the phylochip: a complete microbial community approach to ... · field test: tracking a 764,000...
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The PhyloChip:A complete microbial community
approach to water quality research
Eric Dubinsky, John Hulls*Terry Hazen, Gary Andersen
Lawrence Berkeley National Laboratory, *Marin County
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Immense microbial diversity1,000+ species in human, animal guts
5,000+ species per liter seawater, gram soil
Most bacteria (~99%) resist culturing
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Our goal: Exploit this diversity for definitive waterquality diagnosis and source tracking
Immense microbial diversity1,000+ species in human, animal guts
5,000+ species per liter seawater, gram soil
Most bacteria (~99%) resist culturing
We can now characterize microbial diversity quicklyand cheaply with new molecular methods
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Microbial community analysis: limited
by our (lack of) power to observe?
Low resolution(RFLP, DGGE, PLFA)
Restricted detection,must grow in captivity
Cost/labor prohibitive(DNA sequencing)
Restricted detection(Quantitative PCR)
Culture methods Culture-independent methods
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PhyloChip analysis of microbialcommunities
Detects 50,000 different bacteria and archaea in a single test
Comprehensive census of whole microbial community
Rapid, repeatable and standardized method
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PhyloChipcomprehensive microbial census
High-density oligonucleotide gene microarray
1.1 million DNA probes for most known bacteria andarchaea
Based on entire 16S rRNA gene database(greengenes.lbl.gov)
Analysis of entire pool of community DNA allows detectionof very low abundance taxa, microbial community dynamics
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Archaea
Firmicutes
Bacteroidetes
Proteobacteria
Spirochaetes
Planctomycetes
Verrucomicrobia
Cyanobacteria
Fusobacteria
Acidobacteria
ChloroflexiActinobacteria
Nitrospirae
TM7
OP11
Synergistetes
Aquificae
PhyloChip detects 50,443 bacteria and archaea
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Archaea
Firmicutes
Bacteroidetes
Proteobacteria
Spirochaetes
Planctomycetes
Verrucomicrobia
Cyanobacteria
Fusobacteria
Acidobacteria
ChloroflexiActinobacteria
Nitrospirae
TM7
OP11
Synergistetes
Aquificae
PhyloChip detects 50,443 bacteria and archaea
353 Cyanobacteria184 diatoms/dinoflagellates
2,105 Coliforms
2,272 Bacteroidales
12,098 Clostridia
146 Enterococci
643 Archaea
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Sample preparation
Extract DNA/RNAfrom filtered sample
Amplify 16S rRNAgene with PCR
AnalyzecompositionamplifiedDNA/RNA
Profile of entirecommunity
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PhyloChip analysisPCR amplification of community 16S rRNA genes Fragment and biotin label
Hybridize to arrayWash, stain and scan
www.affymetrix.com
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Generation 3 PhyloChip
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Reveals occurrence and relative abundance 16S rDNAfor 50,000 different bacteria and archaea
Location and intensity offluorescence determines test results
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Clean Beaches Initiative
Goals
Better understand the fate of pollutantmicrobial communities in coastal waters
Identify key indicators of differentsources of fecal contamination
Create more accurate, sensitive tests forroutine testing and source tracking
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PhyloChip water quality projects
Fate of pollutant microbes: What happens to pollutantcommunities once they enter receiving waters?
Source characterization: Which bacteria and archaeadistinguish human and animal fecal sources?
Variation in time and space: How do background microbialcommunities respond to changes in the coastalenvironment?
Epidemiology: Which organisms are associated with humanillness?
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Field test:Tracking a 764,000gallon sewage spillin Richardson Bay
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Spill map
764,000 gallon sewage spill*
*
*
**
*
*
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spill origin
*500 m
Sampling sites
Richardson Bay
February 2009 spill at
Sausalito treatment plant
Sampled 24, 48 and 72 hours
after beginning of the spill
Smaller spill 10 days later
8 onshore/offshore locations
DNA analysis with PhyloChip,
qPCR
Fecal indicator tests
(Enterococci, E. coli, total
coliform)
11-day diffusion chamber
experiment
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PhyloChip ResultsCommunity analysis of 24,748 detected taxa
Sewagesource
Bay samplesHigh FIBBay samples
Low FIB
Nonmetric Multidimensional Scaling ordination
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PhyloChip Results
Microbial community composition strongly
correlated with fecal indicator tests
Enterococcus E. coli Total coliform
Community composition (Axis 1 score)
Popula
tion s
ize
(log [M
PN
/100 m
l])
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Potential molecular indicatorsClostridia
Abundances of 4,625 different taxa found in sewage were strongly
correlated (r>0.9) with fecal indicators
Most correlated taxa were Bacteroidales and Clostridia
PhyloChip Results
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Polymicrobial Source Tracking
Water samples with high fecal indicators fall along a vector toward the
known source community
Illustrates the power of community analysis with microarray to
identify the cause of exceedences when the source is unknown
Ordination (NMS) of community data
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Diffusion chamber experiments
A "cage " for microorganisms: holds captive anything greaterthan 0.2 μm in size. Permeable to water, molecules in solution
Monitor abundance and activity of all bacteria and archaea overtime upon immersion into receiving waters
What is the fate of microbial communities from different fecalwastes in fresh and marine waters?
Photo credits: Janet Fang
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Experiment: fate of cattle waste and septage infresh and marine waters
x 3
x 3
Freshwater(Walker Creek)
Saltwater(Tomales Bay)
x 3
x 3
Sampled at 6 time points: 0, 1, 12, 24, 48, 96hours after immersion
Vacuum filtration through 0.2 μm filtersFrozen on dry ice for DNA/RNA extractionUnfiltered sample for direct counts
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Comparison of diverse communities
Bay Creek
Septage CattleReceiving
waters
Cluster analysis
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Comparison of diverse communities
0-24 hours 96 hours 0-24 hours 96 hoursBay Creek
Septage CattleReceiving
waters
Cluster analysis
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Comparison of diverse communities
0-24 hours 96 hours 0-24 hours 96 hours
BayCreekCreek Bay
Bay Creek
Septage CattleReceiving
waters
Cluster analysis
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Tree Bacteria andarchaea uniqueto each wasteand absent fromreceiving waters
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Effect of time in receivingwaters on waste microbialcommunities
Four day immersion
Distinct differences insurvivability amongtaxonomic groups
Most proteobacteria decreasein relative abundance in bothwastes
Clostridia persist
Some bacilli (Enterococcus)and coliforms in septagepersist
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Composition of fecalbacteria that persist increek versus bay water
Water type has a effects whichfecal bacteria persist over time
Similar response of cattle andseptage communities
Clostridia, -proteobacteria,coliforms favored in creek
-proteobacteria favored inBay
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Ongoing work: characterization of microbialcommunities in potential fecal sources
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Sites
Campbell Cove
Dillon Beach
Miller Park
Green Bridge
Inkwells
Muir Beach
Horseshoe Cove
Baker Beach
Lawsons Landing
*
**
*
**
*
**
San FranciscoTom
ales Bay
Bodega Bay
San Pablo Bay
Drakes Bay
Marin
Sonoma
10 km
AB411 weekly monitoringMolecular analysis: PhyloChips,
qPCRCulturable fecal indicatorsPhysical and chemical properties
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Summary
PhyloChip detects most known bacteria and archaea in asingle test
Enables comprehensive, high resolution surveys ofmicrobial communities
Opportunity to gather much needed information aboutmicrobial communities in pollutant sources and receivingwaters
Potential to identify new sets of indicator taxa to bettertrack coastal pollution
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Thank You
Shariff Osman, Cindy Wu, Yvette Piceno, Bonita Lam,Todd DeSantis
Omar Arias and the Sausalito-Marin City Sanitary District
Counties of Marin, Sonoma and San Francisco
California State Water Resources Board: Clean BeachesInitiative
Rathmann Family Foundation