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

1. DNA Detection Strategies: Sequences, Signatures, and Significance 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

2. 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

3. “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

4. --Events of 9/11/2001 and after • 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

5. The forensic continuum for strain identification “Strain could not have come from…” “Strain did absolutely come from…” Attribution Exclusion Methods Validation Biomarker Stability Differentiation of Strains Extent of Genomic Diversity

6. Food Safety Detection at Genus Level Detection at Species Level Detection at Subspecies Level Detection at Serotype or Serovar Level

7. 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

8. 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.

9. 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. 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 contributions to homeologous recombination in bacteria. The implications for rapid evolution and the emergence of new pathogens are discussed. Cebula LeClerc, 1997 Hypermutability & Homeologous Recombination: Ingredients for Rapid Evolution  Cebula and LeClerc, 1997

10. 265 Microbial Genomes Sequenced • 21 Archaea • 211 Bacteria • 33 Eukaryotes In Progress • 1470 Microbial Genomes www.Genomesonline.org As of June 2, 2005

11. 298 Microbial Genomes Sequenced • 23 Archaea • 236 Bacteria • 39 Eukaryotes In Progress • 1589 Microbial Genomes www.Genomesonline.org As of September 16, 2005

12. 303 Microbial Genomes Sequenced • 24 Archaea • 240 Bacteria • 39 Eukaryotes In Progress • 1608 Microbial Genomes www.Genomesonline.org As of October 20, 2005

13. 313 Microbial Genomes Sequenced • 25 Archaea • 249 Bacteria • 39 Eukaryotes In Progress • 1686 Microbial Genomes www.Genomesonline.org As of October 25, 2005

16. Salmonella mdh gapA icd

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

18. INTRA-SUBSPECIES RECOMBINATION (among S. enterica subspecies I strains) INTRASPECIES RECOMBINATION (among S. enterica subspecies) INTERSPECIES RECOMBINATION E. coli Salmonella SARC E. coli Salmonella SARB mutS mdh mdh mutS ASSORTATIVE NO INTRAGENIC NO I VI II IV IIIB mutS IIIA V ASSORTATIVE NO ASSORTATIVE YES mutS SARC 3333 B21 B21 mdh mutS B20 B64 INTRAGENIC YES B34 B34 B64 B25 B20 B25 B8 B50 B3 B8 B3 B50 INTRAGENIC YES Recombination Genetic Distance

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

20. Salmonella enterica serovar Typhimurium LT2 Genome 4,857 kb 4,596 ORFs Whole Genome DNA Microarrays Salmonella Microarrays Containing ~4,500 PCR-Amplified Salmonella Typhimurium Genes

21. Supplementing the Typhimurium Microarray with Unique Genes from Salmonella Typhi and Salmonella Enteritidis: A Non-Redundant Microarray Representing Related Bacteria 471 Typhi ORFs 284 Enteritidis ORFs • Non-Redundant Salmonella Enteritidis DNA Microarray: • 5184 Unique Genes per Array Spotted in Triplicate • 15,552 Spots Total per Slide Genes Unique to Typhi and Enteritidis added to Array Gene Expression or Genomic Comparison Studies

22. Food Defense Finding a use for a method Is not synonymous with Finding a method that is useful

23. Tiling Microarrays Pyrosequencing Optical Mapping

24. 5.5 Mb Genome - Sampled 1 kb per ~100 kb - Tiled 60 Loci onto Arrays Sampling ~1% of the E.coli O157:H7 Genome at Random Perna, N.T. et al. Nature 409, 529-533 (2001)

25. ~4mm Interrogating 12 Independent Strains in Parallel 1.5 cm ~14,000 Spots (oligos) 2 cm

26. 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.

27. Probes reporting a deletion in the test strain Probes reporting a SNP in the test strain Probes reporting identical sequence between strains Relative Probe Intensity vs. Genome Position

28. AB6 AB1 508 506 AB5

29. Pyrosequencing – Sequencing by Light Polymerase CGT CGT dNTP PP i CGT Sulfurylase CGT ATP CGT Luciferase CGT Light CAT CGT CGT CGT C T Pooled Genomic DNA Allele-Specific PCR 90% C 10% T

31. Phylogenetic mapping of the roi gene s t x I I I A r o i G A 5 0 5 + + 1 8 9 3 9 1 I A 8 6 r o i 5 1 2 + + B r o i 1 2 1 9 + + A B r o i A B 1 + + B r o i 5 5 4 + + E. coli O157:H7 SCCM 7 genes 3232 bp A C B 1 8 9 3 9 1 r o i I I 5 5 8 + + 6 9 B r o i 5 5 9 + + B r o i 9 5 - 0 0 1 + + B r o i 8 6 6 + + I I I r o i - + 5 1 0 1 0 0 O 1 5 7 5 0 6 - - r o i S t r a i n s + - r o i 5 0 9 I V - - 1 2 1 4 r o i - - 4 8 4 r o i A r o i A A 1 0 0 r o i 1 8 9 3 9 1 A B 3 + + A A A r o i r o i 1 8 9 3 9 1 8 6 9 + + V A r o i G A - + r o i 8 6 - 2 4 1 8 9 3 9 1 A 5 4 r o i A C + + 8 6 8 r o i 1 8 9 3 9 1 - - D E C 5 A ( O 5 5 : H 7 ) r o i - - r o i D E C 5 C ( O 5 5 : H 7 ) - - 1 2 1 6 r o i - - 8 8 4 r o i 5 9 + + D E C 7 A ( O 1 5 7 : H 4 3 ) 1 1 9 9 + - 5 2 1 ( O 2 6 : H 1 1 ) 2 7 C r o i 1 2 2 3 - -

32. CLADISTIC BIOMARKERS S Y N A P O M O R P H Y A U T A P O M O R P H Y “CLADE-BREAKING” SEQUENCE-BASED “BINNING” DELINEATION OF PATHOGEN POPULATIONS USING REAL SEQUENCE CHANGES STRAIN-SPECIFIC UNIQUE ATTRIBUTE DELINEATION OF INDIVIDUAL PATHOGENIC STRAINS USING REAL SEQUENCE CHANGES Attribution Exclusion

33. E. coli K12 vs. E. coli O157:H7 Islands or Archipelagos?

34. 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.

35. Optical Mapping: A Single Molecule Technique for Generating Whole Genome Restriction Maps Genome Map

36. 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

37. Multiple Coverage is Necessary for Accurate Map Assembly

38. s#168-169 s#114-115 s#129 Sakai 1276 EDL933 1225 502 533 507 536 AB1 869 1231 Optical mapping;

39. 1,912,000 2,305,000 EC536 Sakai 2,225,000 Optical mapping; Inversions EC536-EDL933-Sakai

40. Optical mapping; 502_EDL933 inversion e#151 EDL#151-161 151 15,289 bp 152 29,558 e#357 EDL#356-7 356 27,972 bp 357 17,040 bp Inverted 502_EDL933

41. Sakai vs EDL933 vs EC533 Sakai vs EDL933 vs inverted map of EC533 Optical mapping; Inversions

42. Optical Maps are Well Suited for Strain Identification and Strain Relatedness Studies

43. “Don’t think to hunt two hares with one dog.” --Ben Franklin