Shiga Toxin-Producing Escherichia coli (STEC) Food Poisoning and its Prevention

October 2019

The shiga toxin producing Escherichia coli (STEC) are important and occasionally lethal foodborne pathogens. Initially nicknamed the ‘hamburger bug’, it is now known that a diverse range of foods can act as vehicles for the transmission of STEC infections, even wheat flour. The STEC comprise many serotypes and can possess a variety of pathogenicity determinants and this complicates their detection in foods, although the O157 serogroup predominates. This information statement is an update providing refreshed coverage of: the clinical picture and epidemiology of STEC infections, outbreaks, control of STEC in the food industry, and testing methods.

Executive summary

Shiga toxin-producing Escherichia coli (STEC) represent a pathotype of the bacterial species E. coli, with a common feature being the production of one or more Shiga-like toxins. While the group is a sub-set of one species, it is diverse in terms of serology and virulence factors expressed. The most important serotype is E. coli O157:H7, which first rose to prominence in the early 1980s to become known, in the USA, as the ‘hamburger bug’. STEC have the capability of producing serious clinical outcomes in humans, including kidney failure and death, albeit rarely. Infection more frequently results in diarrhoea or bloody diarrhoea [hence a sub-set of STEC being referred to as “enterohaemorrhagic” (EHEC)]. Their reservoir is the intestinal contents of ruminant animals, and human infections result from direct or indirect ingestion from this source, via a number of routes. One such route is contaminated food, e.g. raw/undercooked meat products, fresh vegetables, including bean sprouts, unpasteurised milk and fruit juice (‘cider’ in USA). Control in most foods is mediated by standard means such as pasteurisation or cooking, but food such as ready-to-eat (RTE) vegetables and raw drinking milk are not amenable to such treatments. Consumer trends pose additional challenges, for example the demand for ‘rare’ hamburgers that are difficult to produce with an equivalent degree of safety as cooked meat.

Detection in foods is difficult except for the major STEC O157 serogroup. For this purpose, there are many Polymerase Chain Reaction (PCR) and immunological test kits effective for screening food enrichments. The use of immunomagnetic separation helps to decrease the enrichment time necessary for detection. However, for the myriad of other STEC serotypes, detection is much harder since there is such variability within the group. For example, no one single enrichment medium is completely effective for all serotypes. PCR detects genes which may be harboured in non-target bacteria and phages and hence is prone to producing false positive results. This major difficulty, and the low prevalence in foods, makes routine testing for these organisms inefficient and of questionable value, and product testing is best done where HACCP (Hazard Analysis and Critical Control Point) verification is its primary aim.

Key words: foodborne disease, Escherichia coli, shiga-like toxin, pathogen control, detection and identification

Essential Reading:

Overview including hazard identification – FAO/WHO (2018) Shiga toxin-producing Escherichia coli (STEC) and food: attribution, characterization, and monitoring. https://apps.who.int/iris/bitstream/handle/10665/272871/9789241514279-eng.pdf.

Detection methods- Parsons, B.D., Zelyas, N., Berenger, B.M. and Chui, L. (2016) Detection, characterization, and typing of Shiga Toxin-Producing Escherichia coli. Frontiers in Microbiology 7:1.

UK outbreaks - Pennington T. H. (2014). E. coli O157 outbreaks in the United Kingdom: past, present, and future. Infection and drug resistance, 7, 211–222. doi:10.2147/IDR.S49081

Introduction

The species Escherichia coli is an inhabitant of the mammalian gastrointestinal tract and so its presence in other environments has long been used as an indicator of faecal contamination, as reflected in well-known tests such as the eponymous E. coli, faecal coliform and total coliform tests. However, the organism may also cause human disease and there are several pathotypes of E. coli causing different diseases that are recognised, including:

• enteropathogenic (EPEC)

• attaching and effacing (AEEC)

• enterotoxigenic (ETEC)

• enteroinvasive (EIEC)

• enteroaggregative (EAEC)

• shiga toxin-producing/shiga toxigenic E. coli (STEC)/verocytotoxin-producing /verotoxigenic E. coli (VTEC). VTEC produce pathological effects on Vero cells, through the production of Stx1 and/or Stx2. VTEC and STEC describe the same type of pathogenic E. coli.

A sub-set of STEC/VTEC is EHEC, which refers to pathogenic E. coli causing haemorrhagic gastrointestinal disease (bloody diarrhoea) in humans.

Most of these pathotypes cause diseases that are unpleasant but very rarely life-threatening, for example ETEC causes traveller’s diarrhoea. The major exception is the group known as STEC. Foodborne disease caused by this pathogen was first recognised in the early 1980s, in the USA. Disease was particularly associated with burger restaurants hence it became known as the ‘hamburger bug’ which, as it turns out, is quite a misleading term. In 1993 there was an outbreak in USA among people eating at ‘Jack in the Box’ fast food restaurants that resulted in around 700 cases and four deaths. The cause was attributed to the undercooking of burgers and the outbreak heralded a regulatory and food industry focus on the organism which persists to this day.

To complicate matters more, there are approximately 400 serotypes of STEC, some of which can cause disease on a par with STEC O157, and the significance of these STEC non-O157 serogroups continues to grow. In light of this, in the last few years US regulators have increased the list of adulterant STEC strains on certain types of beef, intended for burger production, to include another six serogroups. In the EU there is also a list of serotypes of concern, but it is a different list to the American one. Recent reviews (FAO/WHO STEC Expert Group 2019, National Advisory Committee on Microbiological Criteria for Foods 2019) give data reflecting differences in the types of serogroups that occur between different countries. The ratios of STEC O157:STEC non-O157 clinical cases varied widely, as did the composition of STEC non-O157 serogroups causing cases. In Europe the proportion of STEC non-O157 isolates continues to increase, but this may be the result of enhanced awareness of this group of pathogens.

By definition, STEC produce toxins with a high degree of homology to that produced by Shigella dysenteriae type 1.

Either:

Shiga toxin 1 (Stx1) - differs from Shiga toxin (Stx), produced by S. dysenteriae, by one to seven amino acids

and/or

Shiga toxin 2 (Stx2) - a diverse and heterogenous group of subtypes sharing approximately 60% homology to Stx

The pathogenicity of STEC not only involves the production of toxin(s) but also the adhesion to, and colonisation of, the intestinal tract. The eae (E. coli attachment effacement) gene codes for the production of intimin, which is responsible for attachment to intestinal epithelial cells. Another factor that may contribute towards virulence is the enterohaemolysin (EHly) which is encoded by ehxA. However, there are many putative virulence-associated genes, and these have been used for typing and risk assessment (Brandt et al., 2011).

A large outbreak of infections which occurred in Germany in 2011 (Table 1) was caused by EAEC O104:H4. EAEC characteristically contain the aggR gene which is a transcriptional regulator controlling the expression of virulence genes, including adhesion factors. In this outbreak the EAEC had acquired the ability to express shiga toxin and so a single strain possessed mobile genetic elements from two E. coli pathotypes, and this may have contributed to its virulence (Muniesa et al., 2012).

Recent reviews provide detailed pictures of STEC pathogenicity determinants (FAO/WHO STEC Expert Group 2019, National Advisory Committee on Microbiological Criteria for Foods 2019).

The clinical picture of STEC infections

Although illness caused by STEC is uncommon, with the incidence being low when compared with salmonellosis for example, STEC is regarded as an important pathogen because of the serious complications that may follow infection. Disease symptoms can present as mild diarrhoea to severe bloody diarrhoea [hemorrhagic colitis (HC)]. In some patients, serious sequelae develop, including: haemolytic uraemic syndrome (HUS) with damage to the kidneys resulting in blood in the urine; haemolytic anaemia (loss of red cells); thrombocytopenia (loss of platelets); Thrombotic Thrombocytopenic Purpura (TTP) with loss of platelets and excessive clot formation, leading to kidney and nervous system damage. In some outbreaks, a high proportion of cases develop HUS, which is the most common cause of renal failure in children in the UK. Cases may be fatal, especially in children.

In 2017, 6,260 cases of STEC infection, including 6,073 conﬁrmed cases, were reported in the EU to give an overall incidence of 1.66 cases per 100,000 (EFSA 2018). Countries reporting the highest incidence rates were Ireland, Sweden, and Denmark (at 16.62, 5.07 and 4.57 respectively). Of cases with known outcomes, 37.5% were hospitalised, there were 429 HUS cases, and 20 deaths. There is a distinct seasonal distribution to the occurrence of cases with most occurring in the summer.

The methods used for the testing of human clinical specimens may present a biased picture of the spectrum of STEC serogroups causing human disease, with STEC non-O157 infections potentially being under-reported (Parsons et al., 2016). In the EU in 2017, the most commonly reported serogroup was STEC O157 at 31.9% of cases with typing data, with serogroup O26 the next most frequent at 14.3%, and the next most frequent serogroups identified being O103, O91, O145 and O146 (EFSA 2018).

Epidemiology of STEC infections

The main environmental reservoir for STEC is the bovine intestine, although other ruminants may also harbour the organism. There are four main transmission routes for human infection: foodborne, waterborne, direct or indirect contact with animals, and person-to-person spread. Food vehicles linked to transmission include ground beef, fresh salad vegetables, beansprouts, improperly prepared dried and fermented sausages, milk and milk products and unpasteurised apple juice. Sprouted seeds and water have been the vehicles responsible for some of the largest outbreaks.

A description of the epidemiology of disease in England from 2009 to 2012 (Byrne et al., 2015) identified the following characteristics: the incidence in children 1-4 years of age was around four times the population mean, women and children were more likely to develop HUS, STEC non-O157 cases were more likely to be hospitalised and develop HUS, and contact with livestock faeces was an important risk factor.

In an Australian case control study (McPherson et al., 2009), patients with STEC O157 infections were more likely to have eaten hamburgers, visited restaurants, used antibiotics, or had family exposure to red meat because of their occupation, while eating homegrown fruit, vegetables and herbs was protective. These risk factors were not shared with STEC non-O157 cases who were more likely to have eaten delicatessen meats (cooked chicken or corned beef), been camping, eaten catered meals or been exposed to animals, and disease was negatively associated with eating pork, raw vegetables, and homegrown vegetables, fruit, or herbs.

A case control study of STEC infections, carried out in New Zealand, was unable to associate a food with disease, instead environmental exposures relating to contact with cattle, animal manure and recreational waters were risk factors (Jaros et al., 2013).

Although large outbreaks of STEC infection have occurred in the UK, the majority of cases are sporadic in nature. In England and Wales between 2006 and 2015, the number of laboratory reports of E. coli O157 infections reached a peak of 1182, which is equivalent to an incidence of 2.10 cases/100,000, while the lowest was 664 cases in 2005, an incidence of 1.15 cases/100,000 (Public Health England, 2016).

The incidence of O157 infections is variable throughout the UK, with the highest rate in 2004 being in Scotland (Locking et al., 2006). Outbreaks of STEC are usually small with an average of 8 cases, however, larger outbreaks can occur. A large STEC O157 outbreak occurred over an eight-month period from December 2010 to July 2011. A total of 194 cases were reported in England, with a further 44 cases reported in Scotland and 14 in Wales (Launders et al., 2016).

Non-food routes of infection are important in transmission of STEC infections. For example, in 2000, an estimated 2300 cases (7 deaths) were associated with contaminated drinking water in Walkerton, Ontario, Canada (Hrudey et al., 2003). Cases have also been associated with direct or indirect contact with animals and person-to-person spread. Most cases are not recognised as parts of outbreaks and, where outbreaks do occur, more than one transmission route may be involved. Outbreaks are well-recognised amongst children in nurseries and kindergartens, as well as with visits to open farms, petting zoos and county fairs.

There was evidence of airborne transmission in an incident in the USA. At least 19 people who had visited a county fair in Ohio in 2001 fell ill with STEC that apparently spread through sawdust in the air at an exhibition hall. This was the first time that researchers connected an outbreak to a contaminated building. Testing found E. coli O157 in the rafters, the walls and the sawdust, in some cases 10 months after the fair (Varma et al., 2003).

The Centers for Disease Control and Prevention (CDC) estimate that there are approximately 175,905 domestically acquired foodborne illnesses associated with STEC annually in the USA (Scallan et al., 2011). STEC O157:H7, according to the CDC, is annually responsible for approximately 63,153 (36%) of the domestically acquired US foodborne STEC illnesses. The remainder of the illnesses (112,752 or 64%) are associated with STEC non-O157. While more than 50 STEC non-O157 serogroups have been associated with human illness, 70 to 80 percent of confirmed STEC non-O157 illnesses are caused by six STEC serogroups – O26, O45, O103, O111, O121, and O145. These illnesses can be equivalent in severity to those caused by E. coli O157:H7.

Outbreaks may also be caused by cross-contamination of RTE foods from raw foods or dirty utensils and by post-pasteurisation contamination of milk. Person-to-person spread also occurs and has caused outbreaks in hospitals, day care centres, infant schools and nursing homes. It has not been possible to pinpoint the source of infection in many sporadic cases and small outbreaks. Normal good manufacturing/catering practices should ensure that the chance of cross-contamination occurring is minimised.

Outbreaks have been recorded where the concentration of STEC O157 in the food was very low. For example, in an outbreak involving frozen burgers that caused more than 700 cases and four deaths, the pathogen was present at a median concentration of 1.5 MPN/g, or 67.5 cells per patty (Tuttle et al., 2009).

STEC outbreaks related to contaminated food

The table below gives some examples of STEC outbreaks. It is notable that the term ‘hamburger bug’ is no longer appropriate for either the STEC pathotype, or STEC O157:H7 in particular, since the diversity of implicated foods is wide and includes foods such as flour and soy nut butter. An analysis of outbreaks caused by STEC O157 in the USA between 2003 and 2012 (Heiman et al., 2015) found that, although most outbreaks involved beef as the vehicle (55% of foodborne outbreaks), there was a significant contribution from other food types: leafy vegetables (21%), dairy products (11%), fruit (4%), other types of meat (5%), sprouts, nuts and poultry (all <5%). However, a large proportion of outbreaks had no food vehicle identified. In the EU in 2017 (EFSA, 2018) there were data for only nine “strong evidence” outbreaks with four attributed to bovine meat and its products, two to milk and one each to cheese, dairy products other than cheese and one to meat and meat products.

Table 1: Examples of STEC outbreaks

Implicated Food	Serotype	Clinical Impact	Control Failure	Country	Year
Hamburger patties	O157	732 infections, 171 hospitalised, 4 deaths	Undercooking	USA	1992-1993⁽¹⁾
Raw and cooked meats	O157:H7	503 ill, 20 died. 11% of patients HUS/TTP	Numerous hygiene and training lapses	UK	1996⁽²⁾
White radish sprouts	O157:H7	Approx. 6000 cases, 106 HUS cases	Eaten raw	Japan	1996⁽³⁾
Cooked meats supplied to schools	O157	31 hospitalised, 8 HUS cases, 1 death.	Numerous hygiene and training lapses	UK	2005⁽⁴⁾
Seed sprouts	O104:H4	4321 outbreak cases, including 852 HUS cases and 50 deaths	Contaminated fenugreek seeds	Germany	2011⁽⁵⁾
Unpasteurised milk	O157:H7 sorbitol fermenting	6 children, 2 asymptomatic adults	Raw milk	Finland	2012⁽⁶⁾
Watercress	O157	19 cases	Not described	UK	2013⁽⁷⁾
Raw drinking milk	O157	9 cases	Unpasteurised milk consumed	UK	2014⁽⁸⁾
Mixed salad leaves	O157	161 cases, 7 HUS cases, 2 deaths	Not described	UK	2016⁽⁹⁾
Cheese	O26	25 cases, 9 HUS cases, 3 deaths	Not described. Soft cheese made from pasteurised milk	Europe (various)	2016⁽¹⁰⁾
Alfalfa sprouts	O157:H7	11 cases, 2 hospitalisations	Consumption of raw food	USA	2016⁽¹¹⁾
Wheat flour	O121 and O26	63 cases, 17 hospitalised, 1 HUS case	Consumption of uncooked cake batter etc (raw flour)	USA	2016⁽¹²⁾
Minced beef and pork	O157:H7 sorbitol fermenting	14 cases, 13 HUS cases, 1 death	Exposure to raw meat	Germany	2016 -2017⁽¹³⁾
Leafy greens	O157:H7	25 cases, 9 hospitalisations, 2 HUS cases, 1 death	Consumption of raw food	USA	2017⁽¹⁴⁾
Soya nut butter	O157:H7	32 cases, 12 hospitalised, 9 HUS	Suspected contaminated raw materials	USA	2017⁽¹⁵⁾
Romaine lettuce	O157:H7	210 cases, 96 hospitalised, 5 deaths	Contaminated water (e.g. for irrigation/washing)	USA	2018⁽¹⁶⁾
Flour	O26	17 cases, 3 hospitalised	Not described	USA	2019⁽¹⁷⁾

Table references:

https://www.cdc.gov/mmwr/preview/mmwrhtml/00020219.htm
Dundas, S. et al., (1999) Effectiveness of therapeutic plasma exchange in the Lanarkshire Escherichia coli O157:H7 outbreak. Lancet. 354:1327-1330
Hauswaldt, S. et al., (2013) Lessons learned from outbreaks of Shiga toxin producing Escherichia coli. Current Infectious Disease Reports. 15:4-9.
Pennington, H. (2009) The Public Inquiry into the September 2005 Outbreak of E. coli O157 in South Wales. Aberdeen: HMSO; 2009
Buchholz, U. et al., (2011) German outbreak of Escherichia coli O104:H4 associated with sprouts. New England Journal of Medicine 365:1763-1770
Jaakkonen, A. et al., (2012) Severe outbreak of sorbitol-fermenting Escherichia coli O157 via unpasteurized milk and farm visits, Finland. Zoonoses and Public Health 64:6
Launders N, et al., (2013) Outbreak of Shiga toxin-producing E. coli O157 associated with consumption of watercress, United Kingdom, August to September 2013. Eurosurveillance. 18(44):pii=20624
Butcher, H. et al., (2016). Whole genome sequencing improved case ascertainment in an outbreak of Shiga toxin-producing Escherichia coli O157 associated with raw drinking milk. Epidemiology and Infection 144:2812-2823
https://www.gov.uk/government/news/update-as-e-coli-o157-investigation-continues
https://ecdc.europa.eu/sites/portal/files/media/en/publications/Publications/RRA-Escherichia-coli-O26-Romania-Italy-April2016.pdf
https://www.cdc.gov/ecoli/2016/o157-02-16/index.html
https://www.cdc.gov/ecoli/2016/o121-06-16/index.html
www.eurosurveillance.org/content/10.2807/1560-7917.ES.2017.22.21.30541
https://www.cdc.gov/ecoli/2017/o157h7-12-17/index.html
https://www.cdc.gov/ecoli/2017/o157h7-03-17/index.html
https://www.cdc.gov/ecoli/2018/o157h7-04-18/index.html
https://www.cdc.gov/ecoli/2019/flour-05-19/index.html

The O104:H4 STEC strain responsible for the very large series of outbreaks, notably in Germany, also harboured virulence factors normally associated with EAEC and this pathotype is not normally reported to be associated with cattle or other ruminants. It is therefore possible that the origin of this organism may have been human and/or other animal faecal material causing contamination of seeds.

An outbreak has been described in which the preparation of foods, leeks and potatoes caused the infections (Launders et al., 2016) with 252 cases involved, of which 80 were hospitalised, two suffered HUS and one died.

Control of STEC in the food industry

Cattle are asymptomatic excreters of STEC O157, which are transient members of their gut microflora. The presence of STEC O157 appears to be influenced by the age of the animals and the season. Faecal shedding is usually longer and more intense in calves than in adult cattle and increases after weaning. It is also much higher during the summer period (Caprioli et al., 2005). Faecal carriage rates in cattle worldwide can vary from <1 to 70% of all animals tested (Hussein and Bollinger, 2005). Some animals, known as super-shedders, excrete very large numbers of STEC O157 at certain times, and these animals may be of prime importance in transmission, and maintenance both within herds and the wider environment (Low et al., 2005).

Since faecal contamination may occur, STEC can be transferred onto carcasses at slaughter, or into milk at milking. Raw milk that is to be used to make unpasteurised dairy products must therefore be produced with a high level of hygiene and sourced from selected farms aware of the intended use of the milk. Hygiene should be routinely evaluated by monitoring faecal contamination indicators, and verified by pathogen screening at an appropriate frequency. Raw milk specifically intended for pasteurisation, or where milk production hygiene cannot be verified, is not suitable for consumption as is, or for the production of raw milk dairy products, because of the risk of being sources of STEC infections.

Uncooked produce (including sprouted seeds, vegetables and fruit e.g. lettuce, spinach, bean sprouts, sandwiches, and unpasteurised apple juice) can also become contaminated by faeces and cause outbreaks since commercial washing conditions may be insufficient to remove the organism (Hutchison et al., 2017). Waterborne outbreaks have occurred either because clean water was not available (or had become contaminated with run-off from cattle pastures) or because of chlorination failure. Exposure to recreational water has also caused outbreaks.

It is currently not feasible either to detect all contaminated animals at slaughter or to eliminate the bacterium at source. Quality assurance programmes in slaughterhouses should stress the need to minimise faecal contamination of carcases and to chill meat rapidly. Measures must be taken in slaughterhouses to minimise faecal contamination of carcases. EC Regulation 854/2004 specifies that dirty animals that pose an unacceptable risk of contamination to meat during slaughter cannot be slaughtered for human consumption unless they are cleaned beforehand.

One of the slaughterhouse measures is an assessment by a veterinarian of the fleece/hide cleanliness of animals arriving at the slaughterhouse. Animals are graded from 1 to 5 with the highest score indicating high faecal contamination. The veterinarian has to decide if the animals are to be rejected, cleaned for resubmission to ante-mortem inspection or particular attention paid to hygiene procedures during processing. The use of potable water or steam, decontamination with organic acids of whole carcasses after slaughter can reduce pathogens that originate from faecal contamination.

Elsewhere in the food industry, procedures to ensure that incoming food materials and ingredients are of good quality should be in place and followed. Screening of raw meats for STEC is not an effective control mechanism because contamination rates are low and routine screening insufficiently sensitive.

In 1999, the United States Department of Agriculture (USDA) approved the use of irradiation to treat ground beef, seeds for sprouting, and fresh produce in order to inactivate pathogenic bacteria, particularly STEC. While irradiation would improve the safety of ground beef, adequate cooking remains the most practical and certain way of eliminating STEC infection.

In food manufacture and processing, quality assurance of raw and in-process materials, finished products, and the manufacturing environment should be based on the requirements identified by a HACCP evaluation and the end-product specification. This can include the adoption of operational prerequisite programmes that minimise faecal contamination from animals and contaminated water in the field, effective washing and disinfection during processing, and management of process flow to avoid cross contamination. Following a series of outbreaks of STEC O157 infections associated with unpasteurised apple juice, culminating in the large Odwalla outbreak, the US Food and Drug Administration (FDA) introduced a rule for warning labelling of unpasteurised juice, and in 2001 introduced a rule (finalised in 2004) requiring that juice processors must use FDA-specified HACCP principles for juice processing in accordance with 21. CFR Part 120. Health Canada introduced similar requirements in August 2000. The problem with apple juice arises from use of faecally contaminated fallen apples in orchards where livestock have been grazing.

Following outbreaks associated with fermented meats in the US, the Food Safety Inspection Service produced compliance guidelines for fermented meat products recommending 5-log (or 2-log, where raw materials are tested and no STEC O157 is detected) reductions in STEC O157, based on recommendations by the Blue Ribbon Task Force on Dry Fermented Sausage and E. coli O157:H7, published in 1996.

Development, validation, and implementation of good hygiene practices at processing plants, including verification by testing for microbiological indicators (Enterobacteriaceae and generic E. coli), are likely to be the most effective method for reducing the public health risks from STEC infection. However, compliance with the hygiene criteria does not guarantee the absence of STEC at concentrations sufficient to cause human disease. Therefore, monitoring should take into account compliance with the criteria of the Regulation (EC) No 2073/2005, the presence of STEC in high risk foodstuffs and other risk-based supporting data. Application of efficient, validated HACCP-procedures for production of raw RTE, meat preparations, and other foods is important to reduce the public health risks for STEC infection. Any increases in levels of indicator organisms outside of target levels and tolerances should trigger an active investigation for their increased levels before critical limits are exceeded and unsafe food produced. Effective process control of all cooking/pasteurisation stages is essential to ensure that the correct heating temperatures and times are consistently achieved.

STEC do not have unusual heat resistance with D values at 57ºC to 64ºC between 270 and 9.6 seconds, and so adequate cooking of meat and pasteurisation of milk will protect consumers from infection from these sources. The advice in the UK is that minced beef and minced beef products, including burgers, should be cooked to a minimum internal temperature of 70°C for 2 minutes or equivalent. Industry should provide cooking instructions for burgers to ensure that they are adequately cooked, that the meat juices run clear and there are no pink areas inside cooked products. In USA, the FDA now recommend that ground beef products should be cooked so that all parts of the food are heated to at least 68.3°C for a minimum of 15 seconds. Several of the recorded outbreaks of STEC illness are likely to have been caused by undercooking of burgers and similar products such as blade tenderised steaks in the USA. There is reported to be demand from UK consumers for burgers to be cooked rare (or to appear rare), and the FSA have produced relevant guidelines (FSA, 2013).

The minimum pH for growth of some strains is around pH 4 (Molina et al., 2003). At lower pH values the rate of inactivation is dependent on a number of physicochemical parameters, but temperature is the primary one with increasing temperature causing increasing rates of inactivation at non-growth permissive pH values. The organism can survive fermentation, drying and storage for up to two months in fermented sausage with a pH of 4.5 and, indeed, this food type has been associated with outbreaks. The organism was shown to survive between one and seven weeks in other acidic foods (mayonnaise and apple juice) with pH values between 3 and 4; and these food types have also been associated with STEC outbreaks. STEC are resistant to drying and can survive for long periods under hostile conditions. Some STEC strains may grow at 8°C, but refrigeration at or below 5°C should prevent growth, although any organisms present will likely survive normal refrigeration temperatures and remain viable for several weeks.

Food handlers suffering from STEC O157 infections should be excluded from work until two negative faecal specimens taken at intervals of not less than 48 hours have been obtained (FSA, 2009). It is essential to provide hygienic food handling and good chilled storage conditions to ensure that other foods do not become contaminated and that growth of the organism cannot occur. The FSA produces food safety guidance for food businesses to clarify the steps that they need to take to control the risk of food becoming contaminated by E. coli O157. Guidance has been developed in response to the outbreaks of E. coli O157 infections, for example in Scotland (1996) and Wales (2005) – cases attributed to cross-contamination arising from poor food handling.

It is not likely that STEC can be completely eliminated from raw meats or the cattle population given the present state of technology. However, research aimed at reducing faecal shedding in cattle through changing the gut flora by altering their diet, as well as employing probiotics, competitive gut flora, antimicrobials, and vaccination with Shiga toxoids has been reported (Callaway et al., 2004). Phages may also be used to control STEC on meat (e.g. Hudson et al., 2010), and a wide variety of physical (hot water) and chemical (organic and hypochlorous acids) treatments (Signorini et al., 2018) are available for meat and carcass decontamination, although most are not permitted in the EU. As an example, the application of 4.5% (w/v) lactic acid to beef resulted in a 3.3 log CFU/cm² reduction in concentration of an eight STEC isolate cocktail (Hudson et al., 2019). It has been suggested that super-shedders are of prime importance in transmission and maintenance within herds and the wider environment (Low et al., 2005), and strategies to exclude these animals from the food chain, as well as ‘disinfection’ of their rectal contents by lactoferrin for example, may be effective in reducing the input of STEC into the environment.

Testing for STEC

Widely used standard methods for detection and confirmation of E. coli are not appropriate as many STEC strains grow poorly, or not at all, at 44°C. There are many standardised and sensitive methods to detect STEC O157 in food and animal samples. Testing for other serotypes is more involved, but there is now an ISO method that is being applied across the EU.

Enrichment

All methods require an initial enrichment step to bring the pathogen to detectable concentrations and to ensure that all detections of DNA by molecular means are from viable cells (Hudson, 2017). The most widely used enrichment medium for use with food is Tryptone Soya Broth (TSB), which can be supplemented with selective agents including novobiocin, vancomycin, cefsulodin, cefixime, and bile salts. However, this is not used by the FDA or USDA who, instead use modified Buffered Peptone Water. There is currently no consensus on the optimal incubation temperature (37°C versus 42°C), but recent information suggests that 42°C or 37°C for 6 hours, with completion of incubation at 44°C (total 24 hours) are equally effective (Amagliani et al., 2018). Factors affecting the optimal incubation period include the relative concentration of competing microflora and whether any STEC present may be stressed or sub-lethally injured.

Detection

It is not easy to detect STEC in food enrichments by standard plating, where low levels of the pathogen may be swamped by comparatively high numbers of other bacteria, particularly in unprocessed foods such as raw meats. However, the sensitivity of plating can be improved by Immunomagnetic Separation (IMS) which uses paramagnetic beads, coated with antibodies against the target organism (Calle et al., 2018; Kraft et al., 2017) allowing the pathogen to be concentrated prior to plating. The most widely used solid medium for the detection of non-sorbitol fermenting STEC O157 is sorbitol MacConkey (SMAC) agar. Media that simultaneously indicate sorbitol fermentation and β-glucuronidase activity have also been developed, including chromogenic media, and a range of selective indicative media is commercially available (Fan et al., 2018; Parsons et al., 2016). The selectivity of solid media can be improved by using selective supplements, the most frequently used being cefixime and potassium tellurite (e.g. CT-SMAC) (Zadik et al., 1993). However, some STEC O157 strains are sensitive to cefixime and potassium tellurite and therefore may not be detected on CT-SMAC agar (MacRae et al., 1997), and most STEC non-O157 are sensitive to tellurite (Fan et al., 2018).

Many commercial kits which can be used for the detection of STEC in enrichments are available, including immunoassays, PCR, and real time PCR assays (Bettelheim and Beutin, 2003; Wang et al., 2013).

Presumptive positive isolates should be confirmed as E. coli by biochemical reactions.

Typing

Methods other than serotyping include phage typing (Khakhria et al., 1990), Pulsed Field Gel Electrophoresis (e.g. Jaros et al., 2013) and Whole Genome Sequencing (Lindsey et al., 2016). Data from the latter may also be compatible with multi-locus sequence typing information (Holmes et al., 2015).

Methods – gold standard

The USDA FSIS methods for STEC non-O157 and E. coli O157:H7 differ. One difference is that testing for STEC non-O157 (serogroups O26, O45, O103, O111, O121, O145) involves a two-stage PCR screen, while the methodology for STEC O157:H7 only includes a single-stage PCR screening test. In the STEC non-O157 screen, the first stage detects samples positive for the genes stx1 and/or stx2 and eae [Wang et al. (2013)]. The ISO (International Standards Organisation, 2012) method uses real time PCR for the detection of genes in mTSB+n enrichments. If either stx gene is detected, then further isolation attempts are required.

End-product testing is a quality control rather than a quality assurance approach and does not present an effective control strategy. However, the USDA mandates such testing for certain kinds of beef for both STEC O157 and the six major (‘Big Six’) STEC non-O157 serogroups (O26, O45, O103, O111, O121, and O145). STEC serogroups O157, O26, O103, O111, O145 and O104:H4 are recognised as causing most of the HUS cases occurring in the EU.

Microbiological Criteria

Commission Regulation (EU) No 209/2013 applies to sprouts placed on the market during their shelf life that have not received a treatment effectively eliminating Salmonella and STEC. This stipulates an absence in 25g (n=5, c=0) of serogroups O157, O26, O103, O111, O145 and O104:H4, and the use of PR CEN/ ISO TS 13136 ‘taking into account the most recent adaptation by the EU reference laboratory for E. coli, including Verotoxigenic E. coli (VTEC), for the detection of STEC O104:H4’.

The FSA and Food Standards Scotland developed a working policy on the detection of STEC in foods following the German sprouted seed outbreak caused by a strain with an unusual combination of pathogenicity genes (Food Standards Scotland, 2016). The question addressed was ‘what steps should the regulator take on the detection of an STEC in food?’ The general approach is to divide foods into:

Profile 1 - not going to be subject to a process to kill STEC, prior to consumption. In which case, isolation of a STEC is considered grounds for a recall, even though not all STEC are pathogenic;
Profile 2 - subject to a process to kill STEC prior to consumption. The isolation of a strain capable of causing serious disease would not result in a recall, as long as advice is provided to the consumer in respect to handling instructions, to ensure it is cooked /treated before consumption, enough to remove the STEC risk.

Conclusions

STEC can cause very serious human illness. Numbers of reported cases are usually low (c. 1,000 per annum, in England and Wales) but, because of the serious nature of the illness, individual cases constitute a considerable health burden. The main source of the organism is cattle faeces and it is currently not likely that STEC can be completely eliminated from raw meats, or the cattle population.

Manufacturers, caterers, and consumers need to understand how they can each reduce the chance of causing foodborne STEC illness. The most effective ways of preventing STEC infections are:

thorough cooking of raw meats
pasteurisation of milk
treatment of water supplies
the avoidance of cross-contamination from raw meats, or cattle faeces, to other foods
rigorous personal hygiene.

Foods such as RTE vegetables present challenges, as the options for pathogen control are limited. Generally, the detection of STEC as a group is laborious, and currently there are no simple, inexpensive methods available for routine detection and isolation of all STEC strains. Good hygiene practices at processing plants, including monitoring for microbiological indicators ( and generic E. coli) to determine the effectiveness of those practices, is an effective method for reducing the public health risks of STEC infection.

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