Workshop on Methods for Isolation and Identification of Campylobacter spp.

June 13-17, 2005

Goal: build capacity within the state public health laboratories to effectively identify Campylobacter species and detect outbreaks by:Providing hands-on experience with laboratory procedures for the isolation and characterization of the Campylobacter species commonly isolated from clinical specimens

Pre / Post assessment results

Participant Pre-Test Post-test % increase in score1 57% 82% 25%2 43% 57% 14%3 51% 80% 29%4 44% 69% 25%5 38% 75% 37%6 35% 65% 30%7 37% 59% 22%8 48% 78% 30%9 47% 72% 25%10 53% 87% 34%11 47% 68% 21%12 25% 52% 27%13 44% 67% 23%14 49% 84% 35%15 40% 53% 13%16 32% 81% 49%

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

Bala Swaminathan, Ph.D.Bala Swaminathan, Ph.D.Centers for Disease Control and Centers for Disease Control and

PreventionPrevention

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

PFGE (and other RFLPPFGE (and other RFLP--based methods) are difficult to based methods) are difficult to standardizestandardizeComparability of patterns within and between laboratories Comparability of patterns within and between laboratories requires strict adherence to a standard protocolrequires strict adherence to a standard protocolNormalization of patterns is complexNormalization of patterns is complexPFGE is laborPFGE is labor--intensive and requires high concentrations of a intensive and requires high concentrations of a pure culturepure cultureIn some instances or for some pathogen groups, discrimination In some instances or for some pathogen groups, discrimination may not be adequatemay not be adequate

Requirements for the next generation Requirements for the next generation subtyping method for PulseNetsubtyping method for PulseNet

Broad applicabilityBroad applicabilityRapid results (Rapid results (<< 24 h)24 h)InexpensiveInexpensiveBetter discrimination than PFGEBetter discrimination than PFGEQuantitative relatedness between strainsQuantitative relatedness between strainsAccurate snapshot of the genome diversityAccurate snapshot of the genome diversityBackward compatibility with PFGE dataBackward compatibility with PFGE dataEasy to perform on a routine basis Easy to perform on a routine basis Amenable to automationAmenable to automationResults should be readily comparable within Results should be readily comparable within and between laboratoriesand between laboratories

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

Perna et al., Nature 409:529-533, 2001

Methodologic ApproachesMethodologic Approaches

MultiMulti--locus sequence typing (MLST)locus sequence typing (MLST)Inadequate discrimination for most enteric Inadequate discrimination for most enteric pathogens for outbreak investigationspathogens for outbreak investigationsUseful for Useful for Campylobacter jejuniCampylobacter jejuni

MultiMulti--locus Variablelocus Variable--Number Tandem Repeat Number Tandem Repeat Analysis (MLVA)Analysis (MLVA)

Most promising for nearMost promising for near--term subtypingterm subtypingHigh throughput SNP analysisHigh throughput SNP analysis

Method of choice for the long termMethod of choice for the long term

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 genomeExample: TAACCGExample: TAACCG

Variable numbers of repeat units among isolates of the same Variable numbers of repeat units among isolates of the same speciesspecies

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

TAACCG

TAACCGTAACCG

TAACCGTAACCGTAACCGTAACCGTAACCG

TAACCGTAACCGTAACCGTAACCG

Isolate AIsolate BIsolate CIsolate D

Variable Number Tandem RepeatsVNTRs

Variable Number Tandem RepeatsVariable Number Tandem RepeatsVNTRsVNTRs

InsertionInsertion

DeletionDeletion

Multiple Locus VNTR Analysis can beMultiple Locus VNTR Analysis can bedeveloped from lowdeveloped from low--pass sequence datapass sequence data

Development of Development of E. coliE. coli O157 MLVA O157 MLVA protocolprotocol

Contract awarded to the Massachusetts Contract awarded to the Massachusetts Department of Public Health / State Laboratory Department of Public Health / State Laboratory Institute in fall 2001Institute in fall 2001Collaboration with Dr. Paul Keim (The Collaboration with Dr. Paul Keim (The Northern Arizona University)Northern Arizona University)

E.E. colicoli O157 strains used in the initial O157 strains used in the initial validation at CDCvalidation at CDC

152 isolates analyzed by both MLVA and PFGE 152 isolates analyzed by both MLVA and PFGE using using XbaXbaII

Geographically diverse sporadic isolates with unique Geographically diverse sporadic isolates with unique XbaXbaI I PFGEPFGE patterns (UPP collection) patterns (UPP collection) Outbreak isolates from eight well characterized outbreaksOutbreak isolates from eight well characterized outbreaksEpidemiologically unrelated isolates clustered by PFGEEpidemiologically unrelated isolates clustered by PFGEA subset of 54 isolates were further characterized with A subset of 54 isolates were further characterized with BlnBlnII

Nine VNTR loci included in the optimized Nine VNTR loci included in the optimized

MLVA protocol for MLVA protocol for E. coliE. coli O157O157

Vhec7Vhec7

Vhec2, TR6Vhec2, TR6

TR4TR4

TR7TR7

TR3TR3

Vhec1, TR2Vhec1, TR2

Vhec4, TR1Vhec4, TR1

Vhec3, TR5Vhec3, TR5

Alternative Alternative namename11

YesYes141419193366VNTRVNTR--3737

NoNo141415153377VNTRVNTR--3636

YesYes661010551818VNTRVNTR--3434

NoNo8820201166VNTRVNTR--2525

YesYes7710104466VNTRVNTR--1919

YesYes111118182266VNTRVNTR--1717

YesYes39396868101066VNTRVNTR--1010

NoNo171720205566VNTRVNTR--99

YesYes202023234466VNTRVNTR--33

MaximumMaximumMinimumMinimum

InsideInsideORFORF

No. ofNo. ofallelesalleles

No. of repeatsNo. of repeatsRepeat size Repeat size (bp)(bp)VNTRVNTR

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

Discriminatory power of MLVA Discriminatory power of MLVA compared to PFGEcompared to PFGE

152 isolates152 isolates133 unique MLVA patterns133 unique MLVA patterns126 unique 126 unique XbaXbaI PFGE patternsI PFGE patterns

A subset of 54 isolates were characterized by A subset of 54 isolates were characterized by PFGE using two enzymesPFGE using two enzymes

35 unique MLVA patterns35 unique MLVA patterns39 unique 39 unique XbaXbaII--BlnBlnI PFGE patternsI PFGE patterns

VNTR_valsMLVA_composite

100

80604020

F5733

H6436

G5308

F6141

H2306

01-577

F7382F8751

F8768

F7383

F7384

C9523

C9581

C9815

G5244A7793

F7349

F7350

F7351

F7353

F7354

F6749F6750

A8184

EDL933

EXHX01.0224

EXHX01.0224

EXHX01.0224

EXHX01.0224

EXHX01.0224

EXHX01.0047

EXHX01.0047EXHX01.1264

EXHX01.1264

EXHX01.0047

EXHX01.0047

EXHX01.0001

EXHX01.0001

EXHX01.0001

EXHX01.0001EXHX01.0004

EXHX01.0011

EXHX01.0011

EXHX01.0011

EXHX01.0011

EXHX01.0011

EXHX01.1514EXHX01.0283

EXHX01.0029

EXHX01.0028

EXHA26.0536

EXHA26.0536

EXHA26.0536

EXHA26.0536

EXHA26.0536

EXHA26.0015

EXHA26.0548EXHA26.0015

EXHA26.0015

EXHA26.0250

EXHA26.0250

EXHA26.0001

EXHA26.0001

EXHA26.0001

EXHA26.0001EXHA26.0585

EXHA26.0014

EXHA26.0536

EXHA26.0014

EXHA26.0014

EXHA26.0598

EXHA26.0014EXHA26.0014

EXHA26.0715

EXHA26.0711

GA / Stool

GA / Stool

ME / Environmental

GA / Meat

CT / Stool

VA / Stool

NJ / StoolCO / Stool

CO / Ground beef

NJ / Hamburger

NJ / Fatal case

WA / Sporadic

CA / Outbreak

AZ / Sporadic

WA / SporadicOR / Stool

WI / Stool

WI / Stool

WI / Taco meat

WI / Stool

WI / Stool

NY / Fatal caseNY / Sibling

MI / Stool

MI / Hamburger

1998

1998

1992

1998

1996

2001

20002002

2002

2000

2000

1993

1993

1993

199303-1982

2000

2000

2000

2000

2000

19991999

06-1982

05-1982

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

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

Conclusions from the onConclusions from the on--going going validation of the validation of the E. coliE. coli O157 MLVA O157 MLVA

protocolprotocol

Overall, MLVA slightly less discriminating than Overall, MLVA slightly less discriminating than PFGE with two enzymesPFGE with two enzymesMLVA can further discriminate some of the MLVA can further discriminate some of the most common PFGE patternsmost common PFGE patternsEpidemiological congruence of the MLVA data Epidemiological congruence of the MLVA data appears to be equal to or better than PFGEappears to be equal to or better than PFGEDevelopment of interpretation guidelines may Development of interpretation guidelines may pose a challengepose a challenge

Future plansFuture plans2005: 2005:

JulyJuly: Complete the CDC internal validation of the : Complete the CDC internal validation of the E. E. colicoli O157 MLVA protocolO157 MLVA protocolAugustAugust--SeptemberSeptember: Begin collaborative validation of : Begin collaborative validation of the the E. coliE. coli O157 MLVA protocol by transferring the O157 MLVA protocol by transferring the protocol to PulseNet participating laboratoriesprotocol to PulseNet participating laboratories

PFGE vs MLVA in Outbreak IsolatesPFGE vs MLVA in Outbreak Isolates(Data from Minnesota Dept. of Health)(Data from Minnesota Dept. of Health)

0 (0%)0 (0%)0 (0%)0 (0%)

MST 54 (1)MST 54 (1)MST 85 (9)MST 85 (9)

TM 43TM 43OB6 (n=10)OB6 (n=10)

0 (0%)0 (0%)1 (0.9%)1 (0.9%)

MST 27 (5)MST 27 (5)MST 30 (1)MST 30 (1)

TM 1aTM 1aOB5 (n=6)OB5 (n=6)

4 (3.6%)4 (3.6%)3 (2.7%)3 (2.7%)MST 70MST 70TM 2dTM 2dOB4 (n=8)OB4 (n=8)

0 (0%)0 (0%)0 (0%)0 (0%)0 (0%)0 (0%)

1 (0.9%)1 (0.9%)MST 10 (2)MST 10 (2)MST 8 (2)MST 8 (2)MST 105 (2)MST 105 (2)

TM 5bTM 5bOB3 (n=6)OB3 (n=6)

0 (0%)0 (0%)2 (1.8%)2 (1.8%)0 (0%)0 (0%)0 (0%)0 (0%)

2 (1.8%)2 (1.8%)

MST 62 (1)MST 62 (1)MST 81 (1)MST 81 (1)MST 60 (1)MST 60 (1)MST 61 (29)MST 61 (29)

TM 127TM 127OB2 (n=32)OB2 (n=32)

0 (0%)0 (0%)3 (2.7%)3 (2.7%)0 (0%)0 (0%)0 (0%)0 (0%)

MST 48MST 48TM 14 (4)TM 14 (4)TM 215 (1)TM 215 (1)TM 352 (1)TM 352 (1)

OB1 (n=6)OB1 (n=6)

MLVAMLVAPFGEPFGEMLVAMLVAPFGEPFGEOutbreakOutbreak

Frequency of Outbreak type in Frequency of Outbreak type in Sporadic IsolatesSporadic Isolates

Outbreak TypeOutbreak Type

Discrimination of Phage Typing and MLVA Discrimination of Phage Typing and MLVA Within Common SE PFGE TypesWithin Common SE PFGE Types

4455NANAMSE9MSE9

5544NANAMSE11MSE11

MLVA TypesMLVA Types

1111NANA171713a13a

33NANA111188

77NANA141444

Phage TypesPhage Types

NANA551919SE1B1SE1B1

NANA7*7*1212SE11B6SE11B6

PFGE TypesPFGE Types

No. PFGE TypesNo. PFGE TypesNo. Phage TypesNo. Phage TypesNo. MLVA TypesNo. MLVA TypesMost CommonMost Common

*Includes RDNCData from Minnesota Department of Health

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

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

2528stx2+, Sorbitol+, O157:H-1989Germany493/892529stx1+, stx2+, Mug+, Urea+1993WashingtonG51011264stx2+2002ColoradoF87680224stx1+, stx2+1998GeorgiaF61410390stx2+2001North CarolinaN05870264stx1+, stx2+2001New YorkN03031315stx1+2002ColoradoN04360047stx2+, PFGE type 00472001Virginia01-5770238stx2+, Phage type 311994WashingtonG52890224stx1+, stx2+1998GeorgiaF57330373stx1+, stx2+1996JapanSakai

PFGE patternCharacteristicsYearOriginStrain

SNP (376: GSNP (376: G--A) in gene ECs3157 (1.2Kb, putative sulfatase)A) in gene ECs3157 (1.2Kb, putative sulfatase)

Deletions in gene ECs4864 (4.1Kb, RhsH core protein)Deletions in gene ECs4864 (4.1Kb, RhsH core protein)

Gene absence in ECs2974 (950Gene absence in ECs2974 (950--bp, Shiga toxin I subunit A)bp, Shiga toxin I subunit A)

Summary

1,199 complete chromosomal genes (1,167,510-bp) + pO157 (92,721-bp)

823 backbone genes (22% of 3,729)376 S-loop genes (23% of 1,632)

836 SNPs in 511 genes (42% of 1,199)309 in 9 typical O157:H7 isolatesOn average, 34 SNPs/1,199 genes between two isolatesEstimated ~152 SNPs/5,361 genes between two isolates

Non-synonymous : Synonymous = 499 : 337SNP location: (Backbone vs. S-loop) = 552 (350 loci) : 284 (161 loci)Polymorphism (%): (Backbone vs. S-loop) = 0.125% : 0.143%

http://genome.gen-info.osaka-u.ac.jp/

836 SNPs in 511 Conserved Chromosomal Genes

ConclusionsConclusions

PFGE will continue to be an essential subtyping PFGE will continue to be an essential subtyping method for PulseNet in the near futuremethod for PulseNet in the near futureMLVA may provide additional discrimination for MLVA may provide additional discrimination for E. coliE. coliO157:[H7] and some O157:[H7] and some SalmonellaSalmonella serotypesserotypesWill start transferring MLVA protocol for Will start transferring MLVA protocol for E. coliE. coli O157 O157 :[H7]to state and local public health laboratories in 2005:[H7]to state and local public health laboratories in 2005SNP is the subtyping method of the future; SNP may SNP is the subtyping method of the future; SNP may be used in combination with MLVAbe used in combination with MLVAMuch work remains to be done on new subtyping Much work remains to be done on new subtyping methods for PulseNet; we hope to continue active methods for PulseNet; we hope to continue active participation with public health laboratoriesparticipation with public health laboratories

Bioterrorism Preparedness and Bioterrorism Preparedness and Response Funds for PulseNet ActivitiesResponse Funds for PulseNet Activities

ELC and BT are separate funding streams with ELC and BT are separate funding streams with different (some overlap) goals and objectivesdifferent (some overlap) goals and objectivesELC funds have not increased ELC funds have not increased –– maintenance of maintenance of successful projects; some new projectssuccessful projects; some new projectsBT funds for PulseNetBT funds for PulseNet

Enhance preparedness Enhance preparedness –– accelerated or more informative accelerated or more informative subtypingsubtypingIncrease surge capacityIncrease surge capacityFoodborne pathogen priorities: Foodborne pathogen priorities: E. coliE. coli O157:[H7], O157:[H7], ListeriaListeria