PFGE and Beyond: PulseNet in the Next Decade Bala Swaminathan, Ph.D. Centers for Disease Control and Prevention.

PFGE and Beyond: PFGE and Beyond: PulseNet in the Next PulseNet in the Next

DecadeDecade

Bala Swaminathan, Ph.D.Bala Swaminathan, Ph.D.

Centers for Disease Control Centers for Disease Control and Preventionand Prevention

Why Next Generation Why Next Generation Subtyping Methods?Subtyping Methods?

PFGE (and other RFLP-based methods) are PFGE (and other RFLP-based methods) are difficult to standardizedifficult to standardize

Comparability of patterns within and Comparability of patterns within and between laboratories requires strict between laboratories requires strict adherence to a standard protocoladherence to a standard protocol

Normalization of patterns is complexNormalization of patterns is complex PFGE is labor-intensive and requires high PFGE is labor-intensive and requires high

concentrations of a pure cultureconcentrations of a pure culture In some instances or for some pathogen In some instances or for some pathogen

groups, discrimination may not be adequategroups, discrimination may not be adequate

Clinical isolate clusters Clinical isolate clusters with no demonstrable with no demonstrable epidemiologic links – epidemiologic links –

Example 1Example 1

Clinical isolate clusters Clinical isolate clusters with no demonstrable with no demonstrable epidemiologic links – epidemiologic links –

Example 2Example 2

Requirements for the next Requirements for the next generation subtyping method generation subtyping method

for PulseNetfor PulseNet Broad applicabilityBroad applicability Rapid results (Rapid results (<< 24 h) 24 h) InexpensiveInexpensive Better discrimination than PFGEBetter discrimination than PFGE Quantitative relatedness between strainsQuantitative relatedness between strains Accurate snapshot of the genome diversityAccurate snapshot of the genome diversity Backward compatibility with PFGE dataBackward compatibility with PFGE data Easy to perform on a routine basis Easy to perform on a routine basis Amenable to automationAmenable to automation Results should be readily comparable within and Results should be readily comparable within and

between laboratoriesbetween laboratories

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

Methodologic Methodologic ApproachesApproaches

Multi-locus sequence typing (MLST)Multi-locus sequence typing (MLST) Multi-locus Variable-Number Multi-locus Variable-Number

Tandem Repeat Analysis (MLVA)Tandem Repeat Analysis (MLVA) High throughput SNP analysisHigh throughput SNP analysis

Based on the nucleotide sequence of internal Based on the nucleotide sequence of internal regions of housekeeping lociregions of housekeeping loci

Housekeeping loci should be conserved with only minimal Housekeeping loci should be conserved with only minimal nucleotide changes due to conserved protein functionnucleotide changes due to conserved protein function

Multiple loci are targeted in this subtyping methodMultiple loci are targeted in this subtyping method Sequence variation allows for the assignment of Sequence variation allows for the assignment of

allelesallelesIsolate AIsolate A – ATTCG– ATTCGGGCAT – CAT – allele 1allele 1

Isolate BIsolate B – ATTCG– ATTCGCCCAT – CAT – allele 2allele 2 A combination of alleles for all loci provides an allele A combination of alleles for all loci provides an allele

profile which can then be assigned to a sequence profile which can then be assigned to a sequence type (ST)type (ST)

Isolate A Isolate A (1, 5, 6, 3, (1, 5, 6, 3, 44, 3, 1), 3, 1) ST-5ST-5

Isolate BIsolate B (1, 5, 6, 3, (1, 5, 6, 3, 33, 3, 1), 3, 1) ST-51ST-51 Sequence types are grouped into clonal complexes Sequence types are grouped into clonal complexes

based on similarity to a central allelic profilebased on similarity to a central allelic profile

Multi-Locus Sequence TypingMulti-Locus Sequence Typing

Subtyping Subtyping Campylobacter jujuniCampylobacter jujuni

Three published MLST Three published MLST schemesschemes Dingle at al (2001)Dingle at al (2001)

194 isolates194 isolates 155 sequence types155 sequence types 51 unique ST’s51 unique ST’s

Suerbaum et al (2001)Suerbaum et al (2001) 32 isolates plus NCTC 32 isolates plus NCTC

1116811168 31 unique allele profiles31 unique allele profiles Frequent recombinationFrequent recombination

Manning et al (2003)Manning et al (2003)

Origin of replication

1,641,481 bp

asp

gln

glt

gly

pgm

tkt unc

ddlAasd

eftS

fumC

yphC

nuoH atpAMLST loci

Subtyping Subtyping Campylobacter jujuniCampylobacter jujuni

Sails et al (2003)Sails et al (2003) Comparison of MEE, MLST and PFGEComparison of MEE, MLST and PFGE

MLST is not as discriminatory as PFGEMLST is not as discriminatory as PFGE MLST plus a variable locusMLST plus a variable locus

MLST and MLST and flaAflaA SVR provides similar discrimination SVR provides similar discrimination to PFGEto PFGE

MLST studies with MLST studies with entericsenterics

ListeriaListeria monocytogenes: monocytogenes: Additional variable Additional variable gene targets need to be included in MLST gene targets need to be included in MLST (MLST+) to obtain acceptable (MLST+) to obtain acceptable discriminationdiscrimination Cai et al. 2002Cai et al. 2002 Zhang et al. 2004Zhang et al. 2004

Salmonella entericaSalmonella enterica (Kotetishvili et al, 2002) (Kotetishvili et al, 2002) MLST is more discriminatory than PFGEMLST is more discriminatory than PFGE

EscherichiaEscherichia colicoli (Whittam Laboratory) (Whittam Laboratory) Distinguish pathovars of Distinguish pathovars of E. coli/ShigellaE. coli/Shigella groups groups Distinguish clonal lineages within pathovarsDistinguish clonal lineages within pathovars

E. coliE. coli O157:H7 is too clonal for MLST O157:H7 is too clonal for MLST subtyping (Noller et al, 2003)subtyping (Noller et al, 2003)

Multilocus VNTR AnalysisMultilocus VNTR Analysis(MLVA)(MLVA)

MLVA (MLVA (MMulti ulti LLocus ocus VVNTR NTR AAnalysis)nalysis) VVariable ariable NNumber umber TTandem andem RRepeats (VNTRs)epeats (VNTRs)

Conserved repeat motif found in the genomeConserved repeat motif found in the genome Example: TAACCGExample: TAACCG

Variable numbers of repeat units among isolates of Variable numbers of repeat units among isolates of the same speciesthe same species

MLVA examines the number of repeats at MLVA examines the number of repeats at multiple loci to determine genetic relationships multiple loci to determine genetic relationships

TAACCG

TAACCGTAACCG

TAACCGTAACCGTAACCGTAACCGTAACCG

TAACCGTAACCGTAACCGTAACCG

Isolate A

Isolate B

Isolate C

Isolate D

Development of Development of E. coliE. coli O157 MLVA protocolO157 MLVA protocol

Contract awarded to the Contract awarded to the Massachusetts Department of Public Massachusetts Department of Public Health / State Laboratory Institute in Health / State Laboratory Institute in fall 2001fall 2001

Collaboration with Dr. Paul Keim Collaboration with Dr. Paul Keim (The Northern Arizona University)(The Northern Arizona University)

Development of Development of E. coliE. coli O157 MLVA protocol O157 MLVA protocol (cont’d)(cont’d)

Keys, C., S. Kemper, and P. Keim. 2005. Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing. J. Appl. Microbiol. 98: 928-940.

29 VNTR loci polymorphic in O157:[H7] serotype identified

Development of Development of E. coliE. coli O157 MLVA protocol O157 MLVA protocol (cont’d)(cont’d) MA protocol based on 25 VNTR lociMA protocol based on 25 VNTR loci

Amplified in four multiplex PCR reactionsAmplified in four multiplex PCR reactions Fluorescently labeled PCR amplicons sized Fluorescently labeled PCR amplicons sized

using capillary electrophoresis system using capillary electrophoresis system (CEQ 8000, Beckman Coulter, Fullerton, (CEQ 8000, Beckman Coulter, Fullerton, CA)CA)

Internal validation at the CDC PulseNet Internal validation at the CDC PulseNet Methods Development and Validation Methods Development and Validation Laboratory started in summer 2004Laboratory started in summer 2004

E.E. colicoli O157 strains used in O157 strains used in the initial validationthe initial validation

152 isolates analyzed by both MLVA 152 isolates analyzed by both MLVA and PFGE using and PFGE using XbaXbaII Geographically diverse sporadic isolates Geographically diverse sporadic isolates

with unique with unique XbaXbaI PFGEI PFGE patterns (UPP patterns (UPP collection) collection)

Outbreak isolates from eight well Outbreak isolates from eight well characterized outbreakscharacterized outbreaks

Epidemiologically unrelated isolates Epidemiologically unrelated isolates clustered by PFGEclustered by PFGE

A subset of 54 isolates were further A subset of 54 isolates were further characterized with characterized with BlnBlnII

Nine VNTR loci included in the final Nine VNTR loci included in the final

MLVA protocol for MLVA protocol for E. coliE. coli O157 O157

VNTRVNTR AlternatiAlternative nameve name11

Repeat Repeat size (bp)size (bp) No. of repeatsNo. of repeats

No. No. ofof

allelealleless

InsideInside

ORFORF

MinimuMinimumm

MaximuMaximumm

VNTR-3VNTR-3 Vhec3, Vhec3, TR5TR5 66 44 2323 2020 YesYes

VNTR-9VNTR-9 Vhec4, Vhec4, TR1TR1 66 55 2020 1717 NoNo


VNTR-17VNTR-17 TR3TR3 66 22 1818 1111 YesYes

VNTR-19VNTR-19 TR7TR7 66 44 1010 77 YesYes

VNTR-25VNTR-25 TR4TR4 66 11 2020 88 NoNo


VNTR-36VNTR-36 Vhec7Vhec7 77 33 1515 1414 NoNo

VNTR-37VNTR-37 66 33 1919 1414 YesYes

1 Vhec loci are form Lindstedt et al. (2003); TR loci are from Noller et al. (2003)

MLVA protocol stepsMLVA protocol steps

1.1. Boiled whole cell DNA templates prepared Boiled whole cell DNA templates prepared from overnight culturesfrom overnight cultures

2.2. Nine VNTR sites amplified in three PCR Nine VNTR sites amplified in three PCR reactionsreactions

3.3. Diluted (1:60) PCR products mixed with Diluted (1:60) PCR products mixed with sample loading solution and 600 bp DNA sample loading solution and 600 bp DNA size standardsize standard

4.4. PCR products sized using CEQ 8000 PCR products sized using CEQ 8000 capillary electrophoresis system (Beckman capillary electrophoresis system (Beckman Coulter)Coulter)

5.5. Fragment list exported to BioNumerics Fragment list exported to BioNumerics (Applied Maths, Kortijk, Belgium) for (Applied Maths, Kortijk, Belgium) for analysisanalysis

Discriminatory power of Discriminatory power of MLVA compared to PFGEMLVA compared to PFGE

152 isolates152 isolates 133 unique MLVA patterns133 unique MLVA patterns 126 unique 126 unique XbaXbaI PFGE patternsI PFGE patterns

A subset of 54 isolates were A subset of 54 isolates were characterized by PFGE using two characterized by PFGE using two enzymesenzymes

35 unique MLVA patterns35 unique MLVA patterns 39 unique 39 unique XbaXbaI-I-BlnBlnI PFGE patternsI PFGE patterns

VN

TR

_va

ls

MLVA_composite

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

EC

04P

N0655

G5289L

F5733

H6436

EC

04P

N0477

F6141

F6142

H6039

G5308

EC

04P

N0585

EC

04P

N0586

EC

04P

N0139

EC

04P

N0479

EC

04P

N0454

EC

04P

N0187

EC

05P

N0001

EC

04P

N0519

EC

04P

N0179

EC

04P

N0587

EC

04P

N0660

EC

04P

N0581

EC

04P

N0631

EC

04P

N0632

EC

04P

N0612

EC

04P

N0643

EC

04P

N0503

EC

04P

N0663

EC

04P

N0659

EC

05P

N0024

EC

05P

N0120

H2306

F7383

F7384

K0805

EC

04P

N0137

F7408

K0814

EC

04P

N0618

F7410

F7407

K0803

F8751

F8768

EC

04P

N0640

EC

04P

N0661

01-5

77

F7382

EC

04P

N0547

K0802

EC

04P

N0194

EC

04P

N0198

EC

04P

N0568

493-8

9

EC

04P

N0619

G5101

EC

04P

N0481

EC

04P

N0500

EC

04P

N0191

EC

04P

N0636

EC

04P

N0190

EC

04P

N0113

EC

04P

N0202

EC

05P

N0032

EC

04P

N0615

EC

04P

N0583

EC

04P

N0623

EC

04P

N0456

EC

04P

N0522

EC

04P

N0569

EC

04P

N0548

EC

04P

N0549

EC

04P

N0183

EC

04P

N0656

EC

04P

N0520

EC

04P

N0521

EC

04P

N0580

EC

04P

N0614

EC

04P

N0613

EC

04P

N0452

EC

04P

N0501

F7349

F7350

F7351

F7353

F7354

EC

04P

N0152

EC

04P

N0629

A8184

ED

L933

F6749

F6750

EC

04P

N0630

EC

04P

N0616

EC

05P

N0048

EC

04P

N0114

EC

04P

N0546

EC

04P

N0480

EC

04P

N0582

EC

04P

N0478

EC

04P

N0504

EC

04P

N0634

EC

04P

N0620

F6862

EC

04P

N0644

EC

04P

N0518

EC

04P

N0567

EC

04P

N0639

EC

04P

N0664

EC

04P

N0584

A7793

EC

04P

N0622

EC

04P

N0146

EC

04P

N0628

EC

04P

N0455

C9523

C9581

C9815

G5244

EC

04P

N0658

EC

04P

N0161

EC

04P

N0457

EC

04P

N0626

EC

04P

N0635

EC

04P

N0637

EC

04P

N0499

EC

04P

N0627

EC

04P

N0502

EC

04P

N0458

EC

04P

N0642

EC

04P

N0482

H0706

EC

04P

N0505

EC

04P

N0645

EC

04P

N0570

EC

04P

N0662

EC

04P

N0633

F6854

F6857

EC

04P

N0199

EC

04P

N0203

EC

05P

N0016

EC

04P

N0638

BA

A460

EC

04P

N0624

EC

04P

N0625

EC

04P

N0523

F6939

F6941

F6899

EC

04P

N0153

EC

04P

N0617

EC

04P

N0657

Clustering of 152 E. coli O157:[H7] isolates by MLVA

Cluster I Cluster II

Sakai EDL933

PFGE-BlnI+PFGE-XbaIxba-bln

100

95

90

85

80

75

70

65

xba-blnCDC__01-577F7407/#2DBS__CDC__F8751DBS__CDC__F8768DBS__CDC__F7383DBS__CDC__F7384CDC__K0814cdc__K0805/#1F7382F7408 Purecdc__EC04PN0548cdc__EC04PN0549F7410/#3cdc__EC04PN0585cdc__EC04PN0586cdc__G5308F6141F6899F6939F6941CDC__460-Wcdc__F6749cdc__EC04PN0582DBS_CDC__F6862F6750/#1DBS_CDC__F6854DBS_CDC__F6857DBS_CDC__F7349DBS_CDC__F7351DBS_CDC__F7353F7354/#1cdc__EC04PN0520cdc__EC04PN0521cdc__C9523cdc__C9581cdc__C9815CDC__G5289 lgDBS__CDC__MLVA095DBS_CDC__F7350CDC__EDL933CDC__EC04PN0631CDC__EC04PN0632CDC__493-89

VNTR_valsMLVA_composite

100

90

80

70

60

50

40

30

20

10

F6854F6857BAA460EC04PN0582C9523C9581C9815G5244F6862F6939F6941F6899EC04PN0520EC04PN0521EC04PN0548EC04PN0549F7349F7350F7351F7353F7354F6749F6750EDL933F8751F8768F740701-577F7382F7408K0814F7410F7383F7384K0805G5289LEC04PN0631EC04PN0632493-89EC04PN0585EC04PN0586G5308F6141

Clustering of 43 E. coli O157:[H7] isolates by MLVA and by PFGE using combined XbaI-BlnI

data MLVA II

MLVA Ib MLVA Ia

PFGE III

PFGE I PFGE II

VNTR_vals

MLVA_composite

100

80604020

F5733

H6436

G5308

F6141

H2306

01-577

F7382

F8751

F8768

F7383

F7384

C9523

C9581

C9815

G5244

A7793

F7349

F7350

F7351

F7353

F7354

F6749

F6750

A8184

EDL933

EXHX01.0224

EXHX01.0224

EXHX01.0224

EXHX01.0224

EXHX01.0224

EXHX01.0047

EXHX01.0047

EXHX01.1264

EXHX01.1264

EXHX01.0047

EXHX01.0047

EXHX01.0001

EXHX01.0001

EXHX01.0001

EXHX01.0001

EXHX01.0004

EXHX01.0011

EXHX01.0011

EXHX01.0011

EXHX01.0011

EXHX01.0011

EXHX01.1514

EXHX01.0283

EXHX01.0029

EXHX01.0028

EXHA26.0536

EXHA26.0536

EXHA26.0536

EXHA26.0536

EXHA26.0536

EXHA26.0015

EXHA26.0548

EXHA26.0015

EXHA26.0015

EXHA26.0250

EXHA26.0250

EXHA26.0001

EXHA26.0001

EXHA26.0001

EXHA26.0001

EXHA26.0585

EXHA26.0014

EXHA26.0536

EXHA26.0014

EXHA26.0014

EXHA26.0598

EXHA26.0014

EXHA26.0014

EXHA26.0715

EXHA26.0711

GA / Stool

GA / Stool

ME / Environmental

GA / Meat

CT / Stool

VA / Stool

NJ / Stool

CO / Stool

CO / Ground beef

NJ / Hamburger

NJ / Fatal case

WA / Sporadic

CA / Outbreak

AZ / Sporadic

WA / Sporadic

OR / Stool

WI / Stool

WI / Stool

WI / Taco meat

WI / Stool

WI / Stool

NY / Fatal case

NY / Sibling

MI / Stool

MI / Hamburger

1998

1998

1992

1998

1996

2001

2000

2002

2002

2000

2000

1993

1993

1993

1993

03-1982

2000

2000

2000

2000

2000

1999

1999

06-1982

05-1982

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.

GA water park outbreak

CT apple cider outbreak

CO outbreak

Western States outbreak

WI restaurant outbreak

NY County Fair

MI outbreak

NJ outbreak

Clustering of outbreak isolates and some selected sporadic isolates by MLVA

VNTR_vals

MLVA_composite

100

MLVA_composite

VN

TR

_val

s:V

NT

R_3

VN

TR

_val

s:V

NT

R_3

4

VN

TR

_val

s:V

NT

R_9

VN

TR

_val

s:V

NT

R_1

0

VN

TR

_val

s:V

NT

R_1

9

VN

TR

_val

s:V

NT

R_3

6

VN

TR

_val

s:V

NT

R_2

5

VN

TR

_val

s:V

NT

R_1

7

VN

TR

_val

s:V

NT

R_3

7

11.0 8.0 11.0 24.0 6.0 11.0 4.0 8.0 8.0

11.0 8.0 11.0 24.0 6.0 11.0 4.0 8.0 8.0

11.0 8.0 11.0 24.0 6.0 11.0 4.0 8.0 8.0

11.0 8.0 11.0 24.0 6.0 11.0 4.0 8.0 8.0

11.0 8.0 11.0 24.0 6.0 11.0 4.0 8.0 8.0

11.0 8.0 11.0 24.0 6.0 11.0 4.0 8.0 8.0

11.0 8.0 11.0 24.0 6.0 11.0 4.0 8.0 8.0

11.0 8.0 11.0 24.0 6.0 11.0 4.0 8.0 8.0

K1792

K1793

K1794

K1795

K1796

K1797

K1845

K1846

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Essex / NY

Madison / NY

VA

VA

OH

OH

IN (MI)

MI

10/1/2005

12/11/2005

11/2004

11/2004

01/2005

01/2005

12/2004

12/2004

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.

Clusters 0411ml-1c and 0501ml-1c – PFGE pattern combination EXHX01.0086/EXHA26.0576

Conclusions from the on-Conclusions from the on-going validation of the going validation of the E. coliE. coli

O157 MLVA protocolO157 MLVA protocol Overall, MLVA slightly less Overall, MLVA slightly less

discriminating than PFGE with two discriminating than PFGE with two enzymesenzymes

MLVA can further discriminate some MLVA can further discriminate some of the most common PFGE patternsof the most common PFGE patterns

Epidemiological congruence of the Epidemiological congruence of the MLVA data better than that of PFGEMLVA data better than that of PFGE

Development of interpretation Development of interpretation guidelines may pose a challengeguidelines may pose a challenge

Future plansFuture plans 2005: 2005:

Complete the CDC internal validation of Complete the CDC internal validation of the the E. coliE. coli O157 MLVA protocol O157 MLVA protocol Custom-made 1 kb standard for the locus Custom-made 1 kb standard for the locus

VNTR-10?VNTR-10? Reagent evaluationReagent evaluation Fine-tuning of the BioNumerics scriptsFine-tuning of the BioNumerics scripts

Begin collaborative validation of the Begin collaborative validation of the E. E. colicoli O157 MLVA protocol by transferring O157 MLVA protocol by transferring the protocol to four PulseNet laboratoriesthe protocol to four PulseNet laboratories

Future plans (cont’d)Future plans (cont’d)

20062006 Expand the implementation of the Expand the implementation of the

protocol to at least four more PulseNet protocol to at least four more PulseNet laboratorieslaboratories

Establish a national database with a Establish a national database with a pattern naming strategypattern naming strategy

Establish interpretation criteriaEstablish interpretation criteria

SNP-based Typing of E. coli O157

AAGGTTA

ATGGTTA

SNPs as genotyping markers

• Unambiguous data

• Easy to exchange/compare in database

• Good potential for automation

• Amenable to high-throughput platforms

• Useful for long-term epidemiology/population genetics

• Alternative for typing highly clonal species, serotypes

E. coli O157 genes are highly conserved

• Mosaic genome ~5.59Mb

• Genomic diversity by PFGE & MLVA

• >99.9% homology in orthologous genes

• MLST didn’t work well for typing O157 Noller et al: 7 housekeeping + 2 membrane protein genes 77 isolates, >18 PFGE types, 2 STs (1 SNP in ompA)

Foley et al: 7 virulence + 1 housekeeping genes 92 isolates, 72 PFGE types, 5 STs (2 SNPs in eaeA, 1 in hlyA, 10 in uidA)

In silico genome comparison

http://www.genome.wisc.edu/http://genome.gen-info.osaka-u.ac.jp/http://colibase.bham.ac.uk/http://snpsfinder.lanl.gov/

• Anchor Sakai query EDL933

• Most genes are 100% identical

• ~100 loci bearing SNPs (phageborne, sequencing errors, or paralogous…)

• Need a better strategy to identify novel SNPs

NimbleGen CGR microarray

Singh-Gasson et al. 1999. Nat. Biotechnol. 17:974-978Nuwaysir et al. 2002. Genome Res. 12:1749-1755

Mutation Mapping Resequencing

Selection of genes for CGR

Ohnishi et al. 2002. PNAS. 99:17043-17048

• Conserved among different E. coli O157 isolates

• Single-copy in the genome

• Re-sequencing capacity per slide ~1.2Mb (~1,200 genes)

• 376 O157-specific genes in 95 “size-conserved” S-loops (including many virulence factors)

• ~69 housekeeping genes with putative SNPs

• 754 additional backbone genes randomly-selected throughout the entire genome

• Large virulence plasmid (pO157)

O157 strains for resequencing

Strain Origin Year CharacteristicsPFGE

pattern

Sakai Japan 1996 stx1+, stx2+ 0373

F5733 Georgia 1998 stx1+, stx2+ 0224

G5289 Washington 1994 stx2+, Phage type 31 0238

01-577 Virginia 2001 stx2+, PFGE type 0047 0047

N0436 Colorado 2002 stx1+ 1315

N0303 New York 2001 stx1+, stx2+ 0264

N0587North Carolina

2001 stx2+ 0390

F6141 Georgia 1998 stx1+, stx2+ 0224

F8768 Colorado 2002 stx2+ 1264

G5101 Washington 1993stx1+, stx2+, Mug+, Urea+

2529

493/89 Germany 1989stx2+, Sorbitol+, O157:H-

2528

Total no. of SNPs in test strains = 836Strain

CharacteristicsPFGE

patternTotal no. of

SNPsStrain-specific

SNPs

Sakai stx1+, stx2+ 0373 - -

F5733

stx1+, stx2+ 0224 0 0

G5289

stx2+, Phage type 31 0238 9 1

01-577

stx2+, PFGE type 0047

0047 16 0

N0436

stx1+ 1315 30 4

N0303

stx1+, stx2+ 0264 45 6

N0587

stx2+ 0390 110 21

F6141

stx1+, stx2+ 0224 150 18

F8768

stx2+ 1264 164 25

G5101

stx1+, stx2+, Mug+, Urea+

2529 351 92

493/89

stx2+, Sorbitol+, O157:H-

2528 473 197

No. of unique SNPs common in G5101 & 493/89 =138* Average SNPs between any of two O157:H7 = 65* No. of informative SNPs to differentiate between any of two O157:H7 = 139

Polymorphic genes/regions:

• 836 SNPs in 503 genes, 65 gene >3 SNPs

• ECs1934: backbone, putative exonuclease VIII (RecE) prophage CP-933U 22 SNPs

• ECs1205: Shiga-toxin II subunit A (6 SNPs in 960-bp) ECs1206: Shiga-toxin II subunit B (0 SNPs in 270-bp) ECs2973-2974: Shiga-toxin I (1 SNP in subunit B)

Conserved genes/regions:• S-loops related to adhesion/invasion

• LEE (Locus of enterocyte enfacement) Type III secretion system

• Backbone regions, i.e. between S270-S276

Data analysis in progress:

• Backbone vs. S-loops

• Transition vs. transversion

• Synonymous vs. non-synonymous

• Insertions/deletions

• Phylogenetic analysis

ConclusionsConclusions PFGE will continue to be an essential PFGE will continue to be an essential

subtyping method for PulseNetsubtyping method for PulseNet MLVA may provide additional discrimination MLVA may provide additional discrimination

for for E. coliE. coli O157:[H7] and some O157:[H7] and some SalmonellaSalmonella serotypesserotypes

MLVA protocol for MLVA protocol for E. coliE. coli O157 :[H7] will be O157 :[H7] will be transferred to selected PulseNet laboratories transferred to selected PulseNet laboratories in 2005in 2005

SNP is the subtyping method of the future; SNP is the subtyping method of the future; SNP may be used in combination with MLVASNP may be used in combination with MLVA

Much work needs to be done on new Much work needs to be done on new subtyping methods for PulseNetsubtyping methods for PulseNet

PFGE and Beyond: PulseNet in the Next Decade Bala Swaminathan, Ph.D. Centers for Disease Control and Prevention.

Documents

mlst mlst

mlst subtyping noller

mlst schemesdingle

isolate b attcgccat

generation subtyping

sequence type stisolate

number of repeats

attcggcat allele