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STEC: a worrying zoonotic toxin-producing Escherichia coli, from farm to fork

STEC Escherichia coli.jpeg

Shiga Toxin-producing Escherichia coli (STEC) are rod-shaped pathogenic bacteria belonging to the Enterobacteriaceae Family

 

What are the Shiga Toxin-producing Escherichia coli (STEC)?

Shiga Toxin-producing Escherichia coli (STEC) also known as verocytotoxin-producing E. coli (VTEC) are facultative anaerobic Gram-negative, rod-shaped pathogenic bacteria belonging to the Enterobacteriaceae Family; They produce toxins called Shiga toxins (Stx) or verotoxins (Vtx), respectively because of their similarity with the toxin produced by Shigella dysenteriae or their cytotoxicity for the VERO cells. 

STEC can be differentiated according to their somatic O and flagellar H antigens into serogroups (O) or serotypes (O:H); The E. coli 0157:H7 serotype and the “non-O157” serogroups i) O26, O45, O103, O111, O121, O145 in the USA and ii) O26, O103, O111, and O145 in the EU, are the major pathogenic STEC serogroups linked to severe human infections (CDC, 2012; USDA-MLG 2020; EFSA, 2020).
However, beyond their serogroups or serotypes, the combination and subtypes of genes encoding virulence factors better characterized the potential pathogenicity of the STEC isolates (EFSA, 2013, 2020, 2021). 

The Stx toxins are the primary STEC virulence determinants governing the pathogenicity; stx genes are carried by lambdoid bacteriophages integrated into the E. coli chromosome. There are 2 major stx types (stx1 and stx2) further divided into subtypes (4 for stx1 and 12 for stx2). A strain may carry a stx1 and a stx2 subtype gene, or more than one stx2 subtype.

Additional virulence genes are consistently associated to severe illness (Caprioli et al., 2005; Bolton, 2011), most notably the eae gene coding for intimin production and formation of distinctive lesions on the intestinal cells; Though, this virulence factor is not always essential for severity, suggesting that there are alternative mechanisms of attachment (EFSA, 2020).

Historically, Enterohaemorrhagic E. coli (EHEC) were considered as a subset of a very limited number of STEC serogroups strictly associated with severe outcomes and outbreaks. Typical EHEC were usually stx+, eae+; However, new EHEC serotypes have been emerging such as E. coli O80:H2 (Nurcan et al., 2016) and above all, they have also encompassed atypical eae- strains within unusual serotypes such as O91:H21, O104:H4 and O113:H21, all of which also associated with haemorrhagic colitis (HC) (Caprioli et al., 2005; EFSA, 2020). Hence, the EHEC terminology is now considered as obsolete and should be replaced by STEC (EFSA, 2020). 

 

What are the risks for the consumers?

All STEC strains are pathogenic in humans, capable of causing at least diarrhoea; Depending on the presence of certain stx subtypes and the presence/absence of the eae gene, all STEC subtypes may be associated with severe outcomes, i.e., haemolytic uraemic syndrome (HUS), bloody diarrhoea (BD), kidney failures, hospitalizations and deaths (EFSA, 2020). Some survivors may have permanent disabilities, such as renal insufficiency and neurological deficits (FDA, 2012).

Stx2a showed the highest rates of HUS, BD and hospitalization; however, all other stx subtypes, or combinations thereof, were also associated with at least one of these severe illnesses; The presence of the eae gene is considered as an aggravating factor, with frequent progression to severe disease, such as HUS (FDA, 2012).

The probability of infection upon any STEC exposure is high since the infective dose can be as low as 1-100 cells (Caprioli el al., 2005; EFSA, 2020). Data suggest that individual factors, including age < 5 years, immunosuppression, underlying disease can greatly affect the occurrence and severity of clinical infection (EFSA, 2013, 2020).

Key figures:

In the US  
The CDC estimated, considering the under-diagnosis and under-reporting, that non-O157 STEC cause each year twice the number of foodborne infections in the United States than do E. coli O157:H7 strains (112,752 and 63,153, respectively); Hospitalization / death rates were evaluated respectively at 46 % / 0,5% for O157 and 13% / 0,3% for non-O157 STEC (Scallan et al., 2011).

In 2019, compared to the 2016-2018 period, the STEC incidence (6,3 cases for 100 000) increased significantly (+34%); Hospitalization / death rates were 21% / 0,3 % (CDC, 2020).

In the EU
In 2019, STEC infection was the third most reported zoonosis in humans (2,1 cases for 100,000) and increased from 2015 to 2019; 7,894 cases of STEC infections, including 7,775 confirmed cases, were reported. Hospitalization / death rates were 37,3 % / 0,2 % (EFSA, 2021).

 

How are STEC transmitted?

STEC are zoonotic agents i.e., pathogenic microorganisms transmitted from asymptomatic animals to humans. Ruminants such as cattle, sheep, goats and deer, are the most important reservoirs of STEC.

The consumption of foods contaminated with feces from ruminants is recognized as the main source (60 - 80%) of STEC infection in humans (EFSA 2020; Scallan et al., 2011).

Moreover, environmental fecal contamination of water, direct contact with animals and person-to-person transmission have also been identified as potential routes of transmission (Caprioli et al., 2005; EFSA, 2020).

 

What are all food industries affected by STEC?

‘Bovine meat and products thereof’, ‘raw milk and dairy products thereof’, ‘tap water including well water’ and ‘vegetables, fruit and products thereof’ are considered as the main sources of foodborne STEC outbreaks (FDA, 2012; EFSA, 2020 & 2021).

 

How can STEC be prevented and controlled in the food industry?

EU and US regulations broadly impose to the food chain business operators the prevention of adulteration (EU 178/2002; Federal Food, Drug & Cosmetic Act,1938; FDA-FSMA, 2011; Federal Meat Inspection Act,1906) as well as HACCP- based control systems and Good Hygiene/Manufacturing practices in order to manage the specific food safety risks associated to their processes and products (FDA 21CFR 1 et al.; USDA-FSIS 9CFR 304 et al.; EU 852/2004 & 2073/2005).

Mandatory recalls of any contaminated foodstuff complete the regulatory frameworks applicable to the control of the foodborne risks throughout the food chain (FDA-FSMA, 2011; USDA-FSIS 9CFR 304 et al.; EU 178/2002).

The requirement for strict control of food adulteration by the main pathogenic STEC, deemed essential for public health and consumer protection, has been formally further reinforced by regulatory agencies through specific acts such as:

E. coli O157:H7 was declared as an adulterant in raw ground beef by the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) in 1994 and explicitly included in the Pathogen reduction and HACCP regulations (9CFR304 et al., 1996). Moreover, because of the increasing awareness of the concrete public health impact of the non-O157 STEC, the USDA-FSIS additionally declared in 2011 the top six non-O157 (O26, O45, O103, O111, O121, and O145) STEC serogroups as adulterants in raw, non-intact beef products and raw, intact beef products that are intended for use in raw, non-intact beef products (USDA-FSIS, 2011) and later (USDA-FSIS, 2020) in ground beef, bench trim, and other raw ground beef components.

In addition, for fruits and vegetables grown for human consumption, the Food & Drug Administration (FDA) established science-based minimum standards, integrating the STEC risk, “for the safe growing, harvesting, packing, and holding Produces” (FDA, 2015) which represent significant sources of multistate outbreaks.

In the EU, the monitoring of foodborne disease outbreaks of human STEC infections was made mandatory in 2003 through the Zoonoses Directive 99/2003. In the Hygiene Package Criteria regulation 2073/2005, EU emphasized that “VTEC represents a hazard to public health in raw or undercooked beef and possibly meat from other ruminants, minced meat and fermented beef and products thereof, raw milk and raw milk products, fresh produce, in particular sprouted seeds, and unpasteurized fruit and vegetable juices”. Later, in the wake of the huge 2011 STEC O104:H4 outbreak, the EU finally defined Shiga toxin-producing E. coli O157, O26, O111, O103, O145 and O104:H4 as a food safety criterion for sprouts or spent irrigation water (EU 209/2013).

US and EU food chain business operators and enforcement/regulatory agencies must then pay special attention to the management of the STEC risks where necessary for better control from farm to fork and enhanced consumer/public health protection.

 

How can the presence of STEC be detected in the food industry?

Both EU and US regulations require that food business operators shall perform microbiological testing as appropriate when they are validating or verifying the effectiveness of their HACCP-based control procedures and good hygiene practices (EU 852/2004, 2073/2005; FDA-FSMA, 2011).

Even effective pathogenic STEC- HACCP management procedures may integrate generic E. coli routine monitoring for control of the fecal contamination, complementary regular compliance testing for the main pathogenic STEC serogroups, as do the regulatory/enforcement agencies (USDA-FSIS, 2020; EU 99/2003, 625/2017), is often put in place for verification purposes and prevention of recalls or legal prosecution.

Various standard methods (ISO, FDA-BAM; USDA/FSIS-MLG) have been described:

- for the monitoring of generic E. coli in foods or waters (FDA, 2020a; USDA-FSIS, 2015; US EPA 40CFR136; ISO 16649; ISO 9308),
- for the screening of the main pathogenic STEC (ISO 16654; ISO/TS 13136; FDA 2015 & 2020b; USDA-FSIS-MLG, 2019),

Note: The revision of the Technical Specification ISO/TS 13136:2012 has been initiated aiming at the publication of a full ISO Standard.

Validated (AOAC, EN ISO 16140-2) rapid methods have also been developed for the same purposes, bringing ease of use to the users as well as reduced times to results which add flexibility to the management of the business flows.

BioMérieux provide the food safety managers with proven standardized or validated methods for the effective management of STEC risk along the Food Chain. These solutions can be adapted to the STEC serogroups of interest depending on the different countries and business requirements.

BIOMÉRIEUX SOLUTIONS AND PRODUCTS

Sample and culture media preparation:
- DILUMAT® gravimetric diluter
- SMASHER®  lab blender
- MASTERCLAVE® automated media preparator

Traditional Culture media:
Large range of traditional culture media
- Glutamate broth
- TBX medium 
- SMAC CT Agar
- Cefixime-Tellurite Mixture

Chromogenic culture media for alternative methods:

- CHROMID® Coli Agar - Chromogenic media for detection and enumeration of ß-D-glucuronidase-positive E. coli and other coliforms 
    
- REBECCA™ Agar - Selective medium for the enumeration of β‑D‑glucuronidase‑positive Escherichia coli and Enterobacteriaceae (non E. coli

List of official validations: https://www.biomerieux-industry.com/products/culture-media-food-applications (bottom of the page)

Rapid enumeration solutions:

- TEMPO® EC (E. coli) for the enumeration of Escherichia coli in 22-27 hours

List of official validations: https://www.biomerieux-industry.com/products/tempo-food-quality-indicators (bottom of the page)

Rapid detection solutions: 

VIDAS® Enzyme Linked Fluorescent Assay (ELFA) pathogen detection automated platform:
- VIDAS® UP E. coli O157 (inc. H7) (ECPT) for the detection of potentially enterohemorrhagic E. coli O157

GENE-UP® Molecular pathogen detection automated platform:

- GENE-UP®  E. coli O157:H7 2 (ECO 2) for the detection of potentially enterohemorrhagic E. coli O157:H7

- GENE-UP® STEC – stx and eae 2 (EH1 2) qualitative test for E. coli  stx1 stx2 and eae genes 

- GENE-UP® STEC – Top 6 (EH2)  qualitative test for detection of E. coli  O26, O45, O103, O111, O121, O145 serotypes 

- GENE-UP® EHEC Series (ECO 2 or EH1 2 or EH2 with Immunoconcentration) :
VIDAS® E. coli serogroups (ESPT) automated test for use of the VIDAS® family instruments for the immuno-concentration of E. coli O26, O45, O103, O111, O121, O145 and O157 

- GENE-UP® Pathogenic E. coli : novel approach for the detection of pathogenic STECs (consult us for availability)

- CHROMID® EHEC Agar - Chromogenic medium for the confirmation and presumptive identification of enterohemorrhagic Escherichia coli (EHEC) of serogroups O157, O26, O103, O111, O145, O121 and O45 after an immuno‑concentration step 

- SLIDEX®E. coli - Latex agglutination test for the rapid identification of Escherichia coli serogroups O26, O103, O111, O121, O145 and O157 (presumptive identification of E. coli serogroups isolated on solid media)    

List of official validations (for VIDAS® and GENE-UP® methods)
https://www.biomerieux-industry.com/products/vidas-high-performance-food-pathogen-detection
https://www.biomerieux-industry.com/products/gene-real-time-food-pathogen-detection
(bottom of pages)
 

Identification:
 

- API® galleries (large range)
- VITEK® 2C GN
- VITEK® MS

List of official validations  https://www.biomerieux-industry.com/products/identification
(bottom of the page)

- Large range of culture media and chromogenic culture media for Extended Spectrum β‑Lactamase‑producing enterobacteriaceae (ESBL).

Jean-Pierre Facon
Written by
Jean-Pierre FACON

(PhD), Biotech consultant

Picture Isabelle DESFORGES
Written by
Isabelle DESFORGES

Global Marketing Scientific Manager / Scientific Affairs

Food Business Industry Unit, bioMérieux SA, France

French Delegate of Food Microbiology Standardization committees
(AFNOR V08B, ISO/TC 34/SC 9 and CEN/TC 463)

References

Bolton D.J. Verocytogenic (shiga toxin-producing) Escherichia coli - Virulence factors and pathogenicity in the Farm to Fork paradigm. Foodborne Pathogens and diseases, 2011, 8 (3): 357-365.

Caprioli A. et al. Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission. Vet. Res. 2005, 36: 289–311.

CDC. Preliminary Incidence and Trends of Infections with Pathogens Transmitted Commonly Through Food — Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 2016–2019. MMWR, 2020, 69 (17): 509-514.

EFSA and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control). The European Union One Health 2019 Zoonoses Report. EFSA Journal 2021;19(2):6406, 286 pp.

EFSA. Scientific Opinion on VTEC-seropathotype and scientific criteria regarding pathogenicity assessment. EFSA Journal, 2013;11(4).106 p.

EFSA. Scientific Opinion on the pathogenicity assessment of Shiga toxin-producing Escherichia coli (STEC) and the public health risk posed by contamination of food with STEC. EFSA Journal, 2020;18(1).105 pp.

EU Regulation 178/2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety.

EU Directive 99/2003. on the monitoring of zoonoses and zoonotic agents.

EU Regulation 852/2004 on the hygiene of foodstuffs.

EU Regulation 2073/2005 on microbiological criteria for foodstuffs.

EU REGULATION 209/2013 amending Regulation (EC) No 2073/2005 as regards microbiological criteria for sprouts and the sampling rules for poultry carcasses and fresh poultry meat. 

EU REGULATION 625/2017 on official controls and other official activities performed to ensure the application of food and feed law.

FDA Food Safety Modernization Act (FSMA) – Public Law to amend the Federal Food, Drug, and Cosmetic Act with respect to the safety of the food supply. 2011. 89 p.

FDA. Bad Bug Book. Foodborne Pathogenic Microorganisms and Natural Toxins. Enterohemorrhagic Escherichia coli (EHEC). 2012. 5p.

FDA. 21CFR Parts 1, 11, 16, 106, 110, 114, 117, 120, 123, 129, 179, and 211. Food Safety Modernization Act. Current Good Manufacturing Practice, Hazard Analysis, and Risk-Based Preventive Controls for Human Food. 2015.80(180): 55908-56168.

FDA. Testing Methodologies for E. coli O157:H7 and Salmonella species in Spent Sprout Irrigation Water (or Sprouts). 2015. 39 p. 

FDA 21CFR Parts 11, 16, and 112 - Standards for the Growing, Harvesting, Packing, and Holding of Produce for Human Consumption; Final Rule. 2015.80(228): 74353-74672.

FDA BAM (Bacteriological Analytical Manual) - Chapter 4: Enumeration of Escherichia coli and the Coliform Bacteria. 2020a.
FDA BAM - Chapter 4A: Diarrheagenic Escherichia coli. 2020b.
Federal Meat Inspection Act. 1906. An Act Making appropriations for the Department of Agriculture.

Federal Food, Drug & Cosmetic Act. 1938. To prohibit the movement in interstate commerce of adulterated and misbranded food, drugs, devices, and cosmetics, and for other purposes.

ISO (International Standard Organization) 16654:2001 & AMD 1:2017 - Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O157 & Amendment 1: Annex B: Result of interlaboratory studies.
ISO TS 13136: 2012. Microbiology of food and animal feed - Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups.

ISO 16649-1:2018 - Microbiology of the food chain - Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli - Part 1: Colony-count technique at 44 degrees C using membranes and 5-bromo-4-chloro-3-indolyl beta-D-glucuronide.
ISO 16649-2:2001 - Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli - Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide.
ISO 9308-1:2014 - Water quality — Enumeration of Escherichia coli and coliform bacteria — Part 1: Membrane filtration method for waters with low bacterial background flora.
ISO 9308-2:2012 - Water quality — Enumeration of Escherichia coli and coliform bacteria — Part 2: Most probable number method.
ISO 9308-3:1998 - Water quality — Detection and enumeration of Escherichia coli and coliform bacteria — Part 3: Miniaturized method (Most Probable Number) for the detection and enumeration of E. coli in surface and wastewater.
Nurcan S., et al. Enterohemorrhagic Escherichia coli Hybrid Pathotype O80:H2 as a New Therapeutic Challenge. Emerging Infectious Diseases, 2016; Vol. 22, No. 9. 1604-1612
Scallan E., et al. Foodborne illness acquired in the United State, major pathogens. Emerging Infectious Diseases, 2011; 17:7–15.
US-EPA (Environmental Protection Agency) - 40 CFR 136.3. Identification of test procedures.

USDA-FSIS. 9CFR Parts 416, 417 and 430 - Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products. 2011. 76(182):58157–58165.

USDA-FSIS. Expansion of FSIS Shiga Toxin-Producing Escherichia coli (STEC) testing to Additional Raw Beef Products. 2020. 85(108):34397-34402.

USDA-FSIS 9CFR Parts 304, 308, 310, 320, 327, 381, 416, and 417. Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems; Final Rule.1996. 185p.

USDA-FSIS. Microbiology Laboratory Guidebook MLG-3.02 Quantitative analysis of bacteria in foods as sanitary indicators. 2015. 19p.

USDA-FSIS. MLG 5C.00. Detection, Isolation and Identification of Top Seven Shiga Toxin-Producing Escherichia coli (STECs) from Meat Products and Carcass and Environmental Sponges. 2019. 18p.
 

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