Shiga toxin-Producing Escherichia coli – The Big Six

Kevin J. Allen

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Escherichia coli• Common constituent of mammalian digestive

tracts– Predominant facultative anaerobe

• Essential for our well being…– Improves digestion– Produces essential vitamins– Competitive exclusion

• Strains may have the capacity to cause disease

The other side of E. coli…

(Croxen and Finlay, 2010)

Pathogenically diverse species…

(Nataro and Kaper, 1998)

HUS

Enterically virulent clones

• (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

What are STEC?

• 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)

Are all STEC pathogenic?

• 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

Enterohemorrhagic E. coli (EHEC)

• 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

Pathogenic STEC paradigm shift

EHEC adherence

EAHEC adherence

High Stx2 production

Persistent colonization(i.e. prolonged disease)

Severe disease (HC, HUS)AAF pili (aag)

Stx

• 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

EAHEC – E. coli O104:H4

• Recognised as a foodborne pathogen in 1982 (US)Emergence of E. coli O157

(Rangel et al., 2005)

E. coli O157-related

foodborne vehicles

• 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

Reservoir for E. coli O157

Wild animals serve as natural reservoirs for all 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

Significance of various STEC

• 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?

Non-O157 – The Big 6

Association between disease incidence, severity and serotype

(Karmali et al., 2003)

Big 6

• 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

Differences between O157 & non-O157 STEC

EscR, S T, U, V

Intimin

• 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)

What do we know about non-O157s?

• 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

Why don’t we know more?

Escherichia coli on CT-SMAC medium

E. coli K-12 E. coli O157:H7

O157 and non-O157 on SMAC

Typical O157 Typical non-O157STEC

Issue?

Looks like a generic

E. coli

Not detected! (Bopp, CDC)

Inability to distinguish non-O157 STEC from generic E. coli

• 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??

Current US position on non-O157 STEC

• 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?

Strategies for non-O157 STEC detection

• 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

Moral of the story…

• 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

USDA-MLG Big 6 Assay

What choice do we have?

If we export, we will have to

adopt US policy!

Canada’s position on the Big 6

Can Meat Council (www.cmc-cvc.com)

Likely to be influenced by our beef exports

$1.4 B (201)

• 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?

In the end…

• 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?

Future impact of USDA policy…

• 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

Suggested reading

Questions?

STEC on Rainbow agar

O103 O111 O26 O121 O157 K-12 O103