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)
Everyone loves “no, but…” statements, so here’s mine…

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. AAF pili (aag) High Stx2 production Persistent colonization
EHEC adherence
Stx
EAHEC adherence
Virulence linked to the perfect storm of virulence factors
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 Reservoir…
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. E. coli O157-related foodborne vehicles
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 % 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
EscR, S
T, U, V
Intimin

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 Issue?
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?

25. 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

26. 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

27. 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 (

28. In the end… Focus should be on food safety Short-term…
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

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

30. 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?

31. 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:

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