Download Power Point Presentation - Institute of Food Technologists

First Annual IFT Food Protection
& Defense Research Conference
Atlanta Marriot Marquis November 2-4, 2005
Thomas A. Cebula, Ph.D.
Director, Office of Applied Research
and Safety Assessment
CFSAN
The TIGER Biosensor: Rapid Broad Range Pathogen Detection in
Diagnostics and Food Protection
Lawrence Blyn, Ph.D., Ibis Therapeutics
Improved diagnostic tests for avian influenza surveillance
Blanca Lupiani, Ph.D., Texas A&M University
Efficient nucleic signature development for broad
spectrum pathogen detection
Jason Gans, Ph.D., Los Alamos National Lab
Expanding the Use of Validated Rapid Microbiological Methods
to New Food Matrices
Willis Fedio, Ph.D., New Mexico State University
“Before beginning a Hunt, it is wise
to ask someone what you are looking
for before you begin looking for it.”
--A.A. Milne, 1926, Pooh's Little Instruction Book
The need for:
•Identifying and recognizing patterns
in a disease outbreak
•Communicating those patterns to the
public health community at large
•Determining the pathogen involved
•Containing the outbreak
•Tracing the microbe to its source
--Events of 9/11/2001 and after
The forensic continuum for strain identification
“Strain could
not have come
from…”
“Strain did
absolutely
come from…”
Exclusion
Attribution
Methods Validation
Differentiation
of Strains
Biomarker Stability
Extent of
Genomic
Diversity
Food Safety
Detection at Genus Level
Detection at Species Level
Detection at Subspecies Level
Detection at Serotype or Serovar Level
Food Defense
Detection at Genus Level
Detection at Species Level
Detection at Subspecies Level
Detection at Serotype or Serovar Level
but, for attribution,
Detection at Strain Level
Bacterial Diversity
Whose strains define the universe of
diversity that we study?
There is a genuine lack of appreciation
concerning the extent of diversity that
exists among plant and animal
pathogens.
At this writing, with the complete sequence of four
bacterial genomes already known and a fifth, that of E.
We examine basic tenets of
Cebula LeClerc,
coli, to be unveiled shortly, some still myopically
evolution, i.e., the relative roles
1997
question whether bacteria genomics will offer many
that mutation and recombination
surprises. The Salmonella sequencing project has been
play in instituting the genetic
impacted, hampered by the belief that Salmonella is too
diversity upon which selection
much like E. coli to warrant intense effort. This apathy
works to establish bacteria in
seems steeped in the naive assumption that experiments
particular niches. Specifically, we
conducted in an unnatural setting (the test tube) can be
delve into the importance of
correlated directly with how bacteria behave in their
particular mutator phenotypes and
natural environment. That we do not share this belief is
their potential contributions
obvious—to do so belies an appreciation of their
to homeologous recombination in
differences.
bacteria. The implications for
Homeologous Recombination: Ingredients for Rapid Evolution
rapid Hypermutability
evolution& and
the
emergence of new pathogens
are discussed.
Cebula and LeClerc,
1997
265 Microbial Genomes Sequenced
•
21 Archaea
• 211 Bacteria
•
33 Eukaryotes
In Progress
• 1470 Microbial Genomes
www.Genomesonline.org As of June 2, 2005
298 Microbial Genomes Sequenced
•
23 Archaea
• 236 Bacteria
•
39 Eukaryotes
In Progress
• 1589 Microbial Genomes
www.Genomesonline.org As of September 16, 2005
303 Microbial Genomes Sequenced
•
24 Archaea
• 240 Bacteria
•
39 Eukaryotes
In Progress
• 1608 Microbial Genomes
www.Genomesonline.org As of October 20, 2005
313 Microbial Genomes Sequenced
•
25 Archaea
• 249 Bacteria
•
39 Eukaryotes
In Progress
• 1686 Microbial Genomes
www.Genomesonline.org As of October 25, 2005
100
100
Prior
Agreement 3
Housekeeping
100
100
100
90
100
96
100
100
100
100
74
100
73
99
100
100
95
77
100
100
100
69
100
100
100
100
S3044
S3041
S2979
S2978
S3014
S3013
S3027
S3015
S3057
S2995
S4194
S3333
S2985
S2993
S2983
S2980
EC52
EC64
V
S. bongori
IIIb
VII
IV
S. enterica
I
VI
S. typhimurium
S. typhi
I
III
S. arizonae
E. coli
outgroup
mdh
gapA
icd
HGT “Clouds” Surrounding E. Coli and
S. enterica subspecies I
ASSORTATIVE YES
mdh
B21
B20
B34
B64
B25
B50
B8
B3
B21
B64
B34
B25
B20
B8
B3
B50
mutS
mutS
YES
Recombination
INTRAGENIC
INTERSPECIES
RECOMBINATION
mdh
mutS
I
VI
II
IV
IIIB
IIIA
V
ASSORTATIVE NO
SARC 3333
INTRAGENIC
mutS
YES
E. coli
Salmonella
E. coli
Salmonella
INTRA-SUBSPECIES
RECOMBINATION
(among S. enterica
subspecies I strains)
SARB
INTRASPECIES RECOMBINATION
(among S. enterica subspecies)
SARC
Genetic Distance
mutS
mdh
ASSORTATIVE NO
INTRAGENIC NO
HGT “Clouds” Surrounding E. Coli and
S. enterica subspecies I
Whole Genome DNA Microarrays
Salmonella enterica serovar Typhimurium
LT2 Genome
4,857 kb
4,596 ORFs
Salmonella
Microarrays
Containing
~4,500 PCRAmplified
Salmonella
Typhimurium
Genes
Supplementing the Typhimurium Microarray with Unique
Genes from Salmonella Typhi and Salmonella Enteritidis:
A Non-Redundant Microarray Representing Related
Bacteria
471
Typhi
ORFs
Genes Unique
to Typhi and
Enteritidis
added to Array
284
Enteritidis
ORFs
• NonRedundant
Salmonella
Enteritidis DNA
Microarray:
• 5184 Unique
Genes per Array
Spotted in
Triplicate
• 15,552 Spots
Total per Slide
Gene
Expression or
Genomic
Comparison
Studies
Food Defense
Finding a use for a method
Is not synonymous with
Finding a method that is useful
Tiling Microarrays
Pyrosequencing
Optical Mapping
Sampling ~1% of the E.coli O157:H7
Genome at Random
5.5 Mb Genome
- Sampled 1 kb per ~100 kb
- Tiled 60 Loci onto Arrays
Perna, N.T. et al. Nature 409, 529-533 (2001)
Interrogating 12 Independent Strains in Parallel
1.5 cm
~14,000 Spots (oligos)
~4mm
2 cm
High Density Oligonucleotide Tiling Arrays Provide a
“High Resolution” Snapshot of the Genome
Reference Genome
Test Genome
29-mer Tiling Array
Probes
Mutation
•
Our Tiled Strategy Uses a 5 nt Probe Spacing
•
For a random sampling of ~1% of the genome, 1 kb of genome
sequence was selected at 60 equally spaced regions around the EDL933
chromosome.
Relative Probe Intensity vs. Genome Position
Probes reporting
a deletion in the
test strain
Probes reporting
a SNP in the
test strain
Probes reporting identical
sequence between strains
AB6
AB1
508
AB5
506
Pyrosequencing – Sequencing by Light
CGT
Polymerase
CGT
CGT
CGT
Pooled
Genomic
DNA
AlleleSpecific
PCR
i
Sulfurylase
CGT
CGT
CAT
90% C
Luciferase
CGT
CGT
10% T
CGT
C
T
strain
serotype
4889b
4889a
5210
i559
roi
NG1
NG2
NG7
4777
5096
barcode
EC1214
O157:H7
A
G
C
G
G
del
T
G
G
G
0000010100
EC506
O157:H7
A
G
C
G
A
A
T
C
G
G
0000100000
EC868
O157:H7
A
G
C
G
A
A
T
C
G
G
0000100000
86-24
O157:H7
A
G
C
G
A
A
T
C
G
G
0000100000
EC509
O157:H7
A
G
C
G
A
A
T
C
A
G
0000100010
EC536
O157:H7
A
G
C
G
A
del
T
G
G
G
0000110100
EC484
O157:H7
A
G
C
G
GA
A
T
C
G
G
0000200000
EC869
O157:H7
A
G
C
A
A
del
G
G
G
G
0001111100
AB3
O157:H-
A
G
C
A
A
del
G
G
G
G
0001111100
EC510
O157:H-
A
G
C
A
A
del
-
-
G
G
0001112200
EC554
O157:H7
A
G
T
G
G
A
T
C
G
C
0010000001
EC559
O157:H7
A
G
T
G
G
A
T
C
G
C
0010000001
EC866
O157:H7
A
G
T
G
G
A
T
C
G
C
0010000001
EC1219
O157:H7
A
G
T
G
G
A
T
C
G
C
0010000001
95-01A
O157:H7
A
G
T
G
G
A
T
C
G
C
0010000001
AB1
O157:H-
A
G
T
G
AG
A
T
C
G
C
0010200001
EC558
O157:H7
A
G
T
G
GA
A
T
C
G
C
0010200001
EC505
O157:H7
A
A
C
G
A
A
T
C
A
G
0100100010
EC512
O157:H7
A
A
C
G
A
-
T
C
A
G
0100120010
DEC7A
O157:H43
G
G
C
G
G
del
T
C
G
G
1000010000
EC521
O26:H11
G
G
C
A
G
del
G
G
G
G
1001011100
EC1216
N.D.
G
G
C
A
A
del
-
-
G
G
1001112200
EC884
N.D.
G
G
C
A
AG
del
-
-
G
G
1001212200
DEC5C
O55:H7
G
G
C
-
AG
del
G
-
G
G
1002211200
Phylogenetic mapping of the roi gene
G189A391
+
+
1219
+
+
AB1
+
+
554
+
+
+
+
559
+
+
95-001
+
+
866
+
+
O157
Strains -
+
+
-
1214
-
-
484
-
-
AB3
+
+
+
+
86-24
-
+
868
+
+
DEC5A (O55:H7)
-
-
roi
DEC5C (O55:H7)
-
-
roi
1216
-
-
roi
884
-
-
DEC7A (O157:H43) +
+
521 (O26:H11)
+
-
1223
-
-
A roi
AB roi
B roi
A189C391
B roi
69
B roi
B roi
B roi
roi
506
roi
509
roi
A roi
A189A391
A roi
roi
A189A391
A roi
roi
G189A391
A roi
54
A189C391
roi
roi
11
99
27
558
roi
roi
59
512
510
100
roi
II
+
B roi
100
505
+
86
E. coli O157:H7 SCCM
7 genes
3232 bp
stx
I
A roi
C roi
869
I
II
III
IV
V
-
CLADISTIC BIOMARKERS
“CLADE-BREAKING”
SEQUENCE-BASED
“BINNING”
DELINEATION OF
PATHOGEN
POPULATIONS
USING REAL
SEQUENCE
CHANGES
Exclusion
S
Y
N
A
P
O
M
O
R
P
H
Y
A
U
T
A
P
O
M
O
R
P
H
Y
STRAIN-SPECIFIC
UNIQUE
ATTRIBUTE
DELINEATION OF
INDIVIDUAL
PATHOGENIC
STRAINS
USING REAL
SEQUENCE
CHANGES
Attribution
E. coli K12 vs. E. coli
O157:H7
Islands or
Archipelagos?
Mother Nature is the Quintessential
Terrorist—she has been manipulating
genomes for eons. Man, on the other
hand, has been at it for just a couple
of decades. We should look,
therefore, to the “docking sites” of
recombination that Mother Nature has
used—these sites will be those likely to
be used in strain manipulations.
Optical Mapping: A Single Molecule Technique
for Generating Whole Genome Restriction Maps
Genome Map
Optical Mapping: Image Analysis
Single DNA molecule on Optical Chip after digestion, staining
Image analysis software measures size and order of restriction
fragments Converts “optical” data into digital data - barcodes
Overlapping single molecule maps are aligned to
produce a map assembly covering an entire
chromosome
Multiple Coverage is Necessary for Accurate
Map Assembly
Optical mapping;
s#114-115
Sakai
1276
EDL933
1225
502
533
507
536
AB1
869
1231
s#129
s#168-169
Optical mapping; Inversions
EC536-EDL933-Sakai
1,912,000
2,305,000
EC536
Sakai
2,225,000
Optical mapping;
502_EDL933 inversion
e#151
EDL#151-161
151 15,289 bp
152 29,558
Inverted 502_EDL933
e#357
EDL#356-7
356 27,972 bp
357 17,040 bp
Optical mapping; Inversions
Sakai vs EDL933 vs EC533
Sakai vs EDL933 vs inverted map of EC533
Optical Maps are Well Suited for Strain
Identification and Strain Relatedness Studies
“Don’t think to hunt two
hares with one dog.”
--Ben Franklin