pfge and beyond: pulsenet in the next decade bala swaminathan, ph.d. centers for disease control and...
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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-10VNTR-10 Vhec1, Vhec1, TR2TR2 66 1010 6868 3939 YesYes
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-34VNTR-34 Vhec2, Vhec2, TR6TR6 1818 55 1010 66 YesYes
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
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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
.
.
.
.
.
.
.
.
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