Mapping Microbiomes at the Micron Scale

Whitehead Institute Gary BorisyPartnership for Science Education June 05, 2017

Microbes live in communities

Microbiome—”the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease” Lederberg, 2001

Defining a Healthy MicrobiomeTaxonomy—organism centric viewpoint

Lloyd-Price et al (2016) Genome Medicine 8:51

Defining a Healthy MicrobiomeFunction—gene centric viewpoint

Lloyd-Price et al (2016) Genome Medicine 8:51

Defining a Healthy Microbiome

Lloyd-Price et al (2016) Genome Medicine 8:51

Dynamics—Energy Landscape

The Importance of Mapping Microbiomes at the Micron Scale

• Microbes live and work at the micron scale• Their neighborhoods—microbiomes—are at the micron scale• The DNA revolution has opened new research avenues• Metagenomics—culture-independent analysis• But a gap remains• Need to know “who is next to who”; and “who is next to what”

to understand how a community works• Need to know biogeography at the micron scale

• Imaging• Develop multiplexed, spectral imaging• Go beyond the limitations of band pass filters; use all the

information available in fluorescent probes• Go beyond individual fluorescent probes; use combinatorial

labeling to create fluorescent “signatures”

• Genomics• Use genomic information to design taxon-specific probes • Use metagenomics for culture-independent analysis• Use single-nucleotide resolution to guide imaging priorities

• Putting them together—Imaging Genomics• Correlative imaging and genomics on the same samples

How to Map Microbiomes

Plan of Talk• Introduction to the Oral Microbiome• The importance of single-nucleotide resolution in

microbial taxonomy• Visualizing complexity through spectral imaging • Imaging Proof-of-Principle • Biological Proof-of-Principle • Mapping oral microbiomes• Future prospects

Why to Study the Human Oral Microbiome

Kolenbrander, P.E., et al 2002. Microbiol. Mol. Biol. Rev. 66:486-505

Hypothesis for oral biofilm structure

Leuwenhoek, 1680

• First microbes seen• Accessible, portal to body• Curated database available (HOMD)• ~700 species-level entries• Many taxa cultivated (67%)• Many genomes available (58%)• Human Microbiome Project (HMP)

provides data at 9 oral sites

Oral Microbiome is a good test bed

Microbial Identification Relies on a16S rRNA Gene Barcode

Nature Structural Biology9, 750 - 755 (2002)

Single nucleotide resolution reveals diversity of oral taxa and habitats

SUB

SUP

• 16S RNA gene • V1-V3 region• 493 oligotypes—significant sequence variants• Each oral site has unique oligotype signature

• Eren et al 2014 PNAS 111: E2875-84• Mark Welch et al 2014 Frontiers Microbiol 5: 568• Utter et al 2016 Frontiers Microbiol 7: 564

SUB

SUP

Spectral Imaging Began in Physics

Spectral bleed-through

Problem of Standard Fluorescence Microscopywith Band-Pass Filters

Spectral Image Data Cube3-D data set

Solution is Spectral ImagingFluorescence microscope

spectral detector

Emitted light from specimen

Diffraction grating D

etector

Deconstruct emission signatures by linear unmixing

Proof-of-Principle Test of Spectral Imaging

• Use E. coli as a “test bead”• Label E. coli with 16s rRNA-specific oligonucleotide

conjugated to a single fluor• Label with a binary combination of oligo-fluor probes• n fluorophores; n(n-1)/2 unique binary combinations• Make mixture of differently labeled populations• Spectrally image• Quantify abundance of label types• Compare to input

Bacterial cellrRNA

Fluorescent Oligo Probe

5’- GCT GCC TCC CGT AGG AGT-3’

Probes hybridize against

complementary sequences of the

ribosomal RNA (rRNA)

Fluorescence in situ hybridization (FISH)

Delong, Wickham & Pace 1989. Science 243: 1360-1363

With 8 fluorophores, there exist 28 unique binary combinations

CLASI-FISHCombinatorial Labeling and

Spectral Imaging-Fluorescence in situ Hybridization

Valm et al., 2011 PNAS 108: 4152-57Valm et al., 2012 Syst Appl Microbiol 35: 496-502Valm et al., 2016 PLOS doi: 10.1371/journal.pone.0158495

8 fluorophores

Conjugated to a DNA FISH probe that labels most bacteria

28 tubes of E. coli suspended in hybridization buffer

Add one fluorophore to each tube......then a second to give 28 unique binary combinations

Allow probe to hybridize

Wash cells to remove excess probe

Add an equal volume of each label-type to a single tube

Spot this mixture on a slide and

image

Imaging Proof of Principle with E. coli

spectral mergelabel types

Label Type Assignment and Quantification

Biological Proof-of-Principle with Oral Microbes• Streptococcus

• Prevotella

• Fusobacterium

• Selenomonas

• Rothia

• Gemella

• Actinomyces

• Campylobacter

• Capnocytophaga

• Leptotrichia

• Treponema

• Veillonella

• Porphyromonas

• Pasteurellaceae

• Neisseriaceae

1. Identified 15 of the most abundant genera in the mouth

2. Designed probes for these 15 genera

3. Grew representative species in the lab

4. Validated probes individually and as a set

5. Made an artificial mixture of the 15 species in a test tube

6. Performed CLASI-FISH on the mixture

Actinomyces 476

Rothia 492

Gemella 572

Pasteurellaceae 111

Prevotella 392

Capnocytophaga 371

Veillonella 488

Streptococcus 405

Leptotrichia 568

Selenomonas 60

Porphyromonas 1160

Neisseriaceae 1030

Fusobacterium 714

Treponema 684

Campylobacter 1021

15 Oligonucleotides Targeting Oral Genera Labeled with Binary Combinations of 6 Fluorophores

15 oral taxa visualized simultaneously

Mapping Microbiomes• Dental plaque• Tongue dorsum• Buccal mucosa• Hard palate• Saliva• Mouse gut

Human Dental Plaque Microbiome

20 mm

2016 Feb 9;113(6):E791-800. doi: 10.1073/pnas.1522149113. Epub 2016 Jan 25.

https://www.statnews.com/2016/05/04/mouth-full-bacteria-blooming-beautiful/

LIVE on Air in France: Le Magazine de la Santé

News CoverageDiscovery Channel Daily Planethttp://review.bellmedia.ca/view/962622322

Nature Reviews Microbiology RESEARCH HIGHLIGHTS FISHing in the oral microbiota Published online 8 Feb 2016; doi:10.1038/nrmicro.2016.21

Trends in Microbiology SPOTLIGHTOral Biofilm Architecture at the Microbial ScaleFerrer, D.M & Mira, A. Published online http://dx.doi.org/10.1016/j.tim.2016.02.013

NATURE METHODS | VOL.14 NO.1 | JANUARY 2017 | 39Microbiology: The return of culture

STAT: Bacteria in your mouthBoston Globe, video, 2016

CorynebacteriumStreptococcusCapnocytophagaFusobacteriumLeptotrichiaActinomycesPasteurellaceaeNeisseriaceaePorphyromonas

“Fly-through” plaque structure in 2 mm steps

Step 1

Step 2

Step 3

Step 4

Step 5

CorynebacteriumStreptococcus

Streptococcus perimeter surrounds filamentous Corynebacterium core

CorynebacteriumStreptococcusCapnocytophagaFusobacterium

Capnocytophaga, Fusobacterium are located in a sub-perimeter annulus

CorynebacteriumStreptococcusCapnocytophagaFusobacteriumLeptotrichia

Leptotrichia is internal

CorynebacteriumStreptococcusCapnocytophagaFusobacteriumLeptotrichiaActinomycesHaemophilus/AggrNeisseriaPorphyromonas

Haemophilus/Aggregatibacter, Porphyromonas, Neisseria are at

perimeter

Taxa can be identified to species level

CorynebacteriummatruchotiiStreptococcus cristatus/mitusAggregatibacterPorphyromonaspasteri

8 μm

“Corn-cobs” come in several types

CorynebacteriumStreptococcusHaemophilus/AggrPorphyromonas

1.1 um

0.4 um

0.4 um

1.7 um

1.1 um

toothanoxic

CO2, lactate, acetate, H2O2

O2, saliva, sugars

Crevicular fluid

v

annulus perimeterbase

Corynebacterium Porphyromonas Fusobacterium otherStreptococcus Neisseriaceae LeptotrichiaHaemophilus/Aggr. Capnocytophaga Actinomyces

Map of Plaque Microbiome

Conclusions• Microbiome biogeography matters• Microbiome structures are like “organs”• It matters “who is next to who and to what”• Imaging strategy is generalizable • Applicable to any microbiome• The challenge now is to understand how the

microbiome “organs” work; and how they are formed and maintained.

J. mark welch

D. utter

S. wilbert

M. eren

J. schuhmann

G. borisy

F. dewhirst

A. kempchinsky

Microbiome Team