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
2
The other side of E. coli…
• Strains may have the capacity to cause disease
(Croxen and Finlay, 2010)
Pathogenically diverse species…
Enterically
virulent clones
(Nataro and Kaper, 1998)
HUS
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
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)
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
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?
EHEC adherence
Stx
EAHEC adherence
High Stx2 production
Persistent colonization
(i.e. prolonged disease)
AAF pili (aag)
Severe disease (HC, HUS)
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
Emergence of E. coli O157
• Recognised as a foodborne pathogen in 1982 (US)
E. coli O157related
foodborne
vehicles
(Rangel et al., 2005)
Reservoir for E. coli O157
• Generally considered cattle…
– Low incidence in Can, US and UK before 1982
• Suggests other reservoirs?
• Studies recovered O157 and nonO157 STEC from ruminants
– Food-producing
– Wild animals
• Ruminants
• Birds
Wild animals serve as natural reservoirs for all STEC
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
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?
Association between disease incidence, severity and serotype
Big 6
(Karmali et al., 2003)
Differences between O157 & non-O157 STEC
• Relates to genetic content of virulenceassociated 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
Intimi
n
EscR, S
T, U, V
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)
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
Escherichia coli on CT-SMAC medium
E. coli K-12
E. coli O157:H7
O157 and non-O157 on SMAC
Typical non-O157
STEC
Typical O157
Issue?
Inability to
distinguish nonO157 STEC from
generic E. coli
Looks like
a generic
E. coli
Not
detected!
(Bopp, CDC)
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??
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?
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
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
Canada’s position on the Big 6
Likely to be influenced by our beef exports
$1.4 B (201)
What choice do
we have?
If we export, we
will have to
adopt US policy!
Can Meat Council (www.cmc-cvc.com)
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
In the end…
• Long-term…
– Improved HACCP and processing interventions
• Increased knowledge of STEC ecology/prevalence
– Improved control strategies
– Reduced STEC foodborne disease?
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?
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/Microbiologic
al_Lab_Guidebook/index.asp
Questions?
O103
O111
O26
O121
O157
STEC on Rainbow agar
K-12
O103