Novel Approaches for Detection and Characterization of Foodborne ...
Novel Approaches for Detection and Characterization of Foodborne ...
Novel Approaches for Detection and Characterization of Foodborne ...
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<strong>Novel</strong> <strong>Approaches</strong> <strong>for</strong><br />
<strong>Detection</strong> <strong>and</strong> <strong>Characterization</strong> <strong>of</strong><br />
<strong>Foodborne</strong> Pathogens<br />
Thomas A. Cebula, Ph.D.<br />
Director, Office <strong>of</strong> Applied Research <strong>and</strong> Safety Assessment<br />
Center <strong>for</strong> Food Safety <strong>and</strong> Applied Nutrition<br />
<strong>and</strong><br />
Center <strong>of</strong> Excellence <strong>for</strong> Microbial Forensics <strong>of</strong> Enteric Pathogens
FDA/CFSAN/OARSA<br />
J. Eugene LeClerc<br />
Eric W. Brown<br />
Paula Davis<br />
Kim Dudley<br />
Alice Hay<strong>for</strong>d<br />
Scott Jackson<br />
Michael Kotewicz<br />
Baoguang Li<br />
Tammy Mays<br />
Kristen McCutchan<br />
Mark Mammel<br />
Amit Mukherjee<br />
W. Les Payne<br />
Andrei Perlloni<br />
Richard Raybourne<br />
Saura Sahu<br />
Andrew Shifflet<br />
The Perara Group, Inc.<br />
Junia Jean-Giles<br />
Isha Patel<br />
Dwayne Roberson<br />
Univ. <strong>of</strong> Maryl<strong>and</strong><br />
Suman Mukhopadhyay<br />
Univ. <strong>of</strong> Minnesota &<br />
Minnesota Dept. <strong>of</strong> Health<br />
Michael Osterholm<br />
John Besser<br />
James R. Johnson<br />
Univ. <strong>of</strong> Wisconsin<br />
Fred Blattner<br />
Nicole Perna<br />
DHS/NBFAC<br />
CAPT. James Burans<br />
FBI<br />
Mark Wilson<br />
Bruce Budowle<br />
Sidney Kimmel Cancer<br />
Mike McClell<strong>and</strong><br />
OpGen<br />
Colin Dykes<br />
NimbleGen<br />
Tom Albert
There are no such<br />
things as applied<br />
sciences,<br />
only applications <strong>of</strong><br />
science.<br />
--Louis Pasteur, 9/11/1872
The need <strong>for</strong>:<br />
•Identifying <strong>and</strong> recognizing patterns<br />
in a disease outbreak<br />
•Communicating those patterns to the<br />
public health community at large<br />
•Determing the pathogen involved<br />
•Containing the outbreak<br />
•Tracing the microbe to its source<br />
--Events <strong>of</strong> 9/11/2001 <strong>and</strong> after
Washington Post<br />
12/30/04
The <strong>for</strong>ensic continuum <strong>for</strong> strain identification<br />
“Strain could<br />
not have come<br />
from…”<br />
Exclusion<br />
“Strain did<br />
absolutely<br />
come from…”<br />
Attribution<br />
Differentiation<br />
<strong>of</strong> Strains<br />
Methods Validation<br />
Biomarker Stability<br />
Extent <strong>of</strong><br />
Genomic<br />
Diversity
Food Safety<br />
<strong>Detection</strong> at Genus Level<br />
<strong>Detection</strong> at Species Level<br />
<strong>Detection</strong> at Subspecies Level<br />
<strong>Detection</strong> at Serotype or Serovar Level
Food Defense<br />
<strong>Detection</strong> at Genus Level<br />
<strong>Detection</strong> at Species Level<br />
<strong>Detection</strong> at Subspecies Level<br />
<strong>Detection</strong> at Serotype or Serovar Level<br />
but, <strong>for</strong> attribution,<br />
<strong>Detection</strong> at Strain Level
Reference Strain Collection
Methods <strong>for</strong> Measuring Diversity<br />
Among E. coli O157:H7<br />
FDA Assembled a Diverse Collection <strong>of</strong> ~120 Natural Isolates<br />
<strong>of</strong> E. coli O157:H7<br />
1. Classical microbiological endpoints<br />
2. PCR-based strategies to assess ‘virulence’ genes, incidental markers, variable<br />
repeat regions, <strong>and</strong> the presence <strong>of</strong> IN-DELS occurring in bacterial genomes<br />
3. Comparative sequence analysis <strong>of</strong> multiple loci via conventional sequencing;<br />
Cladistic analyses to assess vertical <strong>and</strong> horizontal inheritance<br />
4. Pyrosequencing to provide real-time sequence data <strong>and</strong> useful in<strong>for</strong>mation <strong>for</strong><br />
confirming <strong>and</strong> identifying the presence <strong>of</strong> SNPs <strong>and</strong> their population frequency<br />
5. Whole genome arrays <strong>for</strong> genotyping <strong>and</strong> expression<br />
6. High density oligonucleotide microarrays that interrogate ~60 kb <strong>of</strong> the genome<br />
<strong>and</strong> precisely identifies polymorphisms occurring in the probed region<br />
7. Optical Mapping<br />
8. Phenotypic microarrays (Biolog) that allow rapid screening <strong>of</strong> up to 1200<br />
phenotypes, providing biological insight to our underst<strong>and</strong>ing <strong>of</strong> diversity
Comparative Genomic Analysis<br />
Of E. coli O157:H7 Using A<br />
<strong>Novel</strong> High-density, High-<br />
Throughput DNA Microarray<br />
Plat<strong>for</strong>m
Sampling ~1% <strong>of</strong> the E.coli O157:H7<br />
Genome at R<strong>and</strong>om<br />
5.5 Mb Genome<br />
- Sampled 1 kb per ~100 kb<br />
- Tiled 60 Loci onto Arrays<br />
Perna, N. T. et al. Nature 409, 529-533 (2001)
High Density Oligonucleotide Tiling Arrays Provide a<br />
“High Resolution” Snapshot <strong>of</strong> the Genome<br />
Reference Genome<br />
Test Genome<br />
29-mer Tiling Array<br />
Probes<br />
Mutation<br />
• Our Tiled Strategy Uses a 5 nt Probe Spacing<br />
• For a r<strong>and</strong>om sampling <strong>of</strong> ~1% <strong>of</strong> the genome, 1 kb <strong>of</strong> genome sequence was<br />
selected at 60 equally spaced regions around the EDL933 chromosome.
Interrogating 12 Independent Strains in Parallel<br />
1.5 cm<br />
~14,000 Spots (oligos)<br />
~4mm<br />
2 cm
Relative Probe Intensity vs. Genome Position<br />
Probes reporting a<br />
deletion in the test<br />
strain<br />
Probes reporting a<br />
SNP in the test<br />
strain<br />
Probes reporting identical<br />
sequence between strains
Validating Tiling Arrays: E.coli K-12 vs E.coli O157:H7<br />
EDL933/MG1655 Hybridizatio Ratio<br />
70.0<br />
65.0<br />
60.0<br />
55.0<br />
50.0<br />
45.0<br />
40.0<br />
35.0<br />
30.0<br />
25.0<br />
20.0<br />
15.0<br />
10.0<br />
5.0<br />
0.0<br />
-5.0<br />
1<br />
92727<br />
276613<br />
369339<br />
553226<br />
645951<br />
829838<br />
1013724<br />
1106450<br />
1290337<br />
1383062<br />
1566949<br />
1750835<br />
1843561<br />
2027448<br />
2120173<br />
2304060<br />
2487946<br />
2580672<br />
2764559<br />
2857284<br />
3041171<br />
3225057<br />
3317783<br />
3501670<br />
3594395<br />
3778282<br />
3962168<br />
4054894<br />
4238781<br />
4331506<br />
4515393<br />
4699279<br />
4792005<br />
4975892<br />
5068617<br />
5252504<br />
5436390<br />
EDL933 Position
Identification <strong>of</strong> Polymorphisms in E.coli K-12<br />
Deletion<br />
SNPs<br />
SNPs<br />
Insertion
• Over the 60 kb region <strong>of</strong> the K-12 genome<br />
interrogated, 814 independent polymorphisms occur in<br />
MG1655 relative to the tiled sequence <strong>of</strong> EDL933.<br />
• Of these, 806 differences were successfully called,<br />
indicating that the accuracy in detecting polymorphisms<br />
by this tiling array was greater than 99%.<br />
Of the eight polymorphisms that were not detected, four were SNPs reported in the Genbank sequence<br />
as either R (purine) or Y (pyrimidine). Two single nucleotide deletions that occur in MG1655 relative to<br />
EDL933 were not detected. Two undetected SNPs would have produced T/C mismatches upon<br />
hybridization, a polymorphism detected at many other sites in the MG1655 genome.
E. coli O157:H7 strain AB5
E. coli O157:H7 strain 508
E. coli O157:H7 strain 506
E. coli O157:H7 strain 488 is EDL933
0<br />
1<br />
2<br />
3<br />
4<br />
5<br />
6<br />
7<br />
8<br />
9<br />
10<br />
11<br />
12<br />
13<br />
14<br />
15<br />
92147<br />
92177<br />
184693<br />
184708<br />
276668<br />
368864<br />
369114<br />
369409<br />
553036<br />
553171<br />
645536<br />
645546<br />
738042<br />
829583<br />
921524<br />
921719<br />
921899<br />
922004<br />
922044<br />
922209<br />
922235<br />
1105925<br />
1105935<br />
1106125<br />
1106650<br />
1290107<br />
1290232<br />
1290582<br />
1474718<br />
1474943<br />
1567059<br />
1658535<br />
1658545<br />
1658575<br />
1658680<br />
1658695<br />
1658805<br />
1658810<br />
1658815<br />
1659015<br />
1659020<br />
1659025<br />
1659160<br />
1659165<br />
1659175<br />
1659215<br />
1659360<br />
1659415<br />
1659420<br />
1659425<br />
1751015<br />
2027993<br />
2027998<br />
2028003<br />
2119318<br />
2119493<br />
2119838<br />
2303920<br />
2303930<br />
2304370<br />
2395876<br />
2488451<br />
2580072<br />
2764289<br />
2764299<br />
2764459<br />
2764759<br />
2765124<br />
2948550<br />
2948775<br />
2949425<br />
3594095<br />
3778462<br />
3778472<br />
3869928<br />
4054614<br />
4146550<br />
4515048<br />
4607109<br />
4699614<br />
4791600<br />
4884216<br />
5067812<br />
SNP Position (EDL933)<br />
# Strains
Pyrosequencing – Sequencing by Light<br />
CGT<br />
CGT<br />
Polymerase<br />
CGT<br />
CGT<br />
Pooled<br />
Genomic<br />
DNA<br />
Allele-<br />
Specific<br />
PCR<br />
CGT<br />
Sulfurylase<br />
CGT<br />
CAT<br />
CGT<br />
90% C<br />
Luciferase<br />
CGT<br />
CGT<br />
10% T<br />
C<br />
T
Pyrosequencing assay <strong>for</strong> SNP identification<br />
GCG<br />
GTG<br />
GCG<br />
Sequence<br />
CAGCGGT<br />
GCG<br />
GCG<br />
GCG<br />
CAGTGGT<br />
GTG<br />
GCG<br />
GTG<br />
GCG<br />
70% CAGCGGT<br />
30% CAGTGGT
strain serotype 4889b 4889a 5210 i559 roi NG1 NG2 NG7 4777 5096<br />
EC1214 O157:H7 A G C G G del T G G G<br />
EC506 O157:H7 A G C G A A T C G G<br />
EC868 O157:H7 A G C G A A T C G G<br />
86-24 O157:H7 A G C G A A T C G G<br />
EC509 O157:H7 A G C G A A T C A G<br />
EC536 O157:H7 A G C G A del T G G G<br />
EC484 O157:H7 A G C G GA A T C G G<br />
EC869 O157:H7 A G C A A del G G G G<br />
AB3 O157:H- A G C A A del G G G G<br />
EC510 O157:H- A G C A A del - - G G<br />
EC554 O157:H7 A G T G G A T C G C<br />
EC559 O157:H7 A G T G G A T C G C<br />
EC866 O157:H7 A G T G G A T C G C<br />
EC1219 O157:H7 A G T G G A T C G C<br />
95-01A O157:H7 A G T G G A T C G C<br />
AB1 O157:H- A G T G AG A T C G C<br />
EC558 O157:H7 A G T G GA A T C G C<br />
EC505 O157:H7 A A C G A A T C A G<br />
EC512 O157:H7 A A C G A - T C A G<br />
DEC7A O157:H43 G G C G G del T C G G<br />
EC521 O26:H11 G G C A G del G G G G<br />
EC1216 N.D. G G C A A del - - G G<br />
EC884 N.D. G G C A AG del - - G G<br />
DEC5C O55:H7 G G C - AG del G - G G<br />
barcode<br />
0000010100<br />
0000100000<br />
0000100000<br />
0000100000<br />
0000100010<br />
0000110100<br />
0000200000<br />
0001111100<br />
0001111100<br />
0001112200<br />
0010000001<br />
0010000001<br />
0010000001<br />
0010000001<br />
0010000001<br />
0010200001<br />
0010200001<br />
0100100010<br />
0100120010<br />
1000010000<br />
1001011100<br />
1001112200<br />
1001212200<br />
1002211200
•Bacterial genomes are known to be mosaic<br />
structures due to extensive recombination <strong>and</strong><br />
horizontal gene transfer occurring throughout their<br />
evolution.<br />
•The overall organization <strong>of</strong> a genome might vary<br />
between two strains observed to be similar in gene<br />
content.<br />
•Genome organization might there<strong>for</strong>e help to<br />
identify <strong>and</strong> discriminate between related strains.
4<br />
0<br />
3<br />
2<br />
0<br />
4<br />
5<br />
2<br />
3<br />
3<br />
1<br />
0<br />
6<br />
1<br />
2<br />
3<br />
0<br />
0<br />
0<br />
1<br />
0<br />
2<br />
1<br />
1<br />
2<br />
2<br />
1<br />
2<br />
0<br />
1<br />
3<br />
2<br />
1<br />
0<br />
4<br />
4<br />
4<br />
8<br />
4<br />
0<br />
1<br />
2<br />
3<br />
4<br />
5<br />
6<br />
7<br />
8<br />
9<br />
86-24<br />
93-111<br />
AB1<br />
AB2<br />
AB4<br />
AB5<br />
AB6<br />
AB8<br />
EC1214<br />
EC1219<br />
EC1220<br />
EC1221<br />
EC1223<br />
EC1227<br />
EC1228<br />
EC1231<br />
EC486<br />
EC488<br />
EC502<br />
EC504<br />
EC505<br />
EC506<br />
EC507<br />
EC508<br />
EC536<br />
EC866<br />
EC868<br />
EC869<br />
EC870<br />
EC871<br />
EC873<br />
EC874<br />
EC875<br />
EC877<br />
EC878<br />
EC881<br />
EC882<br />
EC886<br />
EC887<br />
Strain<br />
# CNPs
Optical Mapping: A Single Molecule Technique<br />
<strong>for</strong> Generating Whole Genome Restriction Maps<br />
Genome Map
Method <strong>for</strong> Generating Optical Maps from E. coli<br />
1. Chromosomal DNA Preparation:<br />
• Intact chromosomal DNA is recovered from E. coli embedded in agarose<br />
plugs.<br />
2. DNA Mounting, Overlay, Digestion, <strong>and</strong> Staining:<br />
• Whole DNA molecules are recovered from agarose plugs <strong>and</strong> mounted on<br />
derivatized glass surfaces<br />
• Immobilized DNA is digested<br />
• DNA is fluorescently stained<br />
3. Image Acquisition <strong>and</strong> Processing:<br />
• Samples are imaged by fluorescence microscopy <strong>and</strong> a high-resolution digital<br />
camera.<br />
4. Map Assembly:<br />
• Individual-molecule restriction maps were overlapped by aligning restriction<br />
sites based on fragment sizes by using specially written s<strong>of</strong>tware.
Optical Mapping: Image Analysis<br />
Single DNA molecule on Optical Chip after digestion, staining<br />
Image analysis s<strong>of</strong>tware measures size <strong>and</strong> order <strong>of</strong> restriction<br />
fragments Converts “optical” data into digital data - barcodes<br />
Overlapping single molecule maps are aligned to<br />
produce a map assembly covering an entire<br />
chromosome
Multiple Coverage is Necessary <strong>for</strong> Accurate<br />
Map Assembly
Alignment <strong>of</strong> Optical Maps to a Reference<br />
Genome Map
Insertions, Deletions, <strong>and</strong> Rearrangements Can be<br />
Identified from Optical Mapping
Optical Map <strong>of</strong> EC536 Shows a Deletion <strong>of</strong> ~65<br />
kb Occurs in EC536 Relative to EDL933<br />
EDL933<br />
1331570bp - 1394263bp<br />
152 158<br />
~63 kb<br />
Deletion<br />
EC536<br />
EDL933 Position 1377163 – 1388578 is O-isl<strong>and</strong> #45<br />
Region <strong>of</strong> the EDL933 chromosome not homologous to E. coli K-12 MG1655
Optical Maps are Well Suited <strong>for</strong> Strain<br />
Identification <strong>and</strong> Strain Relatedness Studies
Cladistic Analysis <strong>of</strong><br />
Escherichia coli O157:H7
S. enterica<br />
mdh Parsimony<br />
I<br />
II<br />
ECOR10<br />
ECOR6<br />
ECOR14<br />
ECOR27<br />
ECOR32<br />
ECOR69<br />
ECOR70<br />
ECOR58<br />
ECOR64<br />
ECOR52<br />
ECOR62<br />
ECOR61<br />
ECOR59<br />
ECOR66<br />
ECOR35<br />
ECOR40<br />
ECOR47<br />
ECOR44<br />
ECOR46<br />
ECOR50<br />
ECOR49<br />
ECOR38<br />
ECOR37<br />
ECOR30<br />
ECOR45<br />
ECOR10<br />
ECOR6<br />
ECOR14<br />
ECOR27<br />
ECOR32<br />
ECOR69<br />
ECOR70<br />
ECOR58<br />
ECOR64<br />
ECOR52<br />
ECOR62<br />
ECOR61<br />
ECOR59<br />
ECOR66<br />
ECOR35<br />
ECOR40<br />
ECOR47<br />
ECOR44<br />
ECOR46<br />
ECOR50<br />
ECOR49<br />
ECOR38<br />
ECOR37<br />
ECOR30<br />
ECOR45<br />
mdh Neighbor-Joining
S. enterica<br />
I<br />
II<br />
mdh Parsimony<br />
ECOR10<br />
ECOR6<br />
ECOR14<br />
ECOR27<br />
ECOR32<br />
ECOR69<br />
ECOR70<br />
ECOR58<br />
ECOR64<br />
ECOR52<br />
ECOR62<br />
ECOR61<br />
ECOR59<br />
ECOR66<br />
ECOR35<br />
ECOR40<br />
ECOR47<br />
ECOR44<br />
ECOR46<br />
ECOR50<br />
ECOR49<br />
ECOR38<br />
ECOR37<br />
ECOR30<br />
ECOR45<br />
ECOR10<br />
ECOR6<br />
ECOR14<br />
ECOR27<br />
ECOR32<br />
ECOR69<br />
ECOR70<br />
ECOR58<br />
ECOR64<br />
ECOR52<br />
ECOR62<br />
ECOR61<br />
ECOR59<br />
ECOR66<br />
ECOR35<br />
ECOR40<br />
ECOR47<br />
ECOR44<br />
ECOR46<br />
ECOR50<br />
ECOR49<br />
ECOR38<br />
ECOR37<br />
ECOR30<br />
ECOR45<br />
mdh Neighbor-Joining
ECOR27<br />
ECOR70<br />
ECOR10<br />
ECOR61<br />
ECOR45<br />
ECOR14<br />
ECOR50<br />
ECOR49<br />
ECOR46<br />
ECOR44<br />
ECOR47<br />
ECOR35<br />
ECOR64<br />
ECOR62<br />
ECOR66<br />
ECOR52<br />
ECOR59<br />
ECOR40<br />
ECOR38<br />
ECOR32<br />
ECOR58<br />
ECOR69<br />
ECOR37<br />
S. enterica<br />
ECOR10<br />
ECOR6<br />
ECOR14<br />
ECOR27<br />
ECOR32<br />
ECOR69<br />
I<br />
II<br />
I<br />
II<br />
ECOR70<br />
ECOR58<br />
ECOR64<br />
ECOR52<br />
ECOR62<br />
ECOR61<br />
ECOR59<br />
ECOR66<br />
ECOR35<br />
ECOR40<br />
ECOR47<br />
ECOR44<br />
ECOR46<br />
ECOR50<br />
ECOR49<br />
ECOR38<br />
ECOR37<br />
mdh<br />
mutS<br />
ECOR30<br />
ECOR6<br />
ECOR30<br />
ECOR45
ECOR27<br />
ECOR70<br />
ECOR10<br />
ECOR61<br />
ECOR30<br />
ECOR45<br />
ECOR6<br />
ECOR14<br />
ECOR50<br />
ECOR49<br />
ECOR46<br />
ECOR44<br />
ECOR47<br />
ECOR35<br />
ECOR64<br />
ECOR62<br />
ECOR66<br />
ECOR52<br />
ECOR59<br />
ECOR40<br />
ECOR38<br />
ECOR32<br />
ECOR58<br />
ECOR69<br />
ECOR37<br />
S. enterica<br />
ECOR10<br />
ECOR6<br />
ECOR14<br />
ECOR27<br />
ECOR30<br />
ECOR45<br />
ECOR32<br />
ECOR69<br />
I<br />
II<br />
I<br />
II<br />
ECOR70<br />
ECOR58<br />
ECOR64<br />
ECOR52<br />
ECOR62<br />
ECOR61<br />
ECOR59<br />
ECOR66<br />
ECOR35<br />
ECOR40<br />
ECOR47<br />
ECOR44<br />
ECOR46<br />
ECOR50<br />
ECOR49<br />
ECOR38<br />
ECOR37<br />
mdh<br />
mutS
Nucleotide at Pos:<br />
CLADE 17 47 95 167 197 218 234 236 264 272 284 296 311 323 341 350 353<br />
1 c/g c c c a c t a t t t t [a] g g t g<br />
2 [t] c c c a [t] t a t t t [c] g g g t g<br />
3 c c c c a c t a t t t t g g g t g<br />
4 c [a] [g] [t] a c t a t [c] t t g [a] [a] [c] [a]<br />
5 c c c c [g] c t a t t t t g g g t g<br />
6 c/g c c c/g a c [c] [g] [c] t [c] t g g g t g<br />
4 (3)<br />
60<br />
11 (6)<br />
99<br />
2 (2)<br />
5 (0)<br />
2 (0)<br />
86<br />
3 (1)<br />
79<br />
4 (0)<br />
92<br />
3 (1)<br />
91<br />
4 (3)<br />
78<br />
1 (1)<br />
S. dysenteriaeATCC20132<br />
1 (1)<br />
S. dys ATCC20133<br />
5 (4) S. dysenteriae ATCC20174<br />
100<br />
S. dysenteriae ATCC20028<br />
3 (1) S. dysenteriae ATCC20011<br />
1 (0)<br />
66<br />
S. dysenteriae FDA567<br />
3 (1)<br />
7 (6) S. dys ATCC20130<br />
1 (1)<br />
96<br />
S. dysenteriae FDA377<br />
2 (0)<br />
E. coli FDA269 (O136-EIEC)<br />
E. coli FDA400 (O26:H11-EHEC)<br />
E. coli FDA329 (O25:K98-ETEC)<br />
E. coli FDA320 (O78:H11-ETEC)<br />
E. coli ATCC35401 (O78:H11-ETEC)<br />
E. coli FDA162 (O152-EIEC)<br />
1(1)<br />
5 (1)<br />
85 E. coli FDA164 (O143-EIEC)<br />
E. coli ATCC43893 (O124:NM-EIEC)<br />
10 (7)<br />
E. coli FDA321 (O55:NM-EPEC)<br />
100<br />
E. coli FDA322 (O127-EPEC)<br />
E. coli ATCC43887 (O111-ETEC)<br />
E. coli FDA319 (O148:H28-ETEC)<br />
1<br />
2<br />
3<br />
4<br />
5<br />
Signature<br />
synapomorphies<br />
unique to individual<br />
clades <strong>of</strong> pathogenic<br />
E. coli<br />
9 (3)<br />
mutS<br />
89<br />
3 (1) E. coli DEC5C (O55:H7-EPEC)<br />
4 (2)<br />
83<br />
4 (2) E. coli DEC5D (O55:H7-EPEC)<br />
97 4 (1) E. coli DEC5E (O55:H7-EPEC)<br />
98<br />
E. coli DEC5B (O55:H7-EPEC)<br />
1 (0)<br />
E. coli 86-24 (O157:H7-EHEC)<br />
E. coli FRIK583 (O157:H7-EHEC)<br />
E. coli FDA486 (O157:H7-EHEC)<br />
E. coli FDA484 (O157:H7-EHEC)<br />
E. coli<br />
4 (0)<br />
FDA488 (O157:H7-EHEC)<br />
2 (0) E. coli FDA536 (O157:H7-EHEC)<br />
38 E. 3 (2) coli 93-111 (O157:H7-EHEC)<br />
E. coli 95-001 (O157:H7-EHEC)<br />
6
mutS<br />
H19<br />
V16<br />
V5<br />
66<br />
H26<br />
V27<br />
PM4<br />
H1<br />
PM5<br />
U3<br />
V3<br />
V19<br />
V1<br />
V22<br />
V24<br />
V6<br />
78<br />
H17<br />
U7<br />
V8<br />
V21<br />
PM3<br />
H5<br />
189<br />
PM2<br />
H9<br />
U4<br />
H7<br />
V31<br />
V7<br />
5-2<br />
U5<br />
PM8<br />
V23<br />
V15<br />
H25<br />
H35<br />
H16<br />
20<br />
77<br />
H15<br />
H38<br />
PM6<br />
168<br />
V29<br />
V20<br />
H2<br />
H18<br />
PM1<br />
V12<br />
U1<br />
H27<br />
V28<br />
V9<br />
PM9<br />
V26<br />
U6<br />
H8<br />
180<br />
V11<br />
V10<br />
V14<br />
51<br />
A<br />
B<br />
C<br />
D<br />
E<br />
F<br />
G<br />
Signature nucleotide synapomorphies<br />
unique to individual mutS clades<br />
Alignment mutS clades a<br />
pos. no. A B C D E F G<br />
9 [A] G G G G G G<br />
39 C C C C C C [T]<br />
45 C A/C A/C [G] C C C<br />
75 G G G G G A A<br />
76 T T T T T C C<br />
93 G G G G G C C<br />
96 T T T T T C C<br />
105 C C C [T] C C C<br />
108 C C C C C T T<br />
111 A A A [C] A A A<br />
117 G G G G G A A<br />
123 A A A [G] A A A<br />
135 C C C [G] C A A<br />
138 G G G G G T T<br />
168 [T] C C C G G [A]<br />
174 T T T T [C] T T<br />
177 T T T T T [C] T<br />
189 G G G [A] G G G<br />
192 T T T T T [C] T<br />
216 C C C C C [T] C<br />
222 C [T] C C C C C<br />
225 C C C [T] C C C<br />
249 A A A A A A [G]<br />
262 T T T T T T [C]<br />
264 G G G G G [A] G<br />
270 T T T [C] T T T<br />
291 C C [T] C C C C<br />
303 T T T [C] T T T<br />
339 A A A A A G G<br />
348 C C C C C T T
E. coli O157:H7 SCCM<br />
7 genes<br />
3232 bp<br />
54<br />
59<br />
100<br />
Phylogenetic mapping <strong>of</strong> the roi gene<br />
27<br />
100<br />
11<br />
G 189 A 391<br />
A 189 C 391 B roi<br />
69<br />
B roi<br />
B roi<br />
B roi<br />
roi<br />
roi<br />
roi<br />
roi<br />
roi<br />
A<br />
roi<br />
roi<br />
A 189 A 391<br />
A roi<br />
roi<br />
A 189 A 391<br />
A roi<br />
roi<br />
G 189 A 391<br />
A roi<br />
roi<br />
A 189 C 391<br />
roi<br />
86<br />
99<br />
roi<br />
roi<br />
roi<br />
A roi<br />
A roi<br />
B roi<br />
AB roi<br />
B roi<br />
C roi<br />
505<br />
512<br />
1219<br />
AB1<br />
554<br />
558<br />
559<br />
95-001<br />
866<br />
510<br />
506<br />
509<br />
1214<br />
484<br />
AB3<br />
869<br />
86-24<br />
868<br />
DEC5A (O55:H7)<br />
DEC5C (O55:H7)<br />
1216<br />
884<br />
DEC7A (O157:H43) + +<br />
521 (O26:H11)<br />
1223<br />
I<br />
II<br />
III<br />
IV<br />
V<br />
O157<br />
Strains<br />
I<br />
stx<br />
+ +<br />
+ +<br />
+ +<br />
+ +<br />
+ +<br />
+ +<br />
+ +<br />
+ +<br />
+ +<br />
II<br />
- +<br />
- -<br />
+ -<br />
- -<br />
- -<br />
+ +<br />
+ +<br />
- +<br />
+ +<br />
- -<br />
- -<br />
- -<br />
- -<br />
+ -<br />
- -
CLADISTIC BIOMARKERS<br />
“CLADE-BREAKING”<br />
SEQUENCE-BASED<br />
“BINNING”<br />
DELINEATION OF<br />
PATHOGEN<br />
POPULATIONS<br />
USING REAL<br />
SEQUENCE<br />
CHANGES<br />
S<br />
Y<br />
N<br />
A<br />
P<br />
O<br />
M<br />
O<br />
R<br />
P<br />
H<br />
Y<br />
A<br />
U<br />
T<br />
A<br />
P<br />
O<br />
M<br />
O<br />
R<br />
P<br />
H<br />
Y<br />
STRAIN-SPECIFIC<br />
UNIQUE<br />
ATTRIBUTE<br />
DELINEATION OF<br />
INDIVIDUAL<br />
PATHOGENIC<br />
STRAINS<br />
USING REAL<br />
SEQUENCE<br />
CHANGES<br />
Exclusion<br />
Attribution
Microbiological Methods I:<br />
‣ Plating on Sorbitol-MacConkey<br />
K-12 Strain 868
Microbiological Methods lI:<br />
‣ Plating on Tellurite<br />
K-12 Strain 86-24
Microbiological Methods Ill:<br />
‣ Plating on MUG <strong>for</strong> β-glucuronidase
Detecting Contaminants:
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