Thomas A. Cebula, Ph.D. Director, Office of Applied Research and Safety Assessment CFSAN

Thomas A. Cebula, Ph.D.Director, Office of Applied Research

and Safety AssessmentCFSAN

First Annual IFT Food Protection & Defense Research Conference

Atlanta Marriot Marquis November 2-4, 2005

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 Before beginning a Hunt, it is wise to ask someone what you wise to ask someone what you are looking for before you begin are looking for before you begin looking for it.”looking for it.”

--A.A. Milne, 1926, Pooh's Little Instruction Book--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

Exclusion Attribution

Differentiation of Strains

Extent of Genomic Diversity

“Strain did absolutely come from…”

“Strain could not have come from…”

Methods Validation

Biomarker Stability

Detection at Genus Level

Detection at Species Level

Detection at Subspecies Level

Detection at Serotype or Serovar Level

Food Safety

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

Food Defense

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.

We examine basic tenets of evolution, i.e., the relative roles that mutation and recombination play in instituting the genetic diversity upon which selection works to establish bacteria in particular niches. Specifically, we delve into the importance of particular mutator phenotypes and their potential contributionsto homeologous recombination in bacteria. The implications for rapid evolution and the emergence of new pathogens are discussed.

Cebula LeClerc, 1997

Cebula and LeClerc, 1997

At this writing, with the complete sequence of four bacterial genomes already known and a fifth, that of E. coli, to be unveiled shortly, some still myopically question whether bacteria genomics will offer many surprises. The Salmonella sequencing project has been impacted, hampered by the belief that Salmonella is too much like E. coli to warrant intense effort. This apathy seems steeped in the naive assumption that experiments conducted in an unnatural setting (the test tube) can be correlated directly with how bacteria behave in their natural environment. That we do not share this belief is obvious—to do so belies an appreciation of their differences.

At this writing, with the complete sequence of four bacterial genomes already known and a fifth, that of E. coli, to be unveiled shortly, some still myopically question whether bacteria genomics will offer many surprises. The Salmonella sequencing project has been impacted, hampered by the belief that Salmonella is too much like E. coli to warrant intense effort. This apathy seems steeped in the naive assumption that experiments conducted in an unnatural setting (the test tube) can be correlated directly with how bacteria behave in their natural environment. That we do not share this belief is obvious—to do so belies an appreciation of their differences.

Hypermutability & Homeologous Recombination: Ingredients for Rapid Evolution

• 21 Archaea

• 211 Bacteria

• 33 Eukaryotes

265 Microbial Genomes Sequenced

In Progress• 1470 Microbial Genomes

www.Genomesonline.org As of June 2, 2005

• 23 Archaea

• 236 Bacteria

• 39 Eukaryotes



www.Genomesonline.org As of September 16, 2005

• 24 Archaea

• 240 Bacteria

• 39 Eukaryotes



www.Genomesonline.org As of October 20, 2005

• 25 Archaea

• 249 Bacteria

• 39 Eukaryotes



www.Genomesonline.org As of October 25, 2005

EC64EC52

100

100

100

100

100

100

99

100

77

74

90

100

100

100 100

100

100

100

100

100

95

69

73

96

100

100

100

100

S3044

S3041

S2979

S2978

S3014

S3013

S3027

S3015

S3057

S2995

S4194

S3333

S2985

S2993

S2983

S2980

V

VII

IV

VI

I

I

IIIb

III

E. colioutgroup

S. bongori

S. arizonae

S. enterica

S. typhimuriumS. typhi

PriorAgreement -3Housekeeping

mdhgapAicd

HGT “Clouds” Surrounding E. Coli and S. enterica subspecies I

Rec

om

bin

atio

n

Genetic Distance

E. coliE. coli

SalmonellaSalmonella

mdhmutS

ASSORTATIVE NOINTRAGENIC NO

INTERSPECIESRECOMBINATION

mdh mutS

mutS

ASSORTATIVE NO

INTRAGENIC YES

IVIIIIVIIIBIIIAV

SARC 3333

INTRASPECIES RECOMBINATION(among S. enterica subspecies)

SARCSARB

INTRA-SUBSPECIES RECOMBINATION(among S. enterica

subspecies I strains)

mdhB21B64B34B25B20B8B3B50

B21B20B34B64B25B50B8B3

mutS

ASSORTATIVE YES

INTRAGENIC YES

mutS

HGT “Clouds” Surrounding E. Coli and S. enterica subspecies I

Salmonella enterica serovar Typhimurium LT2 Genome

4,857 kb4,596 ORFs

Salmonella Microarrays Containing

~4,500 PCR-Amplified

Salmonella Typhimurium

Genes

Whole Genome DNA Microarrays

Supplementing the Typhimurium Microarray with Unique Genes from Salmonella Typhi and

Salmonella Enteritidis: A Non-Redundant Microarray Representing

Related Bacteria

Genes Unique to Typhi and Enteritidis

added to Array

471Typhi ORFs

284 Enteritidis

ORFs

• Non-Redundant Salmonella

Enteritidis DNA Microarray:

• 5184 Unique Genes per Array

Spotted in Triplicate

• 15,552 Spots Total per Slide

Gene Expression or

Genomic Comparison

Studies

Finding a use for a method

Is not synonymous with

Finding a method that is useful

Food Defense

Tiling Microarrays

Pyrosequencing

Optical Mapping

Perna, N.T. et al. Nature 409, 529-533 (2001)

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

Interrogating 12 Independent Strains in Parallel

2 cm

1.5 cm

~4mm

~14,000 Spots (oligos)

Reference Genome

Test Genome

29-mer Tiling Array Probes

Mutation

High Density Oligonucleotide Tiling Arrays Provide a “High Resolution” Snapshot of the

Genome

• 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.

-1.0

1.0

3.0

5.0

7.0

9.0

11.0

13.0

15.0

17.0

19.0

21.0

23.0

25.0

27.0

29.0

31.0

192

142

1842

8327

6423

3685

6446

0705

5528

4664

4986

7371

2782

9268

9214

0910

1354

911

0569

011

9783

112

8997

213

8211

214

7425

315

6639

416

5853

517

5067

518

4281

619

3495

720

2709

821

1923

822

1137

923

0352

023

9566

124

8780

125

7994

226

7208

327

6422

428

5636

429

4850

530

4064

631

3278

732

2492

733

1706

834

0920

935

0135

035

9349

036

8563

137

7777

238

6991

339

6205

340

5419

441

4633

542

3847

643

3061

644

2275

745

1489

846

0703

946

9917

947

9132

048

8346

149

7560

250

6774

251

5988

352

5202

453

4416

554

3630

5

Probes reporting identical sequence

between strains

Probes reporting a

SNP in the test strain

Probes reporting a

deletion in the test strain

Relative Probe Intensity vs. Genome Position

506AB5

508

AB6 AB1

CGT

CGT

CGT

CGT

CGT

CGT

CAT

CGT

CGT

CGT

90% C

10% T

C T

Pooled Genomic

DNA

Allele-Specific

PCR

Polymerase

Sulfurylase

Luciferase

i

Pyrosequencing – Sequencing by LightPyrosequencing – Sequencing by Light

strainstrain serotypeserotype 4889b4889b 4889a4889a 52105210 i559i559 roiroi NG1NG1 NG2NG2 NG7NG7 47774777 50965096

EC1214EC1214 O157:H7O157:H7 AA GG CC GG GG deldel TT GG GG GG

EC506EC506 O157:H7O157:H7 AA GG CC GG AA AA TT CC GG GG

EC868EC868 O157:H7O157:H7 AA GG CC GG AA AA TT CC GG GG

86-2486-24 O157:H7O157:H7 AA GG CC GG AA AA TT CC GG GG

EC509EC509 O157:H7O157:H7 AA GG CC GG AA AA TT CC AA GG

EC536EC536 O157:H7O157:H7 AA GG CC GG AA deldel TT GG GG GG

EC484EC484 O157:H7O157:H7 AA GG CC GG GAGA AA TT CC GG GG

EC869EC869 O157:H7O157:H7 AA GG CC AA AA deldel GG GG GG GG

AB3AB3 O157:H-O157:H- AA GG CC AA AA deldel GG GG GG GG

EC510EC510 O157:H-O157:H- AA GG CC AA AA deldel -- -- GG GG

EC554EC554 O157:H7O157:H7 AA GG TT GG GG AA TT CC GG CC




95-01A95-01A O157:H7O157:H7 AA GG TT GG GG AA TT CC GG CC

AB1AB1 O157:H-O157:H- AA GG TT GG AGAG AA TT CC GG CC

EC558EC558 O157:H7O157:H7 AA GG TT GG GAGA AA TT CC GG CC

EC505EC505 O157:H7O157:H7 AA AA CC GG AA AA TT CC AA GG

EC512EC512 O157:H7O157:H7 AA AA CC GG AA -- TT CC AA GG

DEC7ADEC7A O157:H43O157:H43 GG GG CC GG GG deldel TT CC GG GG

EC521EC521 O26:H11O26:H11 GG GG CC AA GG deldel GG GG GG GG

EC1216EC1216 N.D.N.D. GG GG CC AA AA deldel -- -- GG GG

EC884EC884 N.D.N.D. GG GG CC AA AGAG deldel -- -- GG GG

DEC5CDEC5C O55:H7O55:H7 GG GG CC -- AGAG deldel GG -- GG GG

barcodebarcode

00000101000000010100

00001000000000100000

00001000000000100000

00001000000000100000

00001000100000100010

00001101000000110100

00002000000000200000

00011111000001111100

00011111000001111100

00011122000001112200

00100000010010000001

00100000010010000001

00100000010010000001

00100000010010000001

00100000010010000001

00102000010010200001

00102000010010200001

01001000100100100010

01001200100100120010

10000100001000010000

10010111001001011100

10011122001001112200

10012122001001212200

10022112001002211200

505

512

1219

AB1

554

558

559

95-001

866

AB3

510

506

509

869

1214

484

86-24

868

DEC5A (O55:H7)

DEC5C (O55:H7)

1216

884

DEC7A (O157:H43)

521 (O26:H11)

1223

I

II

III

IV

V

roi

roi

roi

roi

roi

A roi

A roi

A roi

A roi

A roi

A roi

B roi

B roi

B roi

B roi

B roi

B roi

B roiA

C roi

O157Strains

roi

roi

roi

roi

roi

roi

roi

roi

86

69

100

100

54

59

27

11

99

stxI II

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

- +

- -

+ -

- -

- -

+ +

+ +

- +

+ +

- -

- -

- -

- -

+ +

+ -

- -

G189A391

A189C391

G189A391

A189C391

A189A391

A189A391

E. coli O157:H7 SCCM7 genes3232 bp

Phylogenetic mapping of the roi gene

CLADISTIC BIOMARKERS

SYNAPOMORPHY

AUTAPOMORPHY

“CLADE-BREAKING”

SEQUENCE-BASED“BINNING”

DELINEATION OF PATHOGEN

POPULATIONSUSING REAL SEQUENCECHANGES

STRAIN-SPECIFIC

UNIQUEATTRIBUTE

DELINEATION OF INDIVIDUAL

PATHOGENICSTRAINS

USING REAL SEQUENCECHANGES

Exclusion 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

Overlapping single molecule maps are aligned to produce a map assembly covering an entire

chromosome

Single DNA molecule on Optical Chip after digestion, staining

Optical Mapping: Image Optical Mapping: Image AnalysisAnalysis

Image analysis software measures size and order of restriction fragments Converts “optical” data into digital

data - barcodes

Multiple Coverage is Necessary for Accurate Map Assembly

Sakai

1276EDL933

1225

502

533

507

536

AB1

869

1231

s#114-115 s#129 s#168-169Optical mapping;

EC536-EDL933-Sakai

Sakai

EC536

1,912,000 2,305,000

2,225,000

Optical mapping; Inversions

Inverted 502_EDL933

EDL#151-161

151 15,289 bp

152 29,558

EDL#356-7

356 27,972 bp

357 17,040 bp

502_EDL933 inversion

e#151 e#357

Optical mapping;

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 Don’t think to hunt two hares with one dog.”hares with one dog.”

--Ben Franklin--Ben Franklin

Thomas A. Cebula, Ph.D. Director, Office of Applied Research and Safety Assessment CFSAN

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Transcript of Thomas A. Cebula, Ph.D. Director, Office of Applied Research and Safety Assessment CFSAN