MLVA update: E. coli O157:[H7] protocol validation and
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Transcript MLVA update: E. coli O157:[H7] protocol validation and
PFGE and Beyond: PulseNet in
the Next Decade
Bala Swaminathan, Ph.D.
Centers for Disease Control and
Prevention
Why Next Generation Subtyping
Methods?
PFGE (and other RFLP-based methods) are difficult to
standardize
Comparability of patterns within and between
laboratories requires strict adherence to a standard
protocol
Normalization of patterns is complex
PFGE is labor-intensive and requires high
concentrations of a pure culture
In some instances or for some pathogen groups,
discrimination may not be adequate
Clinical isolate clusters with no
demonstrable epidemiologic
links – Example 1
Clinical isolate clusters with no
demonstrable epidemiologic links –
Example 2
Requirements for the next generation
subtyping method for PulseNet
Broad applicability
Rapid results (< 24 h)
Inexpensive
Better discrimination than PFGE
Quantitative relatedness between strains
Accurate snapshot of the genome diversity
Backward compatibility with PFGE data
Easy to perform on a routine basis
..............
Amenable to automation
Results should be readily comparable within
and between laboratories
Methodologic Approaches
Multi-locus sequence typing (MLST)
Multi-locus Variable-Number Tandem Repeat
Analysis (MLVA)
High throughput SNP analysis
Multi-Locus Sequence Typing
Based on the nucleotide sequence of internal regions of
housekeeping loci
Housekeeping loci should be conserved with only minimal
nucleotide changes due to conserved protein function
Multiple loci are targeted in this subtyping method
Sequence variation allows for the assignment of alleles
Isolate A
– ATTCGGCAT –
Isolate B
– ATTCGCCAT –
allele 1
allele 2
A combination of alleles for all loci provides an allele
profile which can then be assigned to a sequence type
(ST)
Isolate A
(1, 5, 6, 3, 4, 3, 1)
ST-5
Isolate B
(1, 5, 6, 3, 3, 3, 1)
ST-51
Sequence types are grouped into clonal complexes based
on similarity to a central allelic profile
Subtyping Campylobacter jujuni
Three published MLST
schemes
Dingle at al (2001)
Suerbaum et al (2001)
194 isolates
155 sequence types
51 unique ST’s
32 isolates plus NCTC 11168
31 unique allele profiles
Frequent recombination
Manning et al (2003)
Origin of replication
glt asp
tkt
MLST loci
unc
nuoH atpA
yphC
pgm
gly
fumC
eftS
1,641,481 bp
asd
ddlA
gln
Subtyping Campylobacter jujuni
Sails et al (2003)
Comparison of MEE, MLST and PFGE
MLST is not as discriminatory as PFGE
MLST plus a variable locus
MLST and flaA SVR provides similar discrimination to
PFGE
MLST studies with enterics
Listeria monocytogenes: Additional variable gene targets
need to be included in MLST (MLST+) to obtain
acceptable discrimination
Salmonella enterica (Kotetishvili et al, 2002)
MLST is more discriminatory than PFGE
Escherichia coli (Whittam Laboratory)
Cai et al. 2002
Zhang et al. 2004
Distinguish pathovars of E. coli/Shigella groups
Distinguish clonal lineages within pathovars
E. coli O157:H7 is too clonal for MLST subtyping
(Noller et al, 2003)
Multilocus VNTR Analysis
(MLVA)
MLVA (Multi Locus VNTR Analysis)
Variable Number Tandem Repeats (VNTRs)
Conserved repeat motif found in the genome
Example: TAACCG
Variable numbers of repeat units among isolates of the same
species
MLVA examines the number of repeats at multiple loci
to determine genetic relationships
Isolate A
TAACCG
Isolate B
TAACCGTAACCG
Isolate C
TAACCGTAACCGTAACCGTAACCG
Isolate D
TAACCGTAACCGTAACCGTAACCGTAACCG
Development of E. coli O157 MLVA
protocol
Contract awarded to the Massachusetts
Department of Public Health / State
Laboratory Institute in fall 2001
Collaboration with Dr. Paul Keim (The
Northern Arizona University)
Development of E. coli O157 MLVA
protocol (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 highresolution molecular typing. J. Appl. Microbiol.
98: 928-940.
29 VNTR loci polymorphic in O157:[H7] serotype
identified
Development of E. coli O157 MLVA
protocol (cont’d)
MA protocol based on 25 VNTR loci
Amplified in four multiplex PCR reactions
Fluorescently labeled PCR amplicons sized using
capillary electrophoresis system (CEQ 8000, Beckman
Coulter, Fullerton, CA)
Internal validation at the CDC PulseNet Methods
Development and Validation Laboratory started
in summer 2004
E. coli O157 strains used in the initial
validation
152 isolates analyzed by both MLVA and PFGE
using XbaI
Geographically diverse sporadic isolates with unique XbaI
PFGE patterns (UPP collection)
Outbreak isolates from eight well characterized outbreaks
Epidemiologically unrelated isolates clustered by PFGE
A subset of 54 isolates were further characterized with
BlnI
Nine VNTR loci included in the final MLVA
protocol for E. coli O157
VNTR
Alternative
name1
Repeat size
(bp)
No. of repeats
Minimum
Maximum
No. of
alleles
Inside
ORF
VNTR-3
Vhec3, TR5
6
4
23
20
Yes
VNTR-9
Vhec4, TR1
6
5
20
17
No
VNTR-10
Vhec1, TR2
6
10
68
39
Yes
VNTR-17
TR3
6
2
18
11
Yes
VNTR-19
TR7
6
4
10
7
Yes
VNTR-25
TR4
6
1
20
8
No
VNTR-34
Vhec2, TR6
18
5
10
6
Yes
VNTR-36
Vhec7
7
3
15
14
No
6
3
19
14
Yes
VNTR-37
1 Vhec loci are form Lindstedt et al. (2003); TR loci are from Noller et al. (2003)
MLVA protocol steps
1.
2.
3.
4.
5.
Boiled whole cell DNA templates prepared
from overnight cultures
Nine VNTR sites amplified in three PCR
reactions
Diluted (1:60) PCR products mixed with sample
loading solution and 600 bp DNA size standard
PCR products sized using CEQ 8000 capillary
electrophoresis system (Beckman Coulter)
Fragment list exported to BioNumerics
(Applied Maths, Kortijk, Belgium) for analysis
Discriminatory power of MLVA
compared to PFGE
152 isolates
133 unique MLVA patterns
126 unique XbaI PFGE patterns
A subset of 54 isolates were characterized by
PFGE using two enzymes
35 unique MLVA patterns
39 unique XbaI-BlnI PFGE patterns
Clustering of 152 E. coli O157:[H7] isolates by MLVA
15
10
25
20
35
30
45
40
55
50
65
60
75
70
85
80
95
90
100
MLVA_composite
EC04PN0655
G5289L
F5733
H6436
EC04PN0477
F6141
F6142
H6039
G5308
EC04PN0585
EC04PN0586
EC04PN0139
EC04PN0479
EC04PN0454
EC04PN0187
EC05PN0001
EC04PN0519
EC04PN0179
EC04PN0587
EC04PN0660
EC04PN0581
EC04PN0631
EC04PN0632
EC04PN0612
EC04PN0643
EC04PN0503
EC04PN0663
EC04PN0659
EC05PN0024
EC05PN0120
H2306
F7383
F7384
K0805
EC04PN0137
F7408
K0814
EC04PN0618
F7410
F7407
K0803
F8751
F8768
EC04PN0640
EC04PN0661
01-577
F7382
EC04PN0547
K0802
EC04PN0194
EC04PN0198
EC04PN0568
493-89
EC04PN0619
G5101
EC04PN0481
EC04PN0500
EC04PN0191
EC04PN0636
EC04PN0190
EC04PN0113
EC04PN0202
EC05PN0032
EC04PN0615
EC04PN0583
EC04PN0623
EC04PN0456
EC04PN0522
EC04PN0569
EC04PN0548
EC04PN0549
EC04PN0183
EC04PN0656
EC04PN0520
EC04PN0521
EC04PN0580
EC04PN0614
EC04PN0613
EC04PN0452
EC04PN0501
F7349
F7350
F7351
F7353
F7354
EC04PN0152
EC04PN0629
A8184
EDL933
F6749
F6750
EC04PN0630
EC04PN0616
EC05PN0048
EC04PN0114
EC04PN0546
EC04PN0480
EC04PN0582
EC04PN0478
EC04PN0504
EC04PN0634
EC04PN0620
F6862
EC04PN0644
EC04PN0518
EC04PN0567
EC04PN0639
EC04PN0664
EC04PN0584
A7793
EC04PN0622
EC04PN0146
EC04PN0628
EC04PN0455
C9523
C9581
C9815
G5244
EC04PN0658
EC04PN0161
EC04PN0457
EC04PN0626
EC04PN0635
EC04PN0637
EC04PN0499
EC04PN0627
EC04PN0502
EC04PN0458
EC04PN0642
EC04PN0482
H0706
EC04PN0505
EC04PN0645
EC04PN0570
EC04PN0662
EC04PN0633
F6854
F6857
EC04PN0199
EC04PN0203
EC05PN0016
EC04PN0638
BAA460
EC04PN0624
EC04PN0625
EC04PN0523
F6939
F6941
F6899
EC04PN0153
EC04PN0617
EC04PN0657
EDL933
Sakai
Cluster II
Cluster I
Clustering of 43 E. coli O157:[H7] isolates by MLVA and by
PFGE using combined XbaI-BlnI data
50
60
70
80
90
100
PFGE-BlnI+PFGE-XbaI
70
xba-bln
65
75
80
85
90
95
100
VNTR_vals
10
20
30
40
MLVA_composite
F6854
F6857
BAA460
EC04PN0582
C9523
C9581
C9815
G5244
F6862
F6939
F6941
F6899
EC04PN0520
EC04PN0521
EC04PN0548
EC04PN0549
F7349
F7350
F7351
F7353
F7354
F6749
F6750
EDL933
F8751
F8768
F7407
01-577
F7382
F7408
K0814
F7410
F7383
F7384
K0805
G5289L
EC04PN0631
EC04PN0632
493-89
EC04PN0585
EC04PN0586
G5308
F6141
PFGE II
PFGE I
PFGE III
MLVA Ia
MLVA Ib
MLVA II
xba-bln
CDC__01-577
F7407/#2
DBS__CDC__F8751
DBS__CDC__F8768
DBS__CDC__F7383
DBS__CDC__F7384
CDC__K0814
cdc__K0805/#1
F7382
F7408 Pure
cdc__EC04PN0548
cdc__EC04PN0549
F7410/#3
cdc__EC04PN0585
cdc__EC04PN0586
cdc__G5308
F6141
F6899
F6939
F6941
CDC__460-W
cdc__F6749
cdc__EC04PN0582
DBS_CDC__F6862
F6750/#1
DBS_CDC__F6854
DBS_CDC__F6857
DBS_CDC__F7349
DBS_CDC__F7351
DBS_CDC__F7353
F7354/#1
cdc__EC04PN0520
cdc__EC04PN0521
cdc__C9523
cdc__C9581
cdc__C9815
CDC__G5289 lg
DBS__CDC__MLVA095
DBS_CDC__F7350
CDC__EDL933
CDC__EC04PN0631
CDC__EC04PN0632
CDC__493-89
80
60
40
20
MLVA_composite
100
Clustering of outbreak isolates and some
selected sporadic isolates by MLVA
VNTR_vals
F5733
EXHX01.0224
EXHA26.0536
GA / Stool
1998
H6436
EXHX01.0224
EXHA26.0536
GA / Stool
1998
G5308
EXHX01.0224
EXHA26.0536
ME / Environmental
1992
F6141
EXHX01.0224
EXHA26.0536
GA / Meat
1998
H2306
EXHX01.0224
EXHA26.0536
CT / Stool
1996
01-577
EXHX01.0047
EXHA26.0015
VA / Stool
2001
F7382
EXHX01.0047
EXHA26.0548
NJ / Stool
2000
F8751
EXHX01.1264
EXHA26.0015
CO / Stool
2002
F8768
EXHX01.1264
EXHA26.0015
CO / Ground beef
2002
F7383
EXHX01.0047
EXHA26.0250
NJ / Hamburger
2000
F7384
EXHX01.0047
EXHA26.0250
NJ / Fatal case
2000
C9523
EXHX01.0001
EXHA26.0001
WA / Sporadic
1993
C9581
EXHX01.0001
EXHA26.0001
CA / Outbreak
1993
C9815
EXHX01.0001
EXHA26.0001
AZ / Sporadic
1993
G5244
EXHX01.0001
EXHA26.0001
WA / Sporadic
1993
A7793
EXHX01.0004
EXHA26.0585
OR / Stool
03-1982
F7349
EXHX01.0011
EXHA26.0014
WI / Stool
2000
F7350
EXHX01.0011
EXHA26.0536
WI / Stool
2000
F7351
EXHX01.0011
EXHA26.0014
WI / Taco meat
2000
F7353
EXHX01.0011
EXHA26.0014
WI / Stool
2000
F7354
EXHX01.0011
EXHA26.0598
WI / Stool
2000
F6749
EXHX01.1514
EXHA26.0014
NY / Fatal case
1999
F6750
EXHX01.0283
EXHA26.0014
NY / Sibling
1999
A8184
EXHX01.0029
EXHA26.0715
MI / Stool
06-1982
EDL933
EXHX01.0028
EXHA26.0711
MI / Hamburger
05-1982
.
GA. water park outbreak
.
CT. apple cider outbreak
.
CO. outbreak
NJ .outbreak
.
.
Western
States outbreak
.
.
.
WI. restaurant
.
outbreak
.
.
.
NY
County Fair
.
.
MI. outbreak
Clusters 0411ml-1c and 0501ml-1c – PFGE pattern
combination EXHX01.0086/EXHA26.0576
VNTR_vals
VNTR_vals:VNTR_37
VNTR_vals:VNTR_17
VNTR_vals:VNTR_25
VNTR_vals:VNTR_36
VNTR_vals:VNTR_19
VNTR_vals:VNTR_10
VNTR_vals:VNTR_9
VNTR_vals:VNTR_34
VNTR_vals:VNTR_3
100
MLVA_composite
MLVA_composite
11.0
8.0
11.0
24.0
6.0
11.0
4.0
8.0
8.0
K1792
..Essex / NY
10/1/2005
11.0
8.0
11.0
24.0
6.0
11.0
4.0
8.0
8.0
K1793
..Madison / NY
12/11/2005
11.0
8.0
11.0
24.0
6.0
11.0
4.0
8.0
8.0
K1794
..VA
11/2004
11.0
8.0
11.0
24.0
6.0
11.0
4.0
8.0
8.0
K1795
..VA
11/2004
11.0
8.0
11.0
24.0
6.0
11.0
4.0
8.0
8.0
K1796
..OH
01/2005
11.0
8.0
11.0
24.0
6.0
11.0
4.0
8.0
8.0
K1797
..OH
01/2005
11.0
8.0
11.0
24.0
6.0
11.0
4.0
8.0
8.0
K1845
..IN (MI)
12/2004
11.0
8.0
11.0
24.0
6.0
11.0
4.0
8.0
8.0
K1846
..MI
12/2004
Conclusions from the on-going
validation of the E. coli O157 MLVA
protocol
Overall, MLVA slightly less discriminating than
PFGE with two enzymes
MLVA can further discriminate some of the
most common PFGE patterns
Epidemiological congruence of the MLVA data
better than that of PFGE
Development of interpretation guidelines may
pose a challenge
Future plans
2005:
Complete the CDC internal validation of the E. coli
O157 MLVA protocol
Custom-made 1 kb standard for the locus VNTR-10?
Reagent evaluation
Fine-tuning of the BioNumerics scripts
Begin collaborative validation of the E. coli O157
MLVA protocol by transferring the protocol to four
PulseNet laboratories
Future plans (cont’d)
2006
Expand the implementation of the protocol to at
least four more PulseNet laboratories
Establish a national database with a pattern naming
strategy
Establish 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
• 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
http://www.genome.wisc.edu/
http://genome.gen-info.osaka-u.ac.jp/
http://colibase.bham.ac.uk/
http://snpsfinder.lanl.gov/
NimbleGen CGR microarray
Mutation Mapping
Resequencing
Singh-Gasson et al. 1999. Nat. Biotechnol. 17:974-978
Nuwaysir et al. 2002. Genome Res. 12:1749-1755
Selection of genes for CGR
• 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)
Ohnishi et al. 2002. PNAS. 99:17043-17048
O157 strains for resequencing
Characteristics
PFGE
pattern
Strain
Origin
Year
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
N0587
North Carolina
2001
stx2+
0390
F6141
Georgia
1998
stx1+, stx2+
0224
F8768
Colorado
2002
stx2+
1264
G5101
Washington
1993
stx1+, stx2+, Mug+, Urea+
2529
493/89
Germany
1989
stx2+, Sorbitol+, O157:H-
2528
Total no. of SNPs in test strains = 836
Strain
Characteristics
PFGE
pattern
Total no. of
SNPs
Strain-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
Conclusions
PFGE will continue to be an essential subtyping
method for PulseNet
MLVA may provide additional discrimination for E. coli
O157:[H7] and some Salmonella serotypes
MLVA protocol for E. coli O157 :[H7] will be
transferred to selected PulseNet laboratories in 2005
SNP is the subtyping method of the future; SNP may
be used in combination with MLVA
Much work needs to be done on new subtyping
methods for PulseNet