average of 4.5 Gb (ranging between 2 and 7.3 Gb) of sequencewas generated for each sample, allowing us to capture most of the

*These authors contributed equally to this work.{Lists of authors and affiliations appear at the end of the paper.

1BGI-Shenzhen, Shenzhen 518083, China. 2European Molecular Biology Laboratory, 69117 Heidelberg, Germany. 3VIB—Vrije Universiteit Brussel, 1050 Brussels, Belgium. 4HagedornResearch Institute, DK 2820 Copenhagen, Denmark. 5Hospital Universitari Val d’Hebron, Ciberehd, 08035 Barcelona, Spain. 6Institut National de la Recherche Agronomique, 78350Jouy en Josas, France. 7School of Software Engineering, South China University of Technology, Guangzhou 510641, China. 8Genome Research Institute, Shenzhen University MedicalSchool, Shenzhen 518000, China. 9Center for Biological Sequence Analysis, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark. 10Commissariat a l’EnergieAtomique, Genoscope, 91000 Evry, France. 11Research Center for Prevention and Health, DK-2600 Glostrup, Denmark. 12The Wellcome Trust Sanger Institute, Hinxton, CambridgeCB10 1SA, UK. 13Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark. 14Institute of Biomedical Sciences, University of Copenhagen & Faculty of HealthScience, University of Aarhus, 8000 Aarhus, Denmark.

Vol 464 | 4 March 2010 | doi:10.1038/nature08821

59Macmillan Publishers Limited. All rights reserved©2010

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Human biogeography

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!!


Population Biology and Variability

!64Huttenhower et al. 2012.

Variability Across People

Extensive Variation in the Microbiome

!65

Yatsunenko et al. 2012. Nature 486, 222–227.

Variation in the Vaginal Microbiome

!66Ravel et al. 2011. PNAS 108(Suppl 1): 4680–4687R

!67Morgan et al. Genome Biology 2012 13:R79 doi:10.1186/gb-2012-13-9-r79

!68Morgan et al. Genome Biology 2012 13:R79 doi:10.1186/gb-2012-13-9-r79

Age Diet Location

Many disease states

ExposurePregnant?

Breast fed? Obese

Almost all (99.96%) of the phylogenetically assigned genes belongedto the Bacteria and Archaea, reflecting their predominance in the gut.Genes that were not mapped to orthologous groups were clusteredinto gene families (see Methods). To investigate the functional con-tent of the prevalent gene set we computed the total number oforthologous groups and/or gene families present in any combinationof n individuals (with n 5 2–124; see Fig. 2c). This rarefaction ana-lysis shows that the ‘known’ functions (annotated in eggNOG orKEGG) quickly saturate (a value of 5,569 groups was observed): whensampling any subset of 50 individuals, most have been detected.However, three-quarters of the prevalent gut functionalities consistsof uncharacterized orthologous groups and/or completely novel genefamilies (Fig. 2c). When including these groups, the rarefaction curveonly starts to plateau at the very end, at a much higher level (19,338groups were detected), confirming that the extensive sampling of alarge number of individuals was necessary to capture this considerableamount of novel/unknown functionality.

Bacterial functions important for life in the gut

The extensive non-redundant catalogue of the bacterial genes fromthe human intestinal tract provides an opportunity to identify bac-terial functions important for life in this environment. There arefunctions necessary for a bacterium to thrive in a gut context (thatis, the ‘minimal gut genome’) and those involved in the homeostasisof the whole ecosystem, encoded across many species (the ‘minimalgut metagenome’). The first set of functions is expected to be presentin most or all gut bacterial species; the second set in most or allindividuals’ gut samples.

To identify the functions encoded by the minimal gut genome weuse the fact that they should be present in most or all gut bacterialspecies and therefore appear in the gene catalogue at a frequencyabove that of the functions present in only some of the gut bacterialspecies. The relative frequency of different functions can be deducedfrom the number of genes recruited to different eggNOG clusters,after normalization for gene length and copy number (Supplemen-tary Fig. 10a, b). We ranked all the clusters by gene frequencies anddetermined the range that included the clusters specifying well-known essential bacterial functions, such as those determined experi-mentally for a well-studied firmicute, Bacillus subtilis27, hypothe-sizing that additional clusters in this range are equally important.As expected, the range that included most of B. subtilis essentialclusters (86%) was at the very top of the ranking order (Fig. 5).Some 76% of the clusters with essential genes of Escherichia coli28

were within this range, confirming the validity of our approach.This suggests that 1,244 metagenomic clusters found within the range(Supplementary Table 10; termed ‘range clusters’ hereafter) specifyfunctions important for life in the gut.

We found two types of functions among the range clusters: thoserequired in all bacteria (housekeeping) and those potentially specificfor the gut. Among many examples of the first category are thefunctions that are part of main metabolic pathways (for example,central carbon metabolism, amino acid synthesis), and importantprotein complexes (RNA and DNA polymerase, ATP synthase, generalsecretory apparatus). Not surprisingly, projection of the range clusterson the KEGG metabolic pathways gives a highly integrated picture ofthe global gut cell metabolism (Fig. 6a).

The putative gut-specific functions include those involved in adhe-sion to the host proteins (collagen, fibrinogen, fibronectin) or inharvesting sugars of the globoseries glycolipids, which are carriedon blood and epithelial cells. Furthermore, 15% of range clustersencode functions that are present in ,10% of the eggNOG genomes(see Supplementary Fig. 11) and are largely (74.3%) not defined(Fig. 6b). Detailed studies of these should lead to a deeper compre-hension of bacterial life in the gut.

To identify the functions encoded by the minimal gut metagenome,we computed the orthologous groups that are shared by individuals ofour cohort. This minimal set, of 6,313 functions, is much larger than theone estimated in a previous study8. There are only 2,069 functionallyannotated orthologous groups, showing that they gravely underesti-mate the true size of the common functional complement among indi-viduals (Fig. 6c). The minimal gut metagenome includes a considerablefraction of functions (,45%) that are present in ,10% of thesequenced bacterial genomes (Fig. 6c, inset). These otherwise rare func-tionalities that are found in each of the 124 individuals may be necessaryfor the gut ecosystem. Eighty per cent of these orthologous groupscontain genes with at best poorly characterized function, underscoringour limited knowledge of gut functioning.

Of the known fraction, about 5% codes for (pro)phage-relatedproteins, implying a universal presence and possible important eco-logical role of bacteriophages in gut homeostasis. The most strikingsecondary metabolism that seems crucial for the minimal metage-nome relates, not unexpectedly, to biodegradation of complex sugarsand glycans harvested from the host diet and/or intestinal lining.Examples include degradation and uptake pathways for pectin(and its monomer, rhamnose) and sorbitol, sugars which are omni-present in fruits and vegetables, but which are not or poorly absorbedby humans. As some gut microorganisms were found to degrade bothof them29,30, this capacity seems to be selected for by the gut ecosystemas a non-competitive source of energy. Besides these, capacity toferment, for example, mannose, fructose, cellulose and sucrose is alsopart of the minimal metagenome. Together, these emphasize the

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Figure 5 | Clusters that contain the B. subtilis essential genes. The clusterswere ranked by the number of genes they contain, normalized by averagelength and copy number (see Supplementary Fig. 10), and the proportion ofclusters with the essential B. subtilis genes was determined for successivegroups of 100 clusters. Range indicates the part of the cluster distributionthat contains 86% of the B. subtilis essential genes.

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Figure 4 | Bacterial species abundance differentiates IBD patients andhealthy individuals. Principal component analysis with health status asinstrumental variables, based on the abundance of 155 species with $1%genome coverage by the Illumina reads in at least 1 individual of the cohort,was carried out with 14 healthy individuals and 25 IBD patients (21 ulcerativecolitis and 4 Crohn’s disease) from Spain (Supplementary Table 1). Two firstcomponents (PC1 and PC2) were plotted and represented 7.3% of wholeinertia. Individuals (represented by points) were clustered and centre ofgravity computed for each class; P-value of the link between health status andspecies abundance was assessed using a Monte-Carlo test (999 replicates).

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• Microbial community different in many disease states compared to healthy individuals

• Unclear if this is cause or effect in most cases

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Cho and Blaser. Nature Reviews Genetics 13, 260-270 (April 2012)

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Mom Knows Best: The Universality of Maternal Microbial Transmission Lisa J. FunkhouserSeth R.

Bordenstein

Milk and the Microbiome

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Microbes from the Built Environment

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ORIGINAL ARTICLE

Architectural design influences the diversity andstructure of the built environment microbiome

Steven W Kembel1, Evan Jones1, Jeff Kline1,2, Dale Northcutt1,2, Jason Stenson1,2,Ann M Womack1, Brendan JM Bohannan1, G Z Brown1,2 and Jessica L Green1,3

1Biology and the Built Environment Center, Institute of Ecology and Evolution, Department ofBiology, University of Oregon, Eugene, OR, USA; 2Energy Studies in Buildings Laboratory,Department of Architecture, University of Oregon, Eugene, OR, USA and 3Santa Fe Institute,Santa Fe, NM, USA

Buildings are complex ecosystems that house trillions of microorganisms interacting with eachother, with humans and with their environment. Understanding the ecological and evolutionaryprocesses that determine the diversity and composition of the built environment microbiome—thecommunity of microorganisms that live indoors—is important for understanding the relationshipbetween building design, biodiversity and human health. In this study, we used high-throughputsequencing of the bacterial 16S rRNA gene to quantify relationships between building attributes andairborne bacterial communities at a health-care facility. We quantified airborne bacterial communitystructure and environmental conditions in patient rooms exposed to mechanical or windowventilation and in outdoor air. The phylogenetic diversity of airborne bacterial communities waslower indoors than outdoors, and mechanically ventilated rooms contained less diverse microbialcommunities than did window-ventilated rooms. Bacterial communities in indoor environmentscontained many taxa that are absent or rare outdoors, including taxa closely related to potentialhuman pathogens. Building attributes, specifically the source of ventilation air, airflow rates, relativehumidity and temperature, were correlated with the diversity and composition of indoor bacterialcommunities. The relative abundance of bacteria closely related to human pathogens was higherindoors than outdoors, and higher in rooms with lower airflow rates and lower relative humidity.The observed relationship between building design and airborne bacterial diversity suggests thatwe can manage indoor environments, altering through building design and operation the communityof microbial species that potentially colonize the human microbiome during our time indoors.The ISME Journal advance online publication, 26 January 2012; doi:10.1038/ismej.2011.211Subject Category: microbial population and community ecologyKeywords: aeromicrobiology; bacteria; built environment microbiome; community ecology; dispersal;environmental filtering

Introduction

Humans spend up to 90% of their lives indoors(Klepeis et al., 2001). Consequently, the way wedesign and operate the indoor environment has aprofound impact on our health (Guenther andVittori, 2008). One step toward better understandingof how building design impacts human healthis to study buildings as ecosystems. Built envi-ronments are complex ecosystems that containnumerous organisms including trillions of micro-organisms (Rintala et al., 2008; Tringe et al., 2008;Amend et al., 2010). The collection of microbiallife that exists indoors—the built environment

microbiome—includes human pathogens and com-mensals interacting with each other and with theirenvironment (Eames et al., 2009). There have beenfew attempts to comprehensively survey the builtenvironment microbiome (Rintala et al., 2008;Tringe et al., 2008; Amend et al., 2010), with moststudies focused on measures of total bioaerosolconcentrations or the abundance of culturable orpathogenic strains (Berglund et al., 1992; Toivolaet al., 2002; Mentese et al., 2009), rather than a morecomprehensive measure of microbial diversity inindoor spaces. For this reason, the factors thatdetermine the diversity and composition of the builtenvironment microbiome are poorly understood.However, the situation is changing. The develop-ment of culture-independent, high-throughputmolecular sequencing approaches has transformedthe study of microbial diversity in a variety ofenvironments, as demonstrated by the recent explo-sion of research on the microbial ecology of aquaticand terrestrial ecosystems (Nemergut et al., 2011)

Received 23 October 2011; revised 13 December 2011; accepted13 December 2011

Correspondence: SW Kembel, Biology and the Built EnvironmentCenter, Institute of Ecology and Evolution, Department of Biology,University of Oregon, Eugene, OR 97405, USA.E-mail: [email protected]

The ISME Journal (2012), 1–11& 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12

www.nature.com/ismej

Microbial Biogeography of Public Restroom SurfacesGilberto E. Flores1, Scott T. Bates1, Dan Knights2, Christian L. Lauber1, Jesse Stombaugh3, Rob Knight3,4,

Noah Fierer1,5*

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, thediversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibitedby bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing ofthe 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla:Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria. The communities clustered into three general categories: thosefound on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched withhands. On toilet surfaces, gut-associated taxa were more prevalent, suggesting fecal contamination of these surfaces. Floorsurfaces were the most diverse of all communities and contained several taxa commonly found in soils. Skin-associatedbacteria, especially the Propionibacteriaceae, dominated surfaces routinely touched with our hands. Certain taxa were morecommon in female than in male restrooms as vagina-associated Lactobacillaceae were widely distributed in femalerestrooms, likely from urine contamination. Use of the SourceTracker algorithm confirmed many of our taxonomicobservations as human skin was the primary source of bacteria on restroom surfaces. Overall, these results demonstrate thatrestroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with clearlinkages between communities on or in different body sites and those communities found on restroom surfaces. Moregenerally, this work is relevant to the public health field as we show that human-associated microbes are commonly foundon restroom surfaces suggesting that bacterial pathogens could readily be transmitted between individuals by the touchingof surfaces. Furthermore, we demonstrate that we can use high-throughput analyses of bacterial communities to determinesources of bacteria on indoor surfaces, an approach which could be used to track pathogen transmission and test theefficacy of hygiene practices.

Citation: Flores GE, Bates ST, Knights D, Lauber CL, Stombaugh J, et al. (2011) Microbial Biogeography of Public Restroom Surfaces. PLoS ONE 6(11): e28132.doi:10.1371/journal.pone.0028132

Editor: Mark R. Liles, Auburn University, United States of America

Received September 12, 2011; Accepted November 1, 2011; Published November 23, 2011

Copyright: ! 2011 Flores et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported with funding from the Alfred P. Sloan Foundation and their Indoor Environment program, and in part by the NationalInstitutes of Health and the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

More than ever, individuals across the globe spend a largeportion of their lives indoors, yet relatively little is known about themicrobial diversity of indoor environments. Of the studies thathave examined microorganisms associated with indoor environ-ments, most have relied upon cultivation-based techniques todetect organisms residing on a variety of household surfaces [1–5].Not surprisingly, these studies have identified surfaces in kitchensand restrooms as being hot spots of bacterial contamination.Because several pathogenic bacteria are known to survive onsurfaces for extended periods of time [6–8], these studies are ofobvious importance in preventing the spread of human disease.However, it is now widely recognized that the majority ofmicroorganisms cannot be readily cultivated [9] and thus, theoverall diversity of microorganisms associated with indoorenvironments remains largely unknown. Recent use of cultiva-tion-independent techniques based on cloning and sequencing ofthe 16 S rRNA gene have helped to better describe these

communities and revealed a greater diversity of bacteria onindoor surfaces than captured using cultivation-based techniques[10–13]. Most of the organisms identified in these studies arerelated to human commensals suggesting that the organisms arenot actively growing on the surfaces but rather were depositeddirectly (i.e. touching) or indirectly (e.g. shedding of skin cells) byhumans. Despite these efforts, we still have an incompleteunderstanding of bacterial communities associated with indoorenvironments because limitations of traditional 16 S rRNA genecloning and sequencing techniques have made replicate samplingand in-depth characterizations of the communities prohibitive.With the advent of high-throughput sequencing techniques, wecan now investigate indoor microbial communities at anunprecedented depth and begin to understand the relationshipbetween humans, microbes and the built environment.

In order to begin to comprehensively describe the microbialdiversity of indoor environments, we characterized the bacterialcommunities found on ten surfaces in twelve public restrooms(six male and six female) in Colorado, USA using barcoded

PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e28132

the stall in), they were likely dispersed manually after women usedthe toilet. Coupling these observations with those of thedistribution of gut-associated bacteria indicate that routine use oftoilets results in the dispersal of urine- and fecal-associated bacteriathroughout the restroom. While these results are not unexpected,they do highlight the importance of hand-hygiene when usingpublic restrooms since these surfaces could also be potentialvehicles for the transmission of human pathogens. Unfortunately,previous studies have documented that college students (who arelikely the most frequent users of the studied restrooms) are notalways the most diligent of hand-washers [42,43].

Results of SourceTracker analysis support the taxonomicpatterns highlighted above, indicating that human skin was theprimary source of bacteria on all public restroom surfacesexamined, while the human gut was an important source on oraround the toilet, and urine was an important source in women’srestrooms (Figure 4, Table S4). Contrary to expectations (seeabove), soil was not identified by the SourceTracker algorithm asbeing a major source of bacteria on any of the surfaces, includingfloors (Figure 4). Although the floor samples contained family-leveltaxa that are common in soil, the SourceTracker algorithmprobably underestimates the relative importance of sources, like

Figure 3. Cartoon illustrations of the relative abundance of discriminating taxa on public restroom surfaces. Light blue indicates lowabundance while dark blue indicates high abundance of taxa. (A) Although skin-associated taxa (Propionibacteriaceae, Corynebacteriaceae,Staphylococcaceae and Streptococcaceae) were abundant on all surfaces, they were relatively more abundant on surfaces routinely touched withhands. (B) Gut-associated taxa (Clostridiales, Clostridiales group XI, Ruminococcaceae, Lachnospiraceae, Prevotellaceae and Bacteroidaceae) were mostabundant on toilet surfaces. (C) Although soil-associated taxa (Rhodobacteraceae, Rhizobiales, Microbacteriaceae and Nocardioidaceae) were in lowabundance on all restroom surfaces, they were relatively more abundant on the floor of the restrooms we surveyed. Figure not drawn to scale.doi:10.1371/journal.pone.0028132.g003

Figure 4. Results of SourceTracker analysis showing the average contributions of different sources to the surface-associatedbacterial communities in twelve public restrooms. The ‘‘unknown’’ source is not shown but would bring the total of each sample up to 100%.doi:10.1371/journal.pone.0028132.g004

Bacteria of Public Restrooms

PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e28132

10 FEBRUARY 2012 VOL 335 SCIENCE www.sciencemag.org 650

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In just that short time, the microbes had begun to take on a “signature” of outside air (more types from plants and soil), and 2 hours after the windows were shut again, the proportion of microbes from the human body increased back to pre-vious levels.

The s tudy, which appeared online 26 Janu-ary in The ISME Journal, found that mechanically ventilated rooms had lower microbial diversity than ones with open win-dows. The availability of fresh air translated into lower proportions of microbes associ-ated with the human body, and consequently, fewer potential pathogens. Although this result suggests that having natural airfl ow may be healthier, Green says answering that question requires clinical data; she’s hoping to convince a hospital to participate in a study to see if the incidence of hospital-acquired infections is associated with a room’s micro-bial community.

For his part, Peccia, who is also a Sloan grantee, is merging microbiology and the

physics of aerosols to look more closely at how the movement of air affects microbes. Peccia says his group is building on work by air-quality engineers and scientists, but “we want to add biology to the equation.”

Bacteria in air behave like other particles; their size dictates how they disperse or settle. Humans in a room not only shed microbes from their skin and mouths, but they also drum up microbial material from the fl oor as

they move around. But to quantify those con-tributions, Peccia’s team has had to develop new methods to collect airborne bacteria and extract their DNA, as the microbes are much less abundant in air than on surfaces.

In one recent study, they used air fi lters to sample airborne particles and microbes in a classroom during 4 days during which students were present and 4 days during which the room was vacant. They measured the abundance and type of fungal and bac-terial genomes present and estimated the microbes’ concentrations in the entire room. By accounting for bacteria entering and leav-

ing the room through ventilation, they calculated that people shed or resuspended about 35 million bacterial cells per person per hour. That number is much higher than the several-hundred-thousand maximum previously estimated to be present in indoor air, Peccia reported last fall at the American Association for Aerosol Research Conference in Orlando, Florida.

His group’s data also suggest that rooms have “memories” of past human inhabitants. By kick-ing into the air settled microbes from the fl oor, occupants expose themselves not just to the microbes of a person coughing next to them, but also possibly to those from a person who coughed in the room a few hours or even days ago.

Peccia hopes to come up with ways to describe the distribution of bacteria indoors that can be used in conjunction with exist-ing knowledge about particulate matter and chemicals in designing healthier buildings. “My hope is that we can bring this enough to the forefront that people who do aerosol sci-ence will fi nd it as important to know biology as to know physics and chemistry,” he says.

Still, even though he’s a willing partici-

pant in indoor microbial ecology research, Peccia thinks that the field has yet to gel. And the Sloan Foundation’s Olsiewski shares some of his con-cern. “Everybody’s gen-erating vast amounts of

data,” she says, but looking across data sets can be diffi cult because groups choose dif-ferent analytical tools. With Sloan support, though, a data archive and integrated analyt-ical tools are in the works.

To foster collaborations between micro-biologists, architects, and building scientists, the foundation also sponsored a symposium on the microbiome of the built environment at the 2011 Indoor Air conference in Austin, Texas, and launched a Web site, MicroBE.net, that’s a clearinghouse of information on the fi eld. Although Olsiewski won’t say how long the foundation will fund its indoor microbial ecology program, she says Sloan is committed to supporting all of the current projects for the next few years. The program’s ultimate goal, she says, is to create a new fi eld of scientifi c inquiry that eventually will be funded by tradi-tional government funding agencies focused on basic biology and environmental policy.

Matthew Kane, a microbial ecologist and program director at the U.S. National Sci-ence Foundation (NSF), says that although there was interest in these questions prior to the Sloan program, the Sloan Foundation has taken a directed approach to funding the research, and “I have no doubt that their investment is going to reap great returns.” So far, though, NSF has funded only one study on indoor microbes: a study of Pseudomonas bacteria in human households.

As studies like Green’s building ecology analysis progress, they should shed light on how indoor environments differ from those traditionally studied by microbial ecologists. “It’s important to have a quantitative under-standing of how building design impacts microbial communities indoors, and how these communities impact human health,” Green says. But it remains to be seen whether we’ll someday design and maintain our build-ings with microbes in mind.

–COURTNEY HUMPHRIES

Courtney Humphries is a freelance writer in Boston and author of Superdove.

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Outside infl uence. Students prepare to sample air outside a class-room in China as part of an indoor ecology study.

Bathroom biogeography. By swabbing different surfaces in public restrooms, researchers determined that microbes vary in where they come from depend-ing on the surface (chart).

Published by AAAS

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Disturbing Normal Assembly

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Restoring the Microbiome?

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Hartman et al. PNAS 2009

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The Importance of History (i.e., Evolution)

History of Ecosystems Important

!86

Vertebrate Microbiomes

Diverse microorganisms and microbial communities are a feature of modern life on the Earth, and have probably been necessary for the evolution of life as we know it1.

Microorganisms formed spatially organized communi-ties as early as 3.25 billion years ago, when some left their mark in the fossil record2. Today, microbial life is found in diverse communities all over the biosphere. The high level of novelty that is necessary for microorganisms to develop a diversity of cell lineages and inhabit a vast range of habitats probably required that whole com-munities exchange innovations1. Comparative studies of microbial communities are starting to reveal which environmental features, such as biogeography, salinity or redox potential, have important effects on the organiza-tion of microbial diversity3–6. These types of analyses are now being extended to the microbial communities that populate a globally ubiquitous but ephemeral habitat: the body surfaces of animals, including those of humans.

Multicellular eukaryotes have existed for at least one-quarter of the Earth’s history, or 1.2 billion years7. Thus, an already long history of interaction between multicel-lular life-forms and microbial communities preceded, and probably shaped, the evolution of vertebrates. The legacy of ancient associations between hosts and their epibiotic microbial communities is evident in the present-day effects that the gut microbiota exerts on host biology, which range from the structure and functions of the gut and the innate and adaptive immune systems, to

host energy metabolism8–11. Host responses to microbial colonization are evolutionarily conserved among diverse vertebrates, including zebrafish, mice and humans12. The underlying factors that dictate our interactions with our microbial partners therefore provide some of the foundations of our Homo sapiens genome.

If microbial communities are, and have always been, so intricately associated with their vertebrate hosts, then how specialized are body-associated microbial lineages to vertebrates and how distinct are they from those that populate the non-living environments of the biosphere? In this Analysis, we place our human gut microbiota in the context of many other diverse microbiotas, from our close relatives the primates, to more distantly related mammals, other metazoans and ‘free-living’ microbial communities. This evolutionary ecology perspective helps put the recently initiated international Human Microbiome Project (see Further information)13 in the context of the biosphere within which humans and their microorganisms have evolved.

Diet and the evolution of modern humansFood is central to the evolution of H. sapiens. During the first half of the evolution of our lineage, Australopithecus species split from prehistoric apes and persisted from ~4.4 Mya (million years ago) until ~2.5 Mya14. This early split has been associated with a dietary shift to seeds and soft fruits, based on comparisons of australopithecine

*Center for Genome Sciences, Washington University School of Medicine, St Louis, Missouri 63108, USA. ‡Department of Microbiology, Cornell University, Ithaca, New York 14850, USA. §Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA. ||Department of Computer Science, University of Colorado, Boulder, Colorado 80309, USA. ¶These authors contributed equally to this work. Correspondence to J.I.G. e-mail: [email protected]

MicrobiotaThe complete set of microbial lineages that live in a particular environment.

Worlds within worlds: evolution of the vertebrate gut microbiotaRuth E. Ley*‡¶, Catherine A. Lozupone*§¶, Micah Hamady||, Rob Knight§ and Jeffrey I. Gordon*

Abstract | In this Analysis we use published 16S ribosomal RNA gene sequences to compare the bacterial assemblages that are associated with humans and other mammals, metazoa and free-living microbial communities that span a range of environments. The composition of the vertebrate gut microbiota is influenced by diet, host morphology and phylogeny, and in this respect the human gut bacterial community is typical of an omnivorous primate. However, the vertebrate gut microbiota is different from free-living communities that are not associated with animal body habitats. We propose that the recently initiated international Human Microbiome Project should strive to include a broad representation of humans, as well as other mammalian and environmental samples, as comparative analyses of microbiotas and their microbiomes are a powerful way to explore the evolutionary history of the biosphere.

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ANALYSIS

Genera that cross the divide. Another way to visualize the vertebrate gut–environment dichotomy is by using a network diagram that displays, in addition to the clus-tering of hosts with similar microbiotas, the bacterial genera they share. In this representation of the data, the vertebrate gut samples are more connected to one another than to the environmental samples (FIG. 4a,b). As in the UniFrac-based analysis, the non-gut human samples also occupy an intermediate position between the free-living and the gut communities. FIGURE 5 shows the phylogenetic classification of operational taxonomic units (OTUs) that are shared between samples: among humans, an over-whelming number of these are from the Firmicutes, with a smaller number from the Bacteroidetes. By contrast, the free-living communities share OTUs from a wider range of phyla. Samples from the guts of obese humans cluster away from the samples of healthy subjects, and most of their shared OTUs are found in the Firmicutes. This obser-vation is consistent with the finding that samples from obese individuals have a higher number of OTUs from Firmicutes than samples from lean subjects31.

Bacterial genera that inhabit both the vertebrate gut-associated microbiotas and the free-living com-munities can be considered to be cosmopolitan. As the analyses discussed above mainly determine the dominant members of a microbiota, these genera are presumed to grow and subsist in the gut environment (autochthonous members) rather than simply passing through as transient members of the gut microbial community (allochthonous members). Among these cosmopolitan groups was the Pseudomonadaceae

family of the gammaproteobacteria class. This fam-ily contained OTUs from both the vertebrate gut and free-living communities in saline and non-saline habitats. Members of the Enterobacteriales order (also from the gammaproteobacteria) were detected in the vertebrate gut, termite gut and other invertebrates, as well as in a surface soil sample and anoxic saline water. Staphylococcaceae family members (from the phylum Firmicutes and class Bacilli) were common in the ver-tebrate gut samples, but were also detected in soil and cultures derived from freshwater and saline habitats. Finally, members of the Fusobacterium genus were detected in salt-water sediments, in addition to the vertebrate gut. The cosmopolitan distribution of these organisms might have made them particularly impor-tant for introducing novel functions during evolution of the gut microbiota, as they could bring new useful genes from the global microbiome into the gut microbiome through horizontal gene transfer. However, it should be noted that some OTUs that are common in humans

Nature Reviews | Microbiology

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Figure 3 | Relative abundance of phyla in samples. Bar graph showing the proportion of sequences from each sample that could be classified at the phylum level. The colour codes for the dominant Firmicutes and Bacteroidetes phyla are shown. For a complete description of the colour codes see Supplementary information S2 (figure). ‘Other humans’ refers to body habitats other than the gut; for example, the mouth, ear, skin, vagina and vulva (see Supplementary information S1 (table)).

Figure 4 | Network analysis of bacterial communities from animal-associated and free-living communities. The panel on the left includes a schematic key that illustrates features of the network analysis and genera keys for panels a and b. Labels are sample nodes. Rounded squares represent operational taxonomic units (OTUs) shared by two or more samples (shown in grey in panels a and b), whereas diamonds represent the set of OTUs that are unique to a sample. Network diagrams are colour coded according to habitat.

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ANALYSIS

782 | OCTOBER 2008 | VOLUME 6 www.nature.com/reviews/micro

ANALYSIS

!87Nat Rev Microbiol. 2008 October ; 6(10): 776–788. doi:10.1038/nrmicro1978.

!88

20 JUNE 2008 VOL 320 SCIENCE www.sciencemag.org1648

REPORTS

Ley et al. 2008.

20 JUNE 2008 VOL 320 SCIENCE www.sciencemag.org1648

REPORTS

Human superorganism

• Human-microbe associations are very old

• Microbial genes on a person >> human genes

• Your microbes are coadapted to each other

• Microbes known to manipulate EVERYTHING imaginable in hosts

!89

Lateral Gene Transfer

Perna et al. 2003!90

!91

!!

Lesson 3: !

Don’t Oversell the Microbiome

Overselling the Microbiome

!92


• Changes in gut bacteria protect against stroke

• Scientists look to mummies for obesity cure

• Good bacteria in the intestine prevent diabetes, study suggests.

!93

http://health.msn.co.nz/healthnews/8443910/scientists-look-to-mummies-for-obesity-cure

http://www.sciencedaily.com/releases/2013/01/130118064731.htm


• Correlation ≠ Causation

• Complexity is astonishing ! 1000s of taxa ! Each with intraspecific variation ! Viruses, bacteria, archaea,

eukaryotes

• Massive risk for false positive associations

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!95

!!

Lesson 4: !

Lots of New Things Happening

American Gut

!96

uBiome

!97

Personal Microbiomes

• How will tests be used?

!98

Personal Genomes

Personal Microbiomes

Family history ++ --

Disease risk ++ --

Treatment ++ --

Research ++ ++

Data returned ++ ++

Citizen Science

Last thoughts

• Microbiome counselors?

• Who owns the microbiome?

• Need 1000s of small studies

• Conservation of the microbiome?

• Openness is critical

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2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

Still Going Up

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 era of the microbiome - talk by jonathan eisen

Health & Medicine

microbiome likely

term microbiome

microbiome elvis

cloud of microbes

culturing microbes

diversity of function

great plate count anomaly

appreciation of diversity