1. MICR 420 Emerging and Re-Emerging Infectious Diseases Lecture 5: E. coli Dr. Nancy McQueen & Dr. Edith Porter

2. Overview Morphology Growth and metabolic characteristics O and H antigens Virulence factors including toxins Diseases Diagnosis Culture Serotyping Cytotoxicity PCR: toxin production Therapy Threats

3. E. coli

4. E. coli: Morphology and Metabolic Characteristics Gram-negative rods LPS: O-antigen Motile Flagella: H-antigen Enterobacteriaceae Facultative anaerobic Nitrate positive Oxidase negative Lactose positive Normal flora in intestine (non-toxigenic strains) TSI Not inoculatedE. coli

5. O- Antigens, H- Antigens and K- Antigens in Enterobacteriaceae Refer to surface structures H-antigen is part of flagella O-antigen located on LPS K or Vi- (Samonella) antigen - capsule High antigenic variability

6. Gram-Negative Cell Wall

7. Lipopolysaccharide (LPS) O-Antigen

8. The Gram-Negative Flagella and Flagellin H-antigen

9. Antigens and Antibodies Antigen is a molecule (free or cell associated) that induces the production of measurable antibody titers. Antibodies are glycoproteins that specifically bind to their antigens.

10. Serotyping: Generation of Specific Antibodies Purified antigens (e.g., O157 or H7) are injected repeatedly into an animal Rabbit Animal produces high levels of antibodies against the specific antigen Blood is drawn and serum prepared Serum contains high concentrations of specific antibodies

11. Examples of Serological Assays Diagnostic tests that are based on antigen- antibody reactions Agglutination Immunofluorescence ELISA Western Immunoblotting

12. E. coli: Virulence Factors and Diseases Pili Adherence to epithelial cells of urethra Urinary tract infection Capsules New born meningitis (K1 serotype) Toxins Various enterotoxins Various types of diarrhea Hemolysins http://library.med.utah.edu/WebPath/CNSHTML/CNS061.html

13. E. coli and Gastroenteritis DesignationToxinSymptoms EnterotoxigenicETECEnterotoxin,watery (traveler’s) diarrhea EnteroinvasiveEIECType III secretion system invasive, bloody diarrhea EnteropathogenicEPECAttachment to enterocytes via pili bundles mucoid diarrhea EnteroaggregativeEAECBiofilm formation Enterotoxin watery diarrhea EnterohemorrhagicEHECShiga-like toxincytotoxic, bloody diarrhea

14. Various types of E. coli

15. Association of Serotype with Pathogenicity Shiga toxin-producing EHEC Escherichia coli serotype O157:H7 Hemorrhagic uremic syndrome (HUS) Acute renal failure in children (and adults) Microangiopathic anemia Thrombotic thrombocytopenia Renal failure Sorbitol negative Phase contrast Anti-O157:H7

16. Main Sympotms of HUS Episode of diarrhea Acute and severe onset Bleeding (hematemesis or melena) Severe oliguria (no urine production) Hematuria (blood in urine) Neurologic changes in 1/3 Hypertension in 50%

17. Pathogenesis of Hemolytic Uremic Syndrome Shigatoxin mediated Locally produced Absorbed across the gut epithelium into the circulation Transported to small vessel endothelial cells Microvascular endothelial damage Inhibition of protein synthesis Apoptosis Platelet activation K. S. Sandhu and C. L. Gyles (2002) Normal Villus +Stx

18. Kidney Damage in Hemolytic Uremic Syndrome http://www.gamewood.net/rnet/renalpath/t14.jpg DiseasedNormal

19. E. coli: Diagnosis Culture using Sorbitol Mac ID using biochemicals Serotyping Cytotoxicity assay PCR to identify toxin genes

20. Therapy Symptomatic Non-specific supportive therapy Hydration Electrolytes Antibiotics should not be used No evidence that treatment with antibiotics is helpful Taking antibiotics may increase the risk of HUS Antidiarrheal agents like Imodium® may also increase that risk http://www.cdc.gov/nczved/dfbmd/disease_listing/stec_gi.html#14

21. Threats by E. coli O157 An estimated 73,000 cases occur annually in the United States 2,100 hospitalizations annually Estimated 61 fatal cases annually Major sources: spinach and undercooked ground beef Easily spread from person to person Nationally reportable

22. The 2006 North American Outbreak of E. coli O157:H7 26 states were affected, with at least 200 cases 3 deaths Traced to organic bagged fresh spinach - sold as conventional produce Originated from irrigation water contaminated with cattle feces or from grazing deer

23. 2009 Multistate Outbreak of E. coli O157:H7 Infections 2009: recall of approximately 545,699 pounds of ground beef products from Fairbank Farms that may be contaminated with E. coli O157:H7. A cluster of individuals including 26 persons from 8 states were infected with matching strains of E. coli O157:H7. Nineteen hospitalized Five developed HUS Two deaths 50% of patients male 38% less than 18 years old Less than 14% of the US population is in this age group

24. Other recent E. coli O157 H7 outbreaks Prepackaged Cookie Dough (June, 2009) 72 infected 34 hospitalized 10 HUS No deaths Beef from JBS Swift Beef Company (July 2009) 23 infected 12 hospitalized 2 HUS No deaths Daycare Center in Washington (March- April, 2010) 4 children hospitalized 1 death

25. Take Home Message E. coli part of normal flora but toxin producing strains can cause great damage Various toxins lead to characteristic diseases. Major public threat is strain O157:H7 because of possible fatal outcome and frequent outbreaks. Note – other strains of E. coli are now reported to produce the Shiga-like toxin and cause HUS

26. Additional Resources ASM Microbe library Madigan & Matinko, Brock Biology of Microorganism, 11 th edition. www.cdc.org