1. Escherichia coli Commensal found in large bowel in most mammals. Certain strains may cause disease: –Urinary tract infections –Sepsis/meningitis –Diarrhea

3. Diarrheagenic groups Enterotoxigenic E. coli (ETEC) Enteroinvasive E. coli (EIEC) Enteropathogenic E. coli (EPEC) Enteroaggregative E. coli (EAEC) Diffusely adherent E. coli (DAEC) Enterohemorrhagic E. coli (EHEC) –Escherichia coli O157:H7

5. EHEC Group defined by those strains that produce shiga-toxin (Stx1, Stx2) and cause hemorrhagic colitis (HC) and/or hemolytic uremic syndrome (HUS).

6. EHEC serotypes O157:H7-50% Non-O157 serotypes –O26:H11-21% –O111:NM-19% –O103:H2-10% –O121-8% –O145-6% –O45-6%

7. Virulence factors Shiga-like toxin –Stx1 and Stx2 –Main virulence factor –associated with HUS Intimin –Mediates attachment EHEC plasmid –Enterohemolysin –Catalase

8. From: Whittam, T.S. 1998. Escherichia coli O157:H7 and other Shiga- Toxin producing E. coli strains. J.B. Kaper and A. D. O’Brien ed.

9. Attaching and effacing (A/E) pathology

13. Shiga Toxin Encoded on Stx bacteriophage Originally discovered in Shigella dysenteriae (Stx1- like) Multiple variants-Stx1, Stx2 (Stx2c, d, e, f, g) AB-5 toxin (5 B components and one A component)

14. Shiga Toxin Toxin enters blood stream 5 B subunits bind to GB3/CD77 glycolipid receptor (Kidney). Translocates A subunit which is cleaved into an A1 peptide A1 peptide has N-glycosidase activity that inhibits protein synthesis through cleavage of 28S ribosomal RNA.

15. Disease associated with EHEC Phase 1: Presymptomatic stage –Acquisition of infection Ingestion of undercooked beef is major risk factor Many other vehicles for infection and reservoirs: water, vegetables, other mammals, etc. Very low infectious dose: 10-100 bacteria. –Incubation period 1-10 days Average ~4 days after ingestion

16. Disease associated with EHEC Phase 2: Symptomatic phase –Before bloody diarrhea Cramp-like abdominal pains Clingliness to a parent-lethargy Irritability and vomiting –Bloody Diarrhea (82%; O157: 38%; non-O157) Supportive therapy to monitor development of HUS HUS (7%; O157: 1.5% non-O157) occurs on average day 6.5 after bloody diarrhea begins.

17. Disease associated with EHEC Phase 3: –Microangiopathic sequelae Development of complete or incomplete HUS Approximately 15% of children with culture confirmed EHEC. Low platelet count is usually first sign of HUS 3-5% mortality rate of patients with HUS

18. Disease associated with EHEC Phase 4: Postsymptomatic stage. –E. coli O157:H7 can be excreted for up to a month. –For a child to return to day care or school, it is recommended that that patient have two negative stool cultures beforehand.

19. Laboratory Detection Methods Culture methods-Sorbital MacConkey agar –Only detects O157:H7 –Does NOT detect other EHEC serotypes Tests to detect shiga-toxin (detects all EHEC serotypes) –EIA (rapid kits available) –PCR (Test available at UNMC-Commercial kits)

21. Public Health Questions-1997 What is the prevalence of E. coli O157:H7 in persons with diarrhea? What is the prevalence of non-O157:H7 STEC in persons with diarrhea? Develop a shiga-toxin PCR test to detect shiga- toxin from stool specimens. Test developed to use at NPHL. Funding from LB-1206

22. Why ask these questions-1997 Some clinical laboratories do not screen for O157:H7 in routine stool cultures. No clinical laboratories screen for non-O157:H7 STEC. Develop a cost-effective method to detect non- O157:H7 STEC from stools.

23. Study Design Collaborated with 9 regional clinical laboratories in Nebraska. NPHL was sent (through NPHL courier system) stool samples from patients with diarrhea. Analysis: –CT-SMAC culture –Meridian EHEC EIA –stx PCR

24. Results -335 specimens were received from May 98-October 98 -5/9 laboratories had positive samples -14 samples were positive by at least one of the methods (4.2%) -Isolates from 13/14 positive samples were obtained -6/13: O157:H7 or O157:NM (1.8%) -7/13: non-O157 serotypes (2.1%)

25. Conclusions of Nebraska Study -4.2% EHEC prevalence rate. -1.8% O157:H7 -2.2% non-O157:H7 -O111:NM, O26:H11, O145:NM, O103:H2 have previously been associated with HUS. -Two O111:NM isolates were indistinguishable by PFGE, which suggests a possible outbreak which was not detected. -Developed a shiga-toxin PCR test which is in use at the NPHL for physician use. Fey et al. 2000 EID

26. Prevalence of other bacterial diarrheal diseases Camplobacteriosis Salmonellosis Shigellosis E. coli O157 Yersiniosis Listeriosis Vibrio EHEC

27. Treatment of EHEC 71 children with culture confirmed O157 infection. –9 patients had HUS –10 patients were treated with antibiotics 5/10 patients receiving antibiotics came down with HUS –4/61 patients not receiving antibiotics came down with HUS. Treatment is supportive, no antibiotics are given Wong et al. 2000 NEJM; 342

28. What is the current Nebraska state protocol? All Microbiology laboratories should be performing shiga-toxin test on routine stool samples for bacterial pathogens. (CDC MMWR 2006) If laboratory does not isolate STEC, then stool sample is sent to NPHL for STEC isolation. –Imperative for molecular epidemiology program

29. Molecular Epidemiology Genomic “Bar Code” Fingerprinting Assesses Relatedness of Different Isolates Is Strain “A” related to Strain “B” 9925234357 0 2

30. Typical questions addressed through molecular epidemiology Are the Escherichia coli O157:H7 isolates obtained from beef the same “strain” as that obtained from the patient(s)? Are the 7 MRSA isolates obtained from the ICU the same “strain?” Pre- and post-treatment isolate…the same strain??

31. Methods used in Molecular Epidemiology First Generation-Plasmid typing Second Generation-Restriction enzymes and probes Third Generation-PCR methods and PFGE Fourth Generation-Sequencing methods PFGE Gold Standard in almost all cases when molecular epidemiology is in question

32. THE CHROMOSOME IS THE MOST FUNDAMENTAL MOLECULE OF IDENTITY IN THE CELL PFGE-Pulsed-Field Gel Electrophoresis

34. Molecular Epidemiology-NPHL Escherichia coli O157:H7 Salmonella, Campylobacter, Listeria. Nosocomial-MRSA, VRE, Pseudomonas aeruginosa, Klebsiella pneumoniae and other enterics.

35. E. coli O157:H7

36. Reporting procedure Step 1--Compare PFGE patterns to Nebraska database –EC157x.001-EC157x.0240

38. Reporting procedure Step 2: Compare Nebraska PFGE pattern with National database at Centers for Disease Control and Prevention (CDC) –Every state laboratory performs PFGE in identical manner—standardized protocol. Detects inter-and intrastate outbreaks. Program called Pulsenet- managed by CDC

39. Reporting procedure Step 3--Send Report to epidemiologists at State level as well as Douglas and Lancaster County –Name, PFGE pattern, site/date of isolation –Has the PFGE pattern been seen in Nebraska recently? –Has the PFGE pattern been seen ever in Nebraska? –Has the PFGE pattern been seen in the US within the last 60 days? –The NPHL receives information from epidemiologists office regarding epidemiological information.

40. Top 5 E. coli O157:H7 PFGE patterns

41. 1-4 5-9 10-14 > 15 2006 fresh spinach outbreak -199 person infected from 26 states -102 were hospitalized (51%) 3 deaths (one from Nebraska) -31 (16%) developed HUS -Both O157:H7 and O26:H11 isolated from ill patients and spinach

42. Richard Goering, Ph.D. Creighton University