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

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

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? a

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)

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

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)

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

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 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

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 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)

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 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)

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

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/Microbiological_Lab_Guidebook/index.asp

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