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Shiga toxin-Producing Escherichia coli – The Big Six

Shiga toxin-Producing Escherichia coli – The Big Six. Kevin J. Allen. Escherichia coli. Common constituent of mammalian digestive tracts Predominant facultative anaerobe Essential for our well being… Improves digestion Produces essential vitamins Competitive exclusion.

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Shiga toxin-Producing Escherichia coli – The Big Six

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  1. Shiga toxin-Producing Escherichia coli – The Big Six Kevin J. Allen

  2. Escherichia coli • Common constituent of mammalian digestive tracts • Predominant facultative anaerobe • Essential for our well being… • Improves digestion • Produces essential vitamins • Competitive exclusion

  3. The other side of E. coli… • Strains may have the capacity to cause disease (Croxen and Finlay, 2010)

  4. Pathogenically diverse species… Enterically virulent clones (Nataro and Kaper, 1998) HUS

  5. What are STEC? • (note: Shiga toxin = Verotoxin; STEC=VTEC) • Definition… • Any E. coli strain producing Shiga toxin (Stx) • stx1 and/or stx2 • Stx2 1000X more toxic! • >400 E. coli serotypes harbour stx genes • Why? • Located on distinct phage elements • Mobile

  6. Are all STEC pathogenic? • Short answer… • No, but… • Stx considered primary virulence factor • Long answer… • Pathogenic STEC require additional virulence factors enabling adherence • Permit colonization of intestinal epithelial cells (enterocytes)

  7. Enterohemorrhagic E. coli (EHEC) • All EHEC are STEC; Prior to German outbreak, EHEC were the only pathogenic STEC • Define EHEC strains based on virulence genes • LEE PAI (41 kb) • Virulence plasmid (pO157) • Possessed stx1 and/or stx2 • Phenotypically • Attaching and effacing phenomenon

  8. Pathogenic STEC paradigm shift • German outbreak marked the “outing” of E. coli O104:H4 • Enteroadherent hemorrhagic E. coli (EAHEC) • Perplexing due to lack of classic EHEC markers • Lacked • LEE PAI • pO157 • Possessed Stx2 (stx2a) • Possessed enteroaggregative E. coli (EAEC) virulence factors • Aggregative adherence factor (AAF) • Extraordinarily virulent… • Why? • a

  9. EHEC adherence Stx EAHEC adherence High Stx2 production Persistent colonization (i.e. prolonged disease) Severe disease (HC, HUS) AAF pili (aag)

  10. EAHEC – E. coli O104:H4 • Emerging pathogen, globally spread • Sporadic infections reported in Asia and across Europe • Endemic in Central Africa • Reservoir… • Not animals! • Humans • Irrigation water contamination may be source of EAHEC

  11. Emergence of E. coli O157 • Recognised as a foodborne pathogen in 1982 (US) E. coli O157-related foodborne vehicles (Rangel et al., 2005)

  12. Reservoir for E. coli O157 • Generally considered cattle… • Low incidence in Can, US and UK before 1982 • Suggests other reservoirs? • Studies recovered O157 and non- O157 STEC from ruminants • Food-producing • Wild animals • Ruminants • Birds

  13. Wild animals serve as natural reservoirs for all STEC

  14. Significance of various STEC • From studies examining human STEC infections • 50-80% identified as E. coli O157:H7 • 30 to 50% are non-O157 STEC!! • Data from Canada and US are similar

  15. Non-O157 – The Big 6 • Non-O157 STEC infections are linked to “Big 6” • 70-75% are caused by Big 6 • O26, O45, O103, O111, O121, O145 • ca. 25-30% other non-O157 STEC… • How much disease potential compared to O157?

  16. Association between disease incidence, severity and serotype Big 6 (Karmali et al., 2003)

  17. Differences between O157 & non-O157 STEC • Relates to genetic content of virulence-associated genes • O157 possess classic EHEC genes • stx1/stx2 , LEE PAI, pO157 • Maximize disease-causing potential • Non-O157… more variable • Stx1 or stx2 • pO157 may be missing • LEE PAI Intimin EscR, S T, U, V

  18. What do we know about non-O157s? • Relatively little compared to O157… • Limited knowledge… • Ecology, reservoirs, transmission, virulence • Understanding derives from O157 research • Why so little knowledge? • Focus has been on O157 since 1982 • USDA-FSIS recognised O157 as an adulterant (1993)

  19. Why don’t we know more? • Inability to effectively detect! • Consider Salmonella testing… • Presence/absence • However, not all E. coli are pathogenic • Major challenge • O157 STEC in North America • Manipulate phenotypic markers • Lack of sorbitol fermentation (37 degrees C after 24 h) • Lack β-glucuronidase

  20. Escherichia coli on CT-SMAC medium E. coli K-12 E. coli O157:H7

  21. O157 and non-O157 on SMAC Typical non-O157 STEC Typical O157 Issue? Looks like a generic E. coli Not detected! Inability to distinguish non-O157 STEC from generic E. coli (Bopp, CDC)

  22. Current US position on non-O157 STEC • September 2011 USDA-FSIS declared “big 6” adulterants • Raw ground beef, trimmings • March 2012 • New policy implementation delayed 90 days • Labs require additional validation period • Issues and consequences??

  23. Strategies for non-O157 STEC detection • Inability to discriminate non-O157 STEC from generic E. coli • Two directions • Shiga toxin genes/toxin • PCR or Stx ELISA-based detection • Serogroup • qPCR/PCR, Immunomagnetic separation (IMS) • Issue… • What does the presence of “Big 6” serotype and/or Stx mean? • Equivalent to E. coli O157?

  24. Moral of the story… • Detection of STEC or STEC-associated serogroups does not correlate with “risk” • Lead to unnecessary product holds/recalls • Require “detection” and “virulence profiling” • Identify isolates possessing disease-causing potential • Proposed USDA-MLG testing incorporates… • qPCR (RT-PCR), IMS, serology, Rainbow agar (RA) • Provide virulence-profiling-based detection

  25. USDA-MLG Big 6 Assay • Overview… • qPCR detection of stx and eae (LEE PAI) • qPCR detection of wzx • Detects Big 6 serogroups • IMS for detected serogroup(s) • Plate on RA • Look for typical phenotype • Reconfirm virulence and serogroup ID • 4 day process for positives

  26. Canada’s position on the Big 6 Likely to be influenced by our beef exports What choice do we have? $1.4 B (201) If we export, we will have to adopt US policy! Can Meat Council (www.cmc-cvc.com)

  27. In the end… • Focus should be on food safety • Considering non-O157 burden of disease • Concept is not without merit! • Short-term… • Issues with testing will be problematic • Increased incidence of STECs, increased recalls? • Tough for industry • Drive safer beef products

  28. In the end… • Long-term… • Improved HACCP and processing interventions • Increased knowledge of STEC ecology/prevalence • Improved control strategies • Reduced STEC foodborne disease?

  29. Future impact of USDA policy… • USDA declaration of E. coli O157 as an adulterant accelerated detection method development and fundamental research… • Big 6 adulterant claim will do the same… • Positive step for food safety • What about non-Big 6 STECs?

  30. Suggested reading • Gill and Gill (2010) Non-O157 verotoxigenic Escherichia coli and beef: A Canadian perspective. Can J Vet Res 74:161-9. • Grant et al. (2011) The significance of non-O157 STEC in food. Food Prot Trends 31:33-45. • USDA-MLG non-O157 detection method: http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/index.asp

  31. Questions? O103 O111 O26 O121 O157 K-12 O103 STEC on Rainbow agar

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