1. Escherichia coli O157:H7 and Shiga toxin-converting bacteriophage Edward G. Dudley, Ph.D Department of Food Science dudley@psu.edu

2. Escherichia coli O157:H7 history and food safety issues

3. http://wwwnc.cdc.gov/eid/article/17/1/pdfs/p2-1101.pdf Foodborne illness affects 48 million annually

4. Escherichia coli O157:H7 is a recently discovered foodborne pathogen

5. E. coli are first characterized by serotyping O-antigen – Somatic antigen – >170 known H-antigen – Flagellar antigen – >60 known E. coli O157:H7 Images from Dr. Erika A. Taylor’s website 5

6. Transmission of O157:H7 to humans usually starts with cattle Nougayrède et al., 2003

7. Control of E. coli O157:H7 in food supply starts at processing plant Beef represents ~50% of foodborne cases of disease

8. Evolution of one of the defining virulence factors of E. coli O157:H7

9. Escherichia coli evolves via genomic insertions and deletions “horizontal gene transfer”

10. Welch R A et al. PNAS 2002;99:17020-17024 ©2002 by National Academy of Sciences <30% of genes are shared in all Escherichia coli strains

11. Temperate phage are drivers of most genomic diversity in Escherichia coli diagram from Dr. Ken Todar

12. Many virulence factors are phage encoded Foodborne pathogens: Clostridium botulinum Staphylococcus aureus Vibrio cholerae Salmonella enterica Shigella flexneri Other Escherichia coli

13. Plunkett III, G., D. J. Rose, T. J. Durfee, and F. R. Blattner. 1999. Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157: H7: Shiga toxin as a phage late-gene product. J. Bacteriol. 181:1767- 1778. Recombination Regulation P R ’ Stx2A/B Lysis Capsid/tail fiber Escherichia coli O157:H7 carry an active phage that encodes a toxin cI = repressor protein

14. DNA damaging agents induce phage and increase toxin production 14 Ciprofloxacin (DNA gyrase)

15. One “A” subunit (319 amino acids) Five “B” subunits (91 amino acids) http://journal.frontiersin.org/Journal/10.3389/fcimb.2012.00081/full Shiga toxin blocks protein synthesis by targeting 28S rRNA AB 5 toxin

16. Do other organisms change amount of toxin produced?

17. Do gut organisms affect virulence of Escherichia coli O157:H7?

18. Experimental setup E. coli O157:H7 strain PA2 Plate counts ELISA for toxin production Commensal (non-pathogenic) +

19. Shiga toxin levels affected by other intestinal E. coli strains

20. Germ free mouse experiments Colonize with E. coli C600 Day -7 Inoculate with E. coli O157:H7 Day 0 Control Test Day 1 Day 6 Plate feces Sacrifice 5 mice

21. Increased virulence of E. coli O157:H7 in presence of non-pathogenic E. coli

22. Growing threats to the food supply

25. The Shiga toxin phage can mobilize to other E. coli, creating new pathogens enteroaggregative Escherichia coli Serotype O104:H4 Ill: 4,321. HUS: ~30% of cases. Dead: 50

26. Several “new” O-groups are of increasing concern YearSerogroupLocationFood/event# ill 2014O111MinnesotaCabbage (Applebees and Yard House restaurants) 13 O1215 StatesSprouts (Jimmy Johns, Pita Pit) 19 2013O1219 States (including PA) Farm Rich Products frozen snacks 35 2012O1455 StatesUnknown18 2011O26OH, PA, ALSprouts (Jimmy Johns)29 2010O1455 States (including PA) Shredded romaine lettuce 33 The “Big Six”: Shiga toxin E. coli of serogroups O26, O45, O103, O111, O121, O145