Foodborne illness caused by shigatoxin-producing Escherichia coli (STEC) - sometimes also referred to as verocytotoxin-producing E. coli (VTEC) or enterohaemorrhagic E. coli (EHEC) - was first recognised in the early 1980s. Although the illness is uncommon, STEC is now regarded as an important pathogen because of the very serious complications which may follow infection. The O157:H7 serotype is the predominant cause of STEC infection in the UK and USA but other serotypes are implicated, particularly in mainland Europe. In comparison with, for example, salmonellosis, numbers of cases appear to be low. Infection may produce mild or severe bloody diarrhoea, as well as severe and sometimes fatal complications including the haemolytic uraemic syndrome (HUS). The infective dose may be very low.
The main reservoir for STEC is the bovine intestine, although other ruminants may also be important. There are four main transmission routes for infection: foodborne, waterborne, direct or indirect contact with animals, and person-to-person spread. Most cases are not recognised as parts of outbreaks and where these do occur, more than one transmission route may be involved. Food vectors linked to transmission include ground beef, poorly-prepared dried and fermented sausages, milk and milk products, apple juice, sprouting seeds, and fresh produce (salads, herbs, etc.) that have become contaminated with animal faeces. Water has been responsible for some of the largest outbreaks.
Control of STEC illness in humans requires good slaughterhouse and kitchen hygiene and heat treatment of raw meat and milk. STEC is destroyed by heat; adequate cooking of meat (an internal temperature of 70°C for 2 minutes) and pasteurisation of milk will protect consumers from infection from these sources. It is essential to provide hygienic food handling and good chilled storage conditions to ensure that other foods do not become contaminated. Infected food handlers should be excluded from working until microbiological clearance of a stool sample has been obtained.
End-product testing for STEC, as for other pathogens, is a quality control rather than a quality assurance approach and thus is not an effective control strategy. However, in the USA, the Department of Agriculture (USDA) has adopted precisely that regulatory strategy for ground beef both for STEC O157 and the six major (‘Big Six’) non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145). STEC serogroups O157, O26, O103, O111, O145 and O104:H4 are recognised to cause most of the Haemolytic Uremic Syndrome (HUS) cases occurring in the EU, and are therefore the subject of forthcoming sampling and testing rules in relation to sprouted seeds.
Contamination rates in suspect foods are low, so the chance of isolating the bacteria from samples in a batch of food is small. The 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 standardised and sensitive methods to detect and isolate STEC O157 from food, and animals. For the other serotypes, there are no universally accepted and validated methods, but USDA’s Food Safety Inspection Serivce (USDA, 2012) has set out methodology for the non-O157 ‘Big Six’.
This Information Statement was written by IFST Scientific Committee and co-authored by Peter McLure.
Escherichia coli is a common organism found in the lower intestinal tract of healthy humans and animals. As a result of contamination via animal faeces or sewage, it is also readily found in the environment, e.g. untreated water, agricultural run-off, soil, vegetation, some moist or wet areas in factories, and abattoirs, etc. There are many types of E. coli, a few of which are potentially pathogenic by a variety of infective and toxin-producing mechanisms. Symptoms vary according to the strain of E. coli encountered, and the resistance of the individual to such illnesses. Infants, young children, elderly and sick people are generally more susceptible to E. coli (and other) infections than healthy older children and adults.
The pathogenic E.coli strain types that may cause human illness are divided into 6 types or pathotypes which are based on the virulence factors and disease causing mechanisms. These pathotypes are usually referred to as diarrheagenic E. coli:
• Enteropathogenic E. coli (EPEC)
• Attaching and effacing E. coli (AEEC)
• Enterotoxigenic E. coli (ETEC)
• Enteroinvasive E. coli (EIEC)
• Enterohaemorrhagic E. coli (EHEC) including Shiga toxin-producing E. coli (STEC) / Verocytotoxin-producing E. coli (VTEC).
• Enteroaggregative E.coli (EAEC)
VTEC or STEC?
Specific to their toxin-producing capabilities, VTEC and STEC E.coli nomenclature commonly refers to strains within the Enterohaemorrhagic pathotype (EHEC).
Shiga toxin-producing E.coli (STEC) strains can produce toxins with a high degree of homology to that produced by Shigella dysenteriae type 1. There are two types of toxin:
• Shiga toxin 1 (Stx1) differs from Shiga toxin (Stx) produced by Shigella dysenteriae by between one and seven amino acids
• Shiga toxin 2 (Stx 2) which is a diverse and heterogenous group of subtypes sharing approximately 60% homology to Stx
These toxins differ from true Shiga toxin (Stx) but as they are considered to belong to the same family, E. coli that produce these are often called STEC.
VTEC is used to refer to both Verocytotoxin-producing E.coli and Verotoxigenic E. coli (they are the same). A vero cell toxicity test can be used to detect either one or both verocytotoxins produced, giving the name VTEC. Verocytotoxins are synonymous with Shiga-like toxins Stx 1 and Stx 2. Therefore both terms (VTEC and STEC) are used to describe the same type of pathogenic E. coli.
The pathogenicity of STEC not only involves the production of a toxin but also the adhesion to, and colonisation of, the intestinal tract and the production of VT. The term EHEC is used to describe pathogenic E. coli that cause haemorrhagic gastrointestinal disease in humans. Clinical symptoms are very variable; some patients are completely asymptomatic whilst in others infection can be fatal or very serious with long-term health impairment. Disease symptoms range from mild diarrhoea to severe bloody diarrhoea (haemorrhagic colitis) and in some patients, particularly children, serious sequelae develop, including the haemolytic uraemic syndrome (HUS, damage to the kidneys resulting in blood in the urine), haemolytic anaemia (loss of red cells), thrombocytopaenia (loss of platelets), and thrombotic thrombocytopaenic purpura (TTP, 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. Most STEC infections in the UK and USA arise from strains of serotype O157, but other serotypes can produce ST and this serotype is in the minority in some mainland European countries.
VTEC O157 colonies are more easily detectable on some types of culture media (CT SMAC medium) than non-O157 VTEC strains, and the detection of these is often limited to a small number of specialized laboratories. The non-O157 VTEC are therefore more often missed in laboratory diagnosis of stool and food specimens which may account for the apparent predominance of this serotype in some countries. However, this probably also reflects true national/regional differences in the distribution of VTEC types in the food chain. It is also the case that some STEC, particularly those not harbouring virulence factors other than Shiga-like toxins, do not cause disease in humans. These are commonly found in cattle and other ruminants.
STEC illness was first recognised in the UK in 1982 and, since then, infections have been reported from more than 30 countries on 6 continents. In 1996, there was an outbreak of gastrointestinal infections with E. coli O157 in Scotland during which 20 people died and just over 500 became ill. Nearly 11% of the patients within the outbreak had a diagnosis of HUS and TTP (Dundas, et al, 1999, Pennington 2000). This outbreak was associated with a raw and cooked meat operation and was investigated by the Pennington Group (1997). This Group identified failures in the training of employees, the use of temperature probes to monitor the cooking process, cleaning schedules, separation of raw and cooked product, provision of lists of the places supplied, and in local authority inspections. This Group proposed far-reaching recommendations, which, inter alia, gave rise to the licensing of butchers’ shops (now discontinued) and Hazard Analysis Critical Control Point (HACCP) training programmes for butchers (mandatory for those handling raw meat and ready-to-eat foods). The outbreak was also the subject of a Fatal Accident Inquiry. The FSA Scotland and the then Scottish Executive set up a Task Force, which reported in June 2001 with 105 recommendations in areas as diverse as waste recycling, access to the countryside, diagnosis, patient care by health professionals, and outbreak control. An Action Plan was established in response to the Task Force Report.
The largest ever outbreak of E. coli O157 in Wales and the second largest in the UK occurred in September 2005 with one fataility; 44 schools across four local authority areas supplied by the butcher were at the centre of the outbreak. 31 people were hospitalised and a child died. The outbreak was caused by cooked meats that had been contaminated with E. coli O157. Investigation of the associated food premises identified that there were serious, and repeated, breaches of Food Safety Regulations. The report identified that the butcher had failed to ensure that critical procedures, such as cleaning and the separation of raw and cooked meats, were carried out effectively. He also falsified certain records that were an important part of food safety practice. The business’HACCP plan was not valid. The report concluded that requirements for food hygiene that were in place at the time of the outbreak should have been sufficient to prevent it (Pennington, 2009). 24 recommendations for systemic improvements and reinforcement were made, which the Government accepted and which drove establishment of the Food Hygiene Rating System, which is, at the time of writing, nearing full participation by Local Authorities across the UK.
Although large outbreaks of STEC infection have occurred in the UK, the majority of cases are sporadic in nature. In England and Wales, the number of laboratory reports of E. coli O157 infections reached a peak of 1182 in 2011 and has ranged from 361 to 1182 infections between 1991 and 2011 (HPA, 2012a).
The incidence of O157 infections is variable throughout the UK with the highest rate in Scotland (Locking et al, 2006). The majority of cases of O157 infection in the UK are sporadic or occur within households. Outbreaks of STEC are usually small with an average of 8 cases, however, larger outbreaks can occur. The largest outbreak O157 in England 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 (HPA, 2012b).
On 21 May 2011, Germany reported an ongoing outbreak of STEC serotype O104:H4. From an initial case control study, the outbreak was associated with the consumption of fresh salad vegetables. Subsequent investigations showed that the risk of infection was significantly associated with the consumption of fresh sprouted seeds rather than with other fresh vegetables. Subsequently, a cluster of patients with bloody diarrhoea was reported, after having participated in a local event in France on 8 June 2011.
Consumption of sprouted seeds was also associated with occurrence of the disease in this cluster. Furthermore, the STEC isolates responsible for the outbreaks in France and Germany were found to be indistinguishable. It was therefore concluded that there was a common source for both outbreaks. A comparison of the back traceability information on the seeds from the French and German outbreaks led to the conclusion that a specific consignment (lot) of fenugreek seeds imported from Egypt was the most likely link between the outbreaks, although it could not be excluded that other lots from the same exporter and importer were also implicated. On 26 July 2011, the Robert Koch Institute declared the outbreak finished. A total of 3911 cases and 51 deaths were reported to the ECDC and WHO, linked to the outbreaks (EFSA, 2011). The O104:H4 STEC strain responsible for these outbreaks also harboured virulence factors normally associated with EAEC and this pathotype is not normally reported to be associated with cattle or other ruminants and the origin of this organism may have been human and/or animal faecal material.
Non-food routes of infection are important in transmission of STEC infections. For example, in 2000 more than 1,000 cases (6 deaths) were associated with contaminated drinking water in Ontario (Woodward et al., 2002). In addition to waterborne sources, cases have 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 in the USA. There was evidence of airborne transmission in the USA. At least 19 people who had visited a county fair in Ohio in 2001 fell ill with E. coli that apparently spread through sawdust in the air at an exhibition hall, the first time researchers have connected an outbreak to a contaminated building. Testing at the building found E. coli O157 in the rafters, the walls and the sawdust, in some cases 10 months after the fair (Varma et al, 2003).
E. coli O157 is a worldwide threat to public health and outbreaks have been reported in Europe, North America, the Far East and Australasia. However VTEC infections are less commonly reported in patients in less industrialised countries.
The Centers for Disease Control and Prevention (CDC) estimates that there are approximately 175,905 domestically acquired foodborne illnesses associated with all STEC producing annually in the USA (Scallan et al, 2011). E. coli 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 associated with STEC (112,752 or 64%) are caused by non-O157 STEC. While more than 50 non-O157 STEC serogroups have been associated with human illness, 70 to 80 percent of confirmed non-O157 STEC 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. In the U.S, at least one outbreak and several sporadic illnesses from non-O157 STEC serogroups have been associated with ground beef products.
It is estimated that E. coli O157 has an infective dose of only 10 to 100 organisms; therefore, with such a small infective dose, cross contamination of high risk foods with raw food is a potential problem in retail outlets and at home. The intestinal tracts of cattle are considered to be the major animal source of STEC that are virulent to humans (Caprioli et al., 2005). STEC O157 and other serotypes associated with human infections have also frequently been isolated from the intestinal content of other ruminant species, including sheep, goat, water buffalo, and wild ruminants, while pigs and poultry have not been identified to be major sources of STEC. Therefore foods most likely to be contaminated are raw meats, particularly beef, as well as raw milk, seeds, and fresh produce that are exposed to animal faeces, e.g. via contaminated water, organic manure, from pests or feral animals.
Cattle are asymptomatic excretors 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 by the season. 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). The reported prevalence of STEC and/or STEC O157 in cattle is also influenced by the sampling and detection methods adopted in the investigations. Faecal carriage rates in cattle worldwide can vary between <1 to 70% of all animals tested (Hussein and Bollinger, 2005). Some animals excrete very large numbers of STEC O157 at certain times and these are referred to as super-shedders. It has been suggested that these super-shedders are of prime importance in transmission and maintenance within herds and the wider environment (Low et al., 2005).
The considerably different isolation rates partly reflect differences in material studied and methods used for isolation and detection. Since faecal contamination occurs, STEC can be transferred onto carcasses at slaughter, or into milk at milking. Since these bacteria are readily killed by heat (including pasteurisation), the consumption of raw milk, cream and cheeses made from raw milk is strongly discouraged because they are a potential source of STEC infections, as well as for other pathogens. 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 hence cause outbreaks.. 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.
Over 200 serotypes producing ST have been identified from all sources and over 100 have been associated with disease in humans. However, in most countries, strains of STEC of serotypes O157 in addition to O26, O103, O111 and O145 constitute the majority of infections. The serotype, however, is only a surrogate marker for the potential to cause human disease but the availability of molecular techniques enables simple direct detection and subtyping of ST genes as well other virulence-associated genes. This has led to the concept of the ‘seropathotype’ that classifies STEC into five groups based on the incidence of serotypes in human disease, associations with outbreaks versus sporadic infection, and their capacity to cause HUS or HC (Karmali, 2003; Wickham et al., 2006). This classification attempts to provide an understanding in differences in virulence of STEC. Seropathotype A strains (STEC O157) have a high relative incidence, commonly cause outbreaks and are associated with HUS. STEC O26, O103 O111 and O145 together with O121 fall into Seropathotype B, as they have a moderate incidence and are uncommon in outbreaks but are associated with HUS. Seropathotype C includes O91, O104 and O113 strains associated with HUS, but these strains were of low incidence and rarely caused outbreaks until the sprouted fenugreek seed-linked O104 outbreak in Germany and France in summer 2011. Seropathotypes D and E are not HUS-associated and are uncommon in man or found only in non-human sources. Surveys targeting isolation of STEC (but not specifically O157) and from non-human sources generally produce isolates from groups C and D.
The organism is heat-sensitive and should be destroyed by the same temperature that is recommended to eliminate Salmonella and Listeria. The advice in the UK is that minced beef and minced beef products, including beefburgers, 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 Food & Drug Administration (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. Pasteurisation of milk also effectively eliminates STEC: thus, at 72°C for 16.2 seconds more than 104 cells/ml will be killed. Several of the recorded outbreaks of STEC illness are likely to have been caused by undercooking of beefburgers and similar products.
The minimum pH for growth of some strains is around or just below pH 4 (Zhao et al., 1993) but some strains of STEC can survive in lower pH products. The organism can survive fermentation, drying and storage for up to 2 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 1 and 7 weeks in other acidic foods (mayonnaise and apple juice) with pH values between 3 and 4; and these food types have been associated with STEC outbreaks. STEC are resistant to drying and can survive for long periods under hostile conditions. However, STEC does not have unusual heat resistance and D values at 57ºC to 64ºC are between 270 and 9.6 seconds. Some STEC strains will grow at 8°C, but refrigeration at or below 5°C should prevent growth. However, any organisms present may survive normal refrigeration temperatures for several weeks.
Large multi-state outbreaks of STEC infection in the USA and EU have drawn attention to fresh produce and sprouted seeds as a food vehicle. In 2006, 205 cases (3 deaths, 103 hospitalisations and 31 cases of HUS) were identified as associated with consumption of spinach in the USA (Anon, 2006a). Furthermore, in the same year, 71 cases (53 hospitalisations and 8 cases of HUS) were associated with consumption of lettuce (Anon 2006b). In both instances, the raw foods were suspected to have become contaminated in the environment, probably via water contaminated with agricultural run-off from cattle farming or via the faeces of feral animals.
Outbreaks may also be caused by cross-contamination of ready-to-eat 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. The measures needed to protect consumers from STEC are the same as those needed to protect against Salmonella, Campylobacter, Listeria and most other non-spore-forming foodborne pathogens.
Food handlers suffering from E. coli O157 infection should be excluded from work until two negative faecal specimens taken at intervals of not less than 48 hours have been obtained (Food Standards Agency, 2009 ).
The Food Standards Agency have produced 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. The guidance has been developed in response to the outbreaks of E. coli O157 in Scotland in 1996 and Wales in 2005, which were attributed to cross-contamination arising from poor handling of food.
STEC have been found in the faeces of healthy cattle, and it is currently not feasible either to detect all contaminated animals 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 vet has to decide if the animals are to be rejected, cleaned for re-submission to ante mortem inspection or particular attention paid to hygiene procedures during processing. The use of potable water or steam as well as decontamination with organic acids in the USA of whole carcasses after slaughter can reduce pathogens (including STEC) 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 should be followed. Screening of raw meats for STEC is not an effective control mechanism because contamination rates are low and routine screening specifically for this organism is considered unlikely to be successful. The value of screening raw meat is being debated widely. Undoubtedly, screening will detect some contaminated material and this can then be designated to a secure heat treatment process but, as screening can never detect all contaminated lots, it is a poor control procedure. Nevertheless, in the USA, the USDA has adopted exactly that regulatory strategy for ground beef, with consequent huge recalls (e.g. Hudson Foods in 1997, ConAgra Foods in 2002) (USDA FSIS Final Rule, 1999).
In 1999, the USDA approved the use of irradiation to treat ground beef in order to inactivate pathogenic bacteria, particularly STEC. While the use of this technique would improve the safety of ground beef, adequate cooking of meat remains the most practical and sure way of eliminating the danger of STEC infection from this source.
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 via contaminated water in the field, effective washing and disinfection during processing, and management of process flow to avoid cross contamination from contaminated products. Following a series of outbreaks of E. coli O157 associated with unpasteurised apple juice, culminating in the large Odwalla outbreak, the US Food and Drug Administration (FDA) in 1998 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 presumed problem with apple juice arises from use of faecally contaminated fallen apples in orchards where livestock have been grazing. A Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables is available from the FDA (FDA, 1998).
Following the outbreaks associated with fermented meats in the US, the Food Safety Inspection Service (FSIS, 1997) produced compliance guidelines for fermented meat products recommending either 5-log or 2-log (where raw materials are tested and no O157 is detected) reductions in O157 STEC, 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 in 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 ready-to-eat meat, 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.
Methods used in medical laboratories to detect the organism from stools are usually more successful, probably because the number of STEC cells present in the stools of someone made ill by the organism is often relatively high in comparison to the background flora. The gold standard for the diagnosis of STEC infection is by detection of specific toxic effects from the ST in a patient’s stool sample by using Vero cells (mammalian cells growing in tissue culture). However, toxin is now more usually detected by the use of immunological methods and STEC can indirectly be diagnosed by examining E. coli strains or samples for the genes encoding ST (stx). Microbiological methods for isolation of STEC are available (see below) and, since a relatively small number of STEC serotypes are responsible for the majority of human STEC infections, serotype-specific detection methods have been developed where strains are isolated on the basis of their O antigen and are subsequently analysed for ST production or presence of stx genes. However, the diagnosis of STEC in medical laboratories is laborious and, currently, there are no simple, inexpensive methods available for routine isolation of all STEC strains.
It is not easy to detect STEC in foods where low levels of E. coli may be swamped by high numbers of other bacteria, particularly in unprocessed foods such as raw meats. STEC are phenotypically very similar to all other E. coli; however, STEC O157 are usually both unable to ferment sorbitol within 24 hours of incubation and lack -glucuronidase activity (March and Ratnam, 1986; Ratnam et al., 1988; Thompson et al., 1990). These characteristics are utilised in the routine selective isolation of STEC O157. 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 -glucuronidase activity have also been developed, including different chromogenic media. A range of selective indicative media is commercially available. The selectivity of the different solid media can be improved by the use of selective supplements, the most frequently used being cefixime, a third generation cephalosporine, 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).
Following incubation of the isolation media, individual colonies suspected to be STEC O157 should be tested for the O157 antigen by using STEC O157 antiserum or latex agglutination reagents. Isolates agglutinating with O157 antiserum should be confirmed as E. coli by biochemical reactions, since other species and STEC non-O157 can cross-react with O157 antiserum. Since not all STEC O157 strains produce ST, it is necessary to confirm ST production or the presence of stx genes.
The FSIS methods for non-O157 STEC and E. coli O157:H7 differ, but both have similar stages when results are communicated by the laboratory (e.g., potential, presumptive, confirmed). One difference between these two methods is that testing for non-O157 STEC involves a two-stage polymerase-chain-reaction (PCR) screening test, while the methodology for E. coli O157:H7 only includes a single-stage PCR screening test. In the non-O157 STEC screening test, the first stage will detect samples positive for the genes stx (Shiga toxin) and eae (intimin) (USDA, 2012).
Since food and environmental samples usually contain low numbers of STEC O157 together with an abundant microbial flora, selective enrichment steps are therefore required. The most widely used media for the enrichment of STEC O157 for food uses is tryptone soya broth (TSB), which can be supplemented with selective agents including novobiocin, vancomycin, cefsulodin, cefixime, and bile salts (Doyle and Schoeni, 1987, Chapman et al., 1994). There is currently no consensus on optimal incubation temperature (37°C versus 42°C) and time (6-8 hours incubation versus overnight incubation) for all types of samples.
The incubation period required will depend on the competing microflora. Standard methods for food include the analysis of both the 6-h and 18-h incubation enrichment cultures. A 6-8 hour incubation of the enrichment broth increases the sensitivity when analysing matrices with a high number of background flora. However, when stressed or sub-lethally injured STEC O157 are present, there are difficulties in reaching a detectable level after 6-8 hours of enrichment. Therefore, this short period of incubation can only be recommended when testing matrices where E. coli has a short-lag time before onset of growth, as for example with minced meat products. This is also relevant to challenge testing (many companies use this to establish safety of their products), where inappropriate recovery conditions can lead the researcher to conclude more rapid die-off than actually occurs. Conditions should be used that allow recovery of injured cells (e.g. some initial recovery on non-selective agar and then transfer via membrane to selective agar).
Following incubation, enriched cultures are subcultured directly onto selective indicative solid media. However, the sensitivity of culture-based enrichment is vastly improved by the use of immunomagnetic separation (IMS) techniques. IMS is a process whereby specific antibodies are linked to paramagnetic beads. These reagents are, for example, mixed with enrichment broths. Following antigen/antibody reactions, separation and concentration of the specific organism on to magnetised areas is achieved which allows the discard of other components (including much of the background microflora) from the broth. IMS allow significant improvements in microbiological enrichment procedures and, originally developed for STEC O157, reagents are now available for other STEC serotypes (O26, O103, O111, and O145) associated with human disease.
The Nordic Committee on Food Analysis (NMKL) and the International Organization for Standardization (ISO) have issued horizontal methods applicable for culture-based detection of STEC O157 in all types of foods and feeding stuffs (NMKL No. 164, 1999, and ISO 16654: 2001). Both methods prescribe the use of TSB supplemented with bile salts and novobiocin (mTSBn) for the pre-enrichment step, with an incubation period of 6-8 hours as well as 18-24 hours at 41.5°C. Further, both methods prescribe to perform an immunomagnetic affinity purification step and to subculture the immunomagnetic particles with adhering bacteria onto CT-SMAC and the user’s choice of a second selective isolation agar.
Forthcoming EU legislation (SANCO, 2012) setting microbiological criteria for ‘sprouts’ that have not received a treatment effective to eliminate Salmonella spp. and STEC stipulates zero tolerance of six STEC serogroups (namely O157, O26, O103, O111, O145 and O104:H4)., and 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. This Standard is in development through a sub-group of Working Group 6 of the Technical Committee 275 of the European Normalisation Committee (CEN TC275/WG6) and based on a PCR-based horizontal method.
STEC can cause very serious illness in humans and the main source of the organism is the faeces of cattle. It is not likely that STEC can be completely eliminated from raw meats or the cattle population. However, research is active to reduce faecal spreading in cattle by changing the gut flora by altering their diet as well as feeding them with probiotics, competitive gut flora as well as antibiotics, phage and even vaccination (Callaway et al., 2004). Since some animals excrete very large numbers of STEC O157 at certain times, it has been suggested that these 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 may be effective in reducing the input of STEC into the environment.
More than 95% of infections detected in Scotland, England and Wales were due to STEC O157 but, in Ireland, this is 86% with the remainder caused by non-O157 strains. However, in continental Europe more than half of the infections were attributed to serotypes other than O157 but there are considerable national differences. O157 is the most commonly detected serotype in Belgium, France, Finland, Hungary, the Netherlands, Sweden and Spain. However, in Denmark, Germany, Italy, Norway and Luxembourg, other serotypes are most commonly recognised. Some of these differences reflect national diagnostic and surveillance strategies; however, they may also reflect natural differences in the disease. Data from Scotland (Locking et al., 2001) indicates that even when diagnostic procedures for the detection of STEC non-O157 are routinely applied to samples of faeces from patients with diarrhoea, STEC non-O157 are rare. In recent years, human infections with sorbitol-fermenting (SF) STEC O157 have been increasingly recognised in some parts of Europe . Although the methods used for STEC O157 are not validated for the detection of SF STEC O157, these organisms can be detected by testing sorbitol-fermenting colonies grown on solid media that do not present typical STEC O157 colonies for the O157 antigen. Because of these national differences, efforts are being made to understand better the epidemiology of the non-O157 VTEC, which includes improving diagnostic tests for diagnosis, identifying risk factors, identifying reservoirs of infection, and developing microbiological detection systems for food and the environment.
Manufacturers, caterers and consumers need to understand how they can each reduce the chance of causing foodborne STEC illness. There is a continued need for education that promulgates the most effective ways of preventing STEC infections, including thorough cooking of raw meats, pasteurisation of milk and the avoidance of cross-contamination from raw meats or cattle faeces to other foods. Because of the associations with fresh produce (especially salad and other leafy vegetables eaten raw), efforts are needed to better understand the control of these types of foods, including tracking the persistence of STEC in the environment, improved water management, especially for private water supplies, and biosecurity.
STEC can cause very serious illness in humans. Numbers of reported cases are usually low (about 1,000 per year in England and Wales) but, because of the serious nature of the illness, are a considerable health burden and concern. The main source of the organism is the faeces of cattle and it is 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. Thorough cooking of raw meats, pasteurisation of milk, treatment of private water supplies, and the avoidance of cross-contamination from raw meats or cattle faeces to other foods are the most effective ways of preventing STEC infections. Generally, the detection of STEC is laborious, and currently there are no simple, inexpensive methods available for routine isolation of all STEC strains. Good hygiene practices at processing plants including monitoring for microbiological indicators (Enterobacteriaceae and in generic E. coli) to determine the effectiveness of those practices is likely to be the most effective method for reducing the public health risks for STEC infection.
REFERENCES AND FURTHER READING:
Adak, G.K., Long, S.M., O’Brien, S.J., (2002). Trends in indigenous foodborne disease and deaths in England and Wales: 1992 to 2000. Gut; 51: 832-841.
Advisory Committee on the Microbiological Safety of Food (1995).
"Report on Verocytotoxin-Producing Escherichia coli" HMSO. ISBN 0113219091.
Anon. (2006a). Ongoing multistate outbreak of Escherichia coli serotype O157:H7 infections associated with consumption of fresh spinach - United States, September 2006. Morb. Mortal. Wkly. Rep 55:1045-1046.
Anon. (2006b). Multistate outbreak of E. coli O157 infection November – December 2006. Available from http://www.cdc.gov/ecoli/2006/december/121406.htm
Caprioli, A., Morabito, S., Brugere, H. and Oswald, E., (2005). Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission. Vet. Res. 36 (3): 289-311.
Chapman, P. A., Wright, D. J., and Siddons, C. A., (1994). A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from bovine faeces. J. Med. Microbiol. 40 (6): 424-427.
Callaway, T.R., Anderson, R.C., Edrington, T.S., Genovese, K.J., Bischoff, K.M., Poole, T.L., Harvey, R.B. and Nisbet, D.J. (2004). What are we doing about Escherichia coli O157:H7 in cattle? J. Anim. Sci. 82: E93-99.
Doyle, M. P., and Schoeni, J. L., (1987). Isolation of Escherichia coli O157:H7 from retail fresh meats and poultry. Appl. Environ. Microbiol. 53 (10): 2394-2396.
Dundas, S., Murphy, J., Soutar, R.L., Jones, G.A., Hutchinson, S.J. and Todd, W.T.A., (1999). Effectiveness of therapeutic plasma exchange in the Lanarkshire Escherichia coli O157:H7 outbreak. Lancet. 354: 1327-1330.
EFSA (2011). Scientific Report of EFSA. Shiga Toxin-Producing E. coli (STEC) O104:H4 2011 Outbreaks In Europe: Taking Stock. European Food Safety Authority (EFSA), Parma, Italy. http://www.efsa.europa.eu/en/efsajournal/doc/2390.pdf (accessed 4/8/12)
Food Standards Agency Scotland and Scottish Executive (2001). “Report of Task Force on E. coli O157” http://www.gov.scot/Resource/Doc/46922/0014164.pdf, accessed 15/06/15.
Food Standards Agency (2009) “Food Handlers: Fitness to Work Regulatory Guidance and Best Practice Advice for Food Business Operators” http://www.food.gov.uk/sites/default/files/multimedia/pdfs/publication/f...
Food Safety Inspection Service (FSIS) 1997 Draft Compliance Guidelines for Ready-to-Eat Meat and Poultry Products. http://www.fsis.usda.gov/OPPDE/rdad/FRPubs/97-013P/RTEGuide.pdf.
International Society for Infectious Diseases (2003). ProMED Report “E. coli: Aerosol Route suspected – USA (Ohio) 2001”.
HPA (2012a). E. coli O157 Annual Totals. https://www.gov.uk/government/publications/escherichia-coli-e-coli-o157-... (accessed 15/6/15)
HPA (2012b). Epidemiology of VTEC in England & Wales. http://webarchive.nationalarchives.gov.uk/20140714084352/http://www.hpa.... (accessed 16/06/15).Hussein, H. S., and Bollinger, L. M., (2005). Prevalence of Shiga toxin-producing Escherichia coli in beef cattle. J. Food Prot. 68 (10): 2224-2241.
Karmali, M. A. (2003). The medical significance of Shiga toxin-producing Escherichia coli infections. An overview. Methods Mol. Med. 73: 1-7.
Locking, M., Allison, L., Rae, L., Pollack, K.G., Hanson, M.F. 2004 VTEC infections and livestock related exposures in Scotland. 2004. Eurosurveillance 2006; 11: available from http://www.eurosurveillance.org/ew/2006/060223.asp#4.
Low, J. C., McKendrick, I. J., McKechnie, I.J., Fenlon, D., Naylor, S.W., Currie, C., Smith, D.G.E., Allison, L. and Gally, D.L., (2005). Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle. Appl. Environ. Microbiol. 71 (1): 93-97.
March, S. B., and Ratnam, S., (1986). Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J. Clin. Microbiol. 23 (5): 869-872.
MacRae, M., Rebate, T., Johnston, M., Ogden, I.D., (1997). The sensitivity of Escherichia coli O157 to some antimicrobials by conventional and conductance assays. Lett. Appl. Microbiol. 25 (2): 135-137.
Microbiological Safety of Food Funders Group (2010). “Publicly Funded Research Relating to enterovirulent Escherichia coli: Update 2010: research covered from 2000 to 2008”.
The Pennington Group (1997). “Report on the circumstances leading to the 1996 outbreak of Infection with E. coli O157 in Central Scotland, the implications for food safety and the lessons to be learned”, Stationery Office Books (8 April 1997)
Pennington, T.H., (2000). VTEC: lessons learned from British outbreaks. J. Appl. Bact. 88: 90S-8S
Pennington, T.H. (2009). The Public Inquiry into the September 2005 Outbreak of
E.coli O157 in South Wales. http://gov.wales/splash?orig=/ecolidocs/3008707/reporten.pdf (accessed 4/8/12)
Perna, N.T.,Plunkett, G., Burland, V., Mau, R., Glasner, J.D., Rose, D.J., Mayhew, G.F., Evans, P.S., Gregor, J., Kirkpatrick, H.A., Posfai, G., Hackett, J., Klink, S., Boutin, A., Shao, Y., Miller, L., Grotbeck, E.J., Davis, N.W., Lim, A., Dimlanta, E.T., Potamousis, K.D., Apodaca, J., Anantharaman, T.S., Lin, J., Yen, G., Schwartz, D.C., Welch, R.A. and Blattner, F.R. (2001). Genome sequence of enterohaemorrhagic E. coli O157:H7, Nature, 409, 529-533.
Ratnam, S., March, S. B., Ahmed, R., Bezanson, G.S. and Kasatiya, S. (1988). Characterization of Escherichia coli serotype O157:H7. J. Clin. Microbiol. 26 (10): 2006-2012.
SANCO (2013). Commission Regulation (EU) No 209/2013, amending Regulation (EC) No 2073/2005 as regards microbiological criteria for sprouts and the sampling rules for poultry carcases and fresh poultry meat. http://faolex.fao.org/docs/pdf/eur121462.pdf, accessed 15/06/15.
Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M.-A., Roy, S.L., Jones, J.L., and Griffin, P.M. 2011. Foodborne illness acquired in the United States – major pathogens. Emerg Infect Dis. 17 (1):7-15.
Scottish Executive and Food Standards Agency: “Response to the Report of the Task Force on E coli O157” http://www.gov.scot/Resource/Doc/46922/0014164.pdf
Scottish Centre for Infection and Environmental Health (SCIEH) Weekly report, 03 June 1997, Volume 31 No.97/22.
Thompson, J. S., Hodge, D. S., and Borczyk, A. A., (1990). Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O157. J. Clin. Microbiol. 28 (10): 2165-2168.
USDA (2012) FSIS Verification Testing for Non-O157 Shiga Toxin-Producing Escherichia coli (Non-O157 Stec) Under Mt60, Mt52, and Mt53 Sampling Programs http://www.fsis.usda.gov/wps/portal/fsis/topics/data-collection-and-repo... (accessed 15/06/15)
US FDA (2004). “Guidance for Industry: Juice HACCP Hazards and Controls Guidance, First Edition: Final Guidance” http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryIn..., accessed 15/6/15.
University of Nebraska, Institute of Agriculture and Natural Resources: “NU Research Shows Vaccine, Bacterial Feed Additive Each Reduce E. coli in Cattle”: http://www.sciencedaily.com/releases/2003/10/031021061818.htm, accessed 15/06/15.
Varma, J. K., Greene, K.D., Reller, M.E., De Long, S.M., Nowicki, S.F., Di Orio, M., Koch, E.M., Bannerman, T.L., York, S.T., Lambert-Fair, M.A.., Wells, J.G. and Mead, P.S. (2003). g”, JAMA, 290: 2709-12.
Wickham, M. E., Lupp, C., Mascarenhas, M., Vazquez, A, Coombs, B.K., Brown, N.F., Coburn, B.A., Deng, W., Puente, J.L., Karmali, M.A. and Finlay, B.B. (2006). Bacterial genetic determinants of non-O157 STEC outbreaks and hemolytic-uremic syndrome after infection. J. Infect. Dis. 194 (6): 819-827.
Woodward, D. L., Clark, C.A., Caldeira, R.A., Ahmed, R. and Rodgers, F.G. (2002) Verotoxigenic Escherichia coli (VTEC): a major public health threat in Canada. Can J Infect Dis 13:321-330.
Zadik, P. M., Chapman, P. A., and Siddons, C. A., (1993). Use of tellurite for the selection of verocytotoxigenic Escherichia coli O157. J. Med. Microbiol. 39 (2): 155-158.
Zhao, T., Doyle, P., Besser, E.R. (1993). Fate of enterohaemorrhagic Escherichia coli O157:H7 in apple cider with and without preservatives. Appl. Env. Microbiol., 59 (8): 2526-2530.
American Medical Association. “Enterotoxigenic Escherichia coli Infection, a Patient Scenario.”
HPA (2007). Vero cytotoxin-producing Escherichia coli (VTEC) O157.
ILSI Europe “Approach to the control of Entero-haemorrhagic Escherichia coli (EHEC)”
Health Protection Scotland http://www.hps.scot.nhs.uk/giz/index.aspx
UK Defra http://www.defra.gov.uk/
Food Standards Agency “E.coli O157: control of cross-contamination http://www.food.gov.uk/business-industry/guidancenotes/hygguid/ecoliguide
US Centers for Disease Control “Disease Information: Escherichia coli O157:H7” http://www.cdc.gov/ncidod/dbmd/diseaseinfo/escherichiacoli_t.htm
USDA Food Safety and Inspection Service: Final Rule “Irradiation of Meat Food Products”, 9 CFR Parts 381 and 424 http://www.fsis.usda.gov/OPPDE/rdad/FRPubs/97-076F.htm
USDA Agricultural Research Service (2002). News Release “A vision of safe meat”
US FDA Guidance for Industry: Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables, 1998. http://www.cfsan.fda.gov/~dms/prodguid.html
Bell, C. and Kyriakides, A. (1998). "E. coli: A practical approach to the organism and its control in foods." Blackie Academic & Professional, 200pp.
The Institute takes every possible care in compiling, preparing and issuing the information contained in IFST Information Statements, but can accept no liability whatsoever in connection with them. Nothing in them should be construed as absolving anyone from complying with legal requirements. They are provided for general information and guidance and to express expert professional interpretation and opinion, on important food-related issues.