HOPE( s) for Qantitative Environm ental Microbiological Studies - - PDF document

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Division of Environmental Science & Engineering

HOPE( s) for Qantitative Environm ental Microbiological Studies

Wen-Tso Liu

AST seminar in Thailand, 11-12 Mar 08

Molecular Biological Tools (MBT)

Nucleic Acid based Whole cell based

• DNA/RNA

extraction

• DNA/RNA

quantification

• Quantitation
• q-PCR
• q-rt PCR
• Fingerprinting
• DGGE
• T-RFLP
• LH-PCR
• microarray
• Sequencing
• Clone library
• Metagenomics
• Cell fixation
• FISH
• NA-based
• immuno-assay
• MAR
• SIP
• Detection System
• Optical microscope
• RAMAN scope
• Flow Cytometry

sample large volume? yes Conc. no purification Intact cell detection nucleic acid/ protein/metab

• lite based

detection cell lysis Sample collection & concentration Sample preparation/ secondary concentration Detection/ analysis

Challenges

• types of targets
• QA/QC
• Reproducibility
• Reliability
• Ease of use
• Sensitivity/specificity
• lowest amount/conc.
• 1-bp
• Quantitation
• Resolution (phylogeny)
• Time
• Cost
• Safety
• lab use v.s. on-site

tools

• clone library v.s.

fingerprinting methods

Other “Omics”

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AST seminar in Thailand, 11-12 Mar 08

B B A A C C

Environmental sample Environmental sample

(1) DNA extraction

Mixture of microbial DNA Mixture of microbial DNA

(3) Restriction enzyme digestion

5’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 3’

• 5’

3’ 5’

1200 bp 300 bp 1100 bp 400 bp 600 bp 800 bp 100 bp

Cut at specific site Cut at specific site

C B A 600 1100 1200 Fluorescent intensity (RFU)

(4) Separation fragments by electrophoresis Fragments on gel after Fragments on gel after electrophoresis electrophoresis (5) Detection of fluorescent fragments Typical Typical electropherograms electropherograms of

• f

T T-

• RFLP fingerprint

RFLP fingerprint

1200 bp 1100 bp 800 bp 600 bp 400 bp 300 bp 100 bp

5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’

Fluorescent Fluorescent PCR products PCR products

• f a specific gene
• f a specific gene

(2) PCR with a fluorescently labeled forward primer

Image analysis Image analysis Cluster analysis Cluster analysis Principal Principal-

• component analysis

component analysis Web Web-

• based database analysis

based database analysis http://rdp.cme.edu http://rdp.cme.edu http://hermes.campus.uidaho.edu http://hermes.campus.uidaho.edu http://trflp.limnology.wisc.edu http://trflp.limnology.wisc.edu http://www.oardc.ohio http://www.oardc.ohio-

• state.edu/trflpfragsort

state.edu/trflpfragsort/ /

(6) Data analysis

Outline of Outline of T T-

• RFLP

RFLP

( Liu et al., 1 9 9 7 ) ( Liu et al., 1 9 9 7 )

AST seminar in Thailand, 11-12 Mar 08

Novel quantitative approach

• Easy
• Fast
• Multiplexing

Constraints of current molecular quantitative techniques

The difficulty to conduct multiplexing analysis is the most severe bottleneck for launching large- scale investigation of microbial abundances.

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Hierarchical Oligonucleotide Primer Extension (HOPE) (Wu and Liu, NAR, 2007) Acknowledgement

• Wu Jer-Horng
• Hong Pei-Ying
• NUS funding

Outline

• concept
• hierarchical primer design
• specificity
• sensitivity
• multiplexing

AST seminar in Thailand, 11-12 Mar 08

Sequence specific fluorescence tag labeling by incorporating dye-terminators to 3’end of primers Clean up dye-terminator-tagged products PCR amplicons (e.g., 16S rRNA gene) Purification

Dye-Terminators (Fluorophore-labeled ddNTPs) ddATP ddTTP ddCTP ddGTP C T G A Primer: 5’ --- 3’ DNA Polymerase

Pol

P1 P2 P3

Measure labeled primers by gel-based DNA sequencer

A C T G

Pol

C

3’

C G T A T A

Pol

3’

G C C

Pol Pol

5’ Target a 5’ Target c

A

3’

C G G C

Pol Pol5’

Target b

G T C

Pol

Intensit y Size Internal standards P1* P2* P3*

3’

C G A T

3’

C G G C

3’

C

5’ Target b 5’ Target a 5’ Target c

30~45 min 30-70 min 40 min

Total time required: 2~3 hr

10 min

C G

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AST seminar in Thailand, 11-12 Mar 08

A C T G G C C 3’ G 5’ Target

Dye-Terminators (Fluorophore-labeled ddNTPs) ddATP ddTTP ddCTP ddGTP C T G A Oligonucleotide Probe DNA Polymerase

Hierarchical Oligonucleotide Primer Extension

• mini-sequencing or single base extension

C

1st cycle

C

2nd cycle

C

n cycle

Intensity Size Internal standards Capillary Electrophoresis-Laser Induced Fluorescence (CE-LIF) Detection

AST seminar in Thailand, 11-12 Mar 08

P1 P2 P3

Hierarchical Oligonucleotide Primer Extension (HOPE)

Dye-Terminators (Fluorophore-labeled ddNTPs) ddATP ddTTP ddCTP ddGTP C T G A Oligonucleotide Probe DNA Polymerase

A C T G G C T A A G T C G C T C C 3’ G A 3’ C 5’ Target a 5’ Target c 3’ G C 5’ Target b G

polyTs Intensity Size Internal standards P1 P2 P3

Capillary Electrophoresis-Laser Induced Fluorescence (CE-LIF) Detection

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Principle of HOPE

C, final concentration of the extended primer, Co, the initial concentration of target template E, the extension efficiency, a constant n, the number of thermal cycles

Cp2 = Co, p2×E2×n Cp3 = Co, p3×E3×n E1, E2, E3, constant, but E1 ≠ E2 ≠ E3 E2 / E1 = a E3 / E1 = b Cp1 = Co, p1×E1×n Cp1=aCp2=bCp3

Calibrated

AST seminar in Thailand, 11-12 Mar 08

1 0.5 1.5 1 0.5 1.5

P1 P2 P3 Template

Hierarchical probes

Ideal Cp1=Cp2=Cp3 Actual Cp1=Cp2=Cp3

Factors to synchronize

Cp1=aCp2=bCp3 Calibrated

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AST seminar in Thailand, 11-12 Mar 08

Escherichia coli Bacteroides fragilis Bacteroides vulgatus Bacteroides distasonis Bifidobacterium Collinsella Eubacterium Low GC EUB338 (79.5% for C*, 9.9% for T*) EUB338Ia (91.6% for T*) BAC303 (C*) Bacteroides thetaiotaomicron BTH584 (C*) BTH274 (C*) (T*)

Primer Design: Fecal Bacteroides as a model system

• obligate anaerobes
• abundant in the faeces of warm-

blooded animals

• predict recent fecal contamination
• distinguish fecal pollution sources

from human and livestock. Indicator for fecal pollution

AST seminar in Thailand, 11-12 Mar 08

10000 20000 30000 40000 50000 60000

10 20 30 40 50 60 70 80 90

EUB338Ia BAC303_5a BTH274_15a BTH584_16a

Size (nt) Fluorescence intensity (rfu)

Probe name Specificity Binding length Poly dA length Total length EUB338Ia Bacteria domain 17 17 BAC303 Bacteroide-Prevotella group 17 5 22 BTH274

• B. thetaiotaomicron
• B. fragilis

17 15 32 BTH584

• B. thetaiotaomicron

20 16 36

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AST seminar in Thailand, 11-12 Mar 08

Probe-to-target molar ratio

100 1000 10000

Conversion factor (v.s EUB338Ia)

2 4 6 8

BAC303-5a BTH274-15a BTH584-16a 20000

Conversion factor = constant?

(Fixed probe concentration but varied template concentrations)

AST seminar in Thailand, 11-12 Mar 08

Specificity

• Target vs. non-target

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AST seminar in Thailand, 11-12 Mar 08

Length BTH584 Species 5-CAACTGACTTAACTGTCCAC C-3

• B. thetaiotaomicron

3-GTTGACTGAATTGACAGGTG G-5

• B. fragilis

3-===========G=T====== G-5

• B. vulgatus

3-============TGT====A G-5

• B. distasonis

3-=GC=========TT=C==C= G-5 BTH274 Species 5-CCCCTATCCATCGAAGG T-3

• B. thetaiotaomicron

3-GGGGATAGGTAGCTTCC A-5

• B. fragilis

3-================= G-5

• B. vulgatus

3-================T G-5

• B. distasonis

3-=============AG== A-5 EUB338 Species 5-GCTGCCTCCCGTAGGAGT T-3

• B. thetaiotaomicron

3-CGACGGAGGGCATCCTCA A-5

• B. fragilis

3-================== A-5

• B. vulgatus

3-================== A-5

• B. distasonis

3-================== A-5 BAC303 Species 5-CCAATGTGGGGGACCTT C-3

• B. thetaiotaomicron

3-GGTTACACCCCCTGGAA G-5

• B. fragilis

3-================= G-5

• B. vulgatus

3-================= G-5

• B. distasonis

3-================= G-5 18 1 19

Primer A tail Allele Total

17 5 1 23

Primer A tail Allele Total

17 15 1 33

Primer A tail Allele Total

20 16 1 37

Primer A tail Allele Total 10000 20000 30000 40000 50000 60000 10 20 30 40 50 60 70 80 90

EUB BAC BTH274 BTH584 Size (nt)

• B. thetaiotamicron

Eub338 (T) BAC303 (C) BTH274 (T) BTH584 (C)

2500 5000 7500 10000 12500 15000

EUB BAC BTH274

• B. fraglis

Eub338 (T) BAC303 (C) BTH274 (C) BTH584

• B. vulgatus

Eub338 (T) BAC303 (C) BTH274 BTH584

2500 5000 7500 10000 12500

EUB BAC 0 10 20 30 40 50 60 70 80 90

5000 10000 15000 20000 25000 30000 35000 40000

10 20 30 40 50 60 70 80 90 EUB BAC

• B. distasonis

Eub338 (T) BAC303 (C) BTH274 BTH584

BTH584

AST seminar in Thailand, 11-12 Mar 08

MM2 Allele Size ddNTP MM2 Allele Size3 ddNTP3 MM Allele Size ddNTP MM Allele Size ddNTP 1 B thetaiotaomicron BCRC10624 A 17.6 T G 19.9 C A 27.0 T G 31.6 C 2

• B. fragilis

BCRC10619 A 17.5 T G 20.0 C G 27.3 C 2 ND ND 3 B. distasonis JCM5825 A 17.7 T G 20.2 C 2 ND ND 6 ND ND 4

• B. vulgatus

BCRC12903 A 17.5 T G 19.9 C 7 ND ND 4 ND ND 5 R. albus DSMZ20455 G 16.8 C 6 ND ND 7 ND ND 8 ND ND 6

• C. aerofaciens

JCM10188 G 16.9 C 6 ND ND 7 ND ND 10 ND ND 7 L. acidophilus DSMZ20079 A 17.5 T 5 ND ND 7 ND ND 7 ND ND 8

• B. adolescentis

BCRC14606 G 16.9 C 6 ND ND 5 ND ND 9 ND ND 9 P. productus DSM2950 G 16.9 C 6 ND ND 8 ND ND 9 ND ND 10 C. leptum BCRC14522 G 16.9 C 6 ND ND 7 ND ND 10 ND ND 11 R. bromii ATCC27255 G 17.0 C 6 ND ND 7 ND ND 8 ND ND 12 B. longum BCRC11847 G 17.0 C 6 ND ND 5 ND ND 9 ND ND 13 E. faecium BCRC10067 A 17.5 T 5 ND ND 6 ND ND 7 ND ND 14 C. clostridiiforme BCRC14545 A 17.5 T 6 ND ND 6 ND ND 8 ND ND 15 B. longum DSMZ20088 G 17.1 C 6 ND ND 5 ND ND 9 ND ND 16 R. obeum ATCC29174 G 17.0 C 6 ND ND 7 ND ND 6 ND ND 17 E. biforme DSMZ3989 A 17.5 T 6 ND ND 5 ND ND 9 ND ND 18 F. prausnitzii ATCC27768 G 17.0 C 5 ND ND 6 ND ND 6 ND ND 19 R. callidus ATCC27760 G 16.8 C 5 ND ND 8 ND ND 7 ND ND 20 E. coli NCIMB10083 G 16.4 C 6 ND ND 8 ND ND 5 ND ND 17.0 C/T 20.0 C 27.2 C/T 32.0 C 0.30 0.14 0.21

• #

Species Source1 Eub338 (18nt) BAC303-5a(22nt) BTH274-15a(32nt) BTH584-16a(36nt) Standard deviation: Averaged size (nt) and Terminators:

Table 1 Muliplexing single terminator extension analysis of twenty fecal bacterial strains with one set of hierarchical oligonucleotide probes.

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AST seminar in Thailand, 11-12 Mar 08

Multiplexing

• 4 Plexing
• 6 Plexing
• 7 Plexing
• Multiple tube approach

AST seminar in Thailand, 11-12 Mar 08

Multiplexing – theoretical

n (length type) x m (fluorescence type)

ATGCGGGCGC TTGCATGCCG TCCATGCGGG AGTGCGCGCG ATGCGGGCGCGCC TTGCATGCCGTTA TCCATGCGGGTGG AGTGCGCGCGAAT ATGCGGGCGATCTT TTGCATGCCGGGCG TCCATGCGGGAATT AGTGCGCGCGCCAT A T G C A T G C A T G C 11 14 17

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AST seminar in Thailand, 11-12 Mar 08

Escherichia coli Bacteroides fragilis Bacteroides vulgatus Bacteroides distasonis Bifidobacterium Collinsella Eubacterium Low GC EUB338 (79.5% for C*, 9.9% for T*) EUB338Ia (91.6% for T*) BAC303 (C*) Bacteroides thetaiotaomicron BTH584 (C*) BTH274 (C*) (T*)

Primer Design: Fecal Bacteroides as a model system

• obligate anaerobes
• abundant in the faeces of warm-

blooded animals

• predict recent fecal contamination
• distinguish fecal pollution sources

from human and livestock. Indicator for fecal pollution

AST seminar in Thailand, 11-12 Mar 08

Model microbial communities.

15.3 ± 1.2 21.8 17.4 ± 0.4 21.8 53.1 ± 2.9 46.2 AC2 + M. barkeri (12.8 fmol/µl) 3.4 ± 1.0 5.2 2.8 ± 0.3 5.2 9.7 ± 0.8 11.1 AC2 + E. coli (15.8 fmol/µl) 14.9 ± 1.0 21.5 14.4 ± 0.9 21.5 14.8 ± 1.0 21.5

3AC3

18.1 ± 1.4 21.8 17.6 ± 0.7 21.8 49.2 ± 3.6 46.2

2AC2

18.5 ± 1.2 22.1 18.4 ± 0.7 22.1 77.0 ± 3.9 72.7

1AC1

Observed Theoretical Observed Theoretical Observed Theoretical BTH584-16a (for B. thetaiotaomicron) BTH274-15a (for B. thetaiotaomicron) BAC303-5a (for Bacteroides) % of the probes for specific groups with respect to probe EUB338Ia for domain Bacteria (mean ± sd, n=6) Artificial communities

1AC1 contained 16S rRNA gene amplicons of B. thetaiotaomicron (2.2 fmol/µl), B. distasonis (2.5 fmol/µl), B. vulgatus (2.5 fmol/µl) and L.

acidophilus (2.7 fmol/µl).

2AC2 contained 16S rRNA gene amplicons of B. thetaiotaomicron (2.2 fmol/µl), B. distasonis (2.5 fmol/µl), L. acidophilus (2.7 fmol/µl) and E.

faecium (2.7 fmol/µl).

3AC3 contained 16S rRNA gene amplicons of B. thetaiotaomicron (2.2 fmol/µl), L. acidophilus (2.7 fmol/µl) and E. faecium (2.7 fmol/µl), P.

productus (2.7 fmol/µl).

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AST seminar in Thailand, 11-12 Mar 08

• BTT1250(T*)

Bacteroides tectus (tRF 496) Bacteroides pyogenes (tRF 496) Bacteroides suis (tRF 496) Bacteroides denticanum (tRF 494) Bacteroides nordii (tRF 161) Bacteroides salyersiae (tRF 59) Bacteroides thataiotaomicron (tRF 167) Bacteroides finegoldii (tRF 495) Bacteroides finegoldii (tRF 495) Bacteroides ovatus (tRF 495) Bacteroides fragilis (tRF 165) Bacteroides caccae (tRF 166) Bacteroides acidifaciens (tRF 495) Bacteroides acidifaciens (tRF 167) Bacteroides intestinalis (tRF 489) Bacteroides intestinalis (tRF 489) Bacteroides helcogenes Bacteroides uniformis (tRF 165/493) Bacteroides gallinarum (tRF 165) Bacteroides gallinarum (tRF 165) Bacteroides stercoris (tRF 165) Bacteroides eggerthii (tRF 165) Bacteroides eggerthii (tRF 165) Bacteroides vulgatus (tRF 165) Bacteroides vulgatus (tRF 165) Bacteroides dorei (tRF 161) Bacteroides dorei (tRF 161) Bacteroides massiliensis (tRF 494) Bacteroides massiliensis (tRF 494) Bacteroides coprocola (tRF 494) Bacteroides plebeius (tRF 494) Bacteroides barnesiae (tRF 107) Parabacteroides distasonis (tRF 141) Parabacteroides merdae (tRF 165) Parabacteroides merdae (tRF 165) Parabacteroides johnsonii (tRF 165) Bacteroides merdae (tRF 165) Parabacteroides goldsteinii Bacteroides splanchnicus (tRF 489) 0.10 ○● EUB338Ia(T*) ○● BAC303(C*)

• ■ BFRG602(C*)

○●■ BTH274(T*) ■BUFM1018(T*) ■BFG1024(C*) ○BTH584(C*) ■BADF1037(G*) ■BITT141(T*) 4-plex ○ 6-plex ● 7-plex ■ ○●■BTH274(C*) ○●■BTH274(C*) AST seminar in Thailand, 11-12 Mar 08

(a)

BTH274-15a EUB338Ia-23a BFRG602-19a C* T* G* 500 1000 1500 2000 2500 3000 3500 4000 4500

10 20 30

50 60 70 80 90

Size (nt) Dye intensity (rfu)

BTT1250 BAC303-5a BTH274-15a

40

• 6plex tube

(b)

2500 5000 7500 10000 12500 15000 17500 10 20 30 50 BITT141 BFG1024 BADF1037-9a BTH274-15a BFRG602-19a BUFM1018-18a 40 Dye intensity (rfu) ■ 7plex tube Size (nt)

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Quantifying relative abundance of specific targets in the influent and effluent from sewage treatment plant.

ND ND ND ND

• B. acidifaciens

ND ND ND ND

• B. intestinalis

ND ND 21.6±1.1 19.7±1.0

• B. uniformis

ND ND 5.1±1.2 4.9±0.7 BFRG602- related group

• B. fragilis

ND ND ND ND BTT1250-related group ND ND 0.3±0.3 0.5±0.1 BTH274t-related group2 ND ND 0.8±0.2 1.1±0.1 BTH274c-related group1 0.1±0.1 ND 3.6±0.5 4.5±0.4 BFRG602-related group 1.1±0.9 1.6±0.1 10.0±0.3 11.1±1.4 Bacteria (EUB338Ia-23a) Bacteroidales Sample 2 (n=3) Sample 1 (n=3) Sample 2 (n=3) Sample 1 (n=3) group Target Effluent (%) Influent (%) Target abundance within a group

1 The group detected by primer extension with ddCTP of BTH274-15a. 2 The group detected by primer extension with ddTTP of BTH274-15a.

Calibration factors obtained for the 6-plexing, EUB338Ia-23a: BAC303-5a: BFRG602-19a: BTH274-15a(C): BTH274-15a(T): BTT1250=1: 6.1: 11.2: 14.4: 3.1: 2.4, and 7-plexing, BFRG602-19a: BUFM1018-18a: BTH274-15a(C): BTH274-15a(T): BFG1024: BITT141= 1: 0.8: 1.2: 0.4: 1.7: 0.1. Bacterial strains used include B. thetaiotaomicron, B. fragilis, B. acidifaciens (JCM10556), B. intestinalis (JCM13266), B. uniformis (JCM5828), Bacteroides. tectus (JCM10003), and Bacteroides pyogenes (JCM6294). ND, not detected.

AST seminar in Thailand, 11-12 Mar 08

Future (of) HOPE(s)

Targets

• HOPE for RNA
• HOPE for genomic DNA

Applications

• HOPE for Bacteroides spp. (gut flora study)
• HOPE for microbial source tracking
• HOPE for quantification of nitrifying populations
• many others more…

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AST seminar in Thailand, 11-12 Mar 08

Gut flora

∆ Reaction one:

Bth274 [T]_15dA Pg_dtc732_20dA Bsls1016_10dA Bnd136 Bth584_16dA

■ Reaction two:

Bfrg602_19dA Bth274_15dA Bfrg1026_20dA O Reaction three: Bfrg602_19dA Bcc1066_15dA Badf1009 Bitt141

• Reaction four:

Bfrg602_19dA Bufm1018_20dA Begt999 Bhcg171_8dA

Reaction five:

Bfrg602_19dA Bvg1016 Bmsl1000_19dA Bcpc1015_24dA

# Reaction six:

U1390_17dA Bac303_5dA Bfrg602_19dA Bdts_ gp980 Bth274 [C]_15dA Bth274 [T]_15dA Bovine-specific uncultivated Bacteroidales Swine-specific uncultivated Bacteroidales Canines/Felines-specific uncultivated Bacteroidales 0.02

∆ Pg_dtc732 ∆ Bsls1016 ∆ Bnd136 ∆ Bth584

■ Bfrg1026

O Bcc1066 O Badf1009 O Bitt141

• Bufm1018
• Begt999
• Bhcg171

Bvg1016 Bmsl1000 Bcpc1015

∆, # Bth274 [T]

■,O, ●, , # Bfrg602 # Bac303 # U1390 # Bdts_gp980 Bacteroides tectus Bacteroides pyogenes Bacteroides salyersiae Bacteroides nordii

• B. thetaiotaomicron

Bacteroides fragilis Bacteroides caccae Bacteroides acidifaciens Bacteroides intestinalis Bacteroides uniformis Bacteroides eggerthii Bacteroides helcogenes Bacteroides vulgatus Bacteroides massiliensis Parabacteroides goldsteinii Parabacteroides distasonis Parabacteroides merdae Bacteroides coprocola

Other functionally important bacterial targets? Bifidobacterium spp. Clostridium leptum subgroup Butyrate producing Firmicutes Bacteroides and Parabacteroides spp.

AST seminar in Thailand, 11-12 Mar 08

H1 H2 H3 P1, P2, P3 Swine WW C1, C2, C3 Municipa l WW Stress: 0

Human Pig Cow Legend: 10 20 30 40 50 60

Others

• B. fragilis
• B. caccae
• B. acidifaciens
• B. intestinalis
• B. uniformis
• B. eggerthii
• B. helcogenes
• B. vulgatus
• B. massiliensis
• B. coprocola

Relative abundance (%)

• B. fragilis cluster

Hong et al. AEM, 2008 (in press)

• 10

10 20 30 40 50 Municipal WW Swine WW Pig Cow Human

Others

• B. fragilis cluster
• P. distasonis cluster
• B. fragilis subcluster [extends with ddCTP]
• B. fragilis subcluster [extends wtih ddTTP]

Relative abundance (%)

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AST seminar in Thailand, 11-12 Mar 08

Fecal source tracking

Fecal contamination in drinking water sources and recreational bodies

○Adverse health effects and liability issues

Conventional monitoring and detection

○24 - 48h FC test ○Cultivation bias ○Not able to pinpoint the fecal

• rigin

AST seminar in Thailand, 11-12 Mar 08

Human? Cow? Pig? Dog? Host-specific primers Predominantly in human feces Bacteroides caccae (Bcc1066) Bacteroides fragilis (Bfrg1024) Bacteroides vulgatus (Bvg1016) Bacteroides uniformis (Bufm1018) Bovine-specific Uncultivated bovine-specific Bacteroidales (C367) Swine-specific Uncultivated swine-specific Bacteroidales (P163) Canine-specific Uncultivated canine-specific Bacteroidales (D475) 1. Presence and absence of extended host-specific primer (library-independent) 2. Relative abundance of each microbial target (library-dependent)

HOPE for microbial source tracking

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AST seminar in Thailand, 11-12 Mar 08

Cow Pig Human Dog

C2C3 C4 C6 C7 C8 C9 C10 C11 C12 C13 C15 C16 C17 C18 C19 P1 P2 P3 P4 P5 P9 P10 P11 P12 P13 P14 P16 P17 P18 P19 H4 H8 H10 H11 H12 H13 H15 H16 H17 H18 H19 H22 H23 H24 H25 H15N D1 D2 D4 D5 D6 D7 D8 D9 D10 D12 D13 D14 D18 c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 p1 p2 p3 p4 p5 p6 p7 p8 h1 h2 h3 h4 h5 h6 h7 h8 d1 d2 d3 d4 d5d6 d7 d8

Stress: 0.15

By establishing a HOPE-based library obtained from feces of known origin, a new sample contaminated with unknown sources can potentially be identified based on its location on this plot. Hong et al. (unpublished)

Decide based on which type of host- specific primer extended

AST seminar in Thailand, 11-12 Mar 08

Thank You!

More HOPEs…