Strategies for the detection of Escherichia coli O157:H7 in foods

Summary, 419 Introduction, 419 Epidemiology and occurrences, 420 Methods of detection, 420 Conventional methods, 420 Immunomagnetic separation, 421 Immunological detection, 421 Enzyme-linked immunosorbent assay, 421 Nonenzymatic immunoassays, 421 PCR-based detection methods, 422 The BAX® automated PCR system, 423 Biosensors, 423 Fluorescence and microscopy, 424 Emerging technologies, 424 Microarrays, 424 Molecular beacons, 424 Integrated systems, 425 Concluding remarks, 425 References, 427 This review assesses the various methods used to detect Escherichia coli O157:H7 in foods. As this organism has been involved in many outbreaks of disease, it is essential to develop a rapid, yet reliable, method of detection. Conventional methods such as culturing and biochemical tests are covered, followed by a discussion of immunological methods. Both enzymatic and nonenzymatic approaches are discussed, and commercially available kits based on these principles are described. PCR has allowed the rapid amplification of very small numbers of organisms and standard PCR along with multiplex and real-time PCR are discussed. Biosensors and microarrays can provide real-time detection and the current status of each of these is reviewed. It is believed that molecular beacons and integrated systems (lab-on-a-chip) can offer potential advantages for the detection of this pathogen and both are analysed. Drawbacks and advantages of each method described are considered throughout the article. Escherichia coli was first isolated in 1885 from children's faeces by the German bacteriologist Theodor Escherich. It is a normal commensal organism of the gastrointestinal tract of human beings and, although, generally harmless, it can cause a number of infections such as Gram-negative sepsis, urinary tract infection, pneumonia in immunocompromised patients and meningitis (Adams and Moss 1995). Strains of E. coli were first recognized as a cause of enteritis by workers in England investigating summer diarrhoea in infants in the early 1940s (Adams and Moss 1995). Until 1982, three major strains were described: enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC). In 1982, E. coli serotype O157:H7 was implicated in two outbreaks of haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). This organism has been classified as verocytotoxigenic E. coli (VTEC). Haemorrhagic colitis is characterized by abdominal pain, watery diarrhoea followed by bloody diarrhoea. HUS occurs in all age groups but is more common in infants and young children. It is the most important complication of infection by E. coli O157:H7 and is characterized by the sudden onset of haemolytic anaemia with fragmentation of red blood cells, thrombocytopenia and acute renal failure after acute gastroenteritis. The gastrointestinal disease may be severe, with HC being presented, and the central nervous system, pancreas, lungs and heart also being affected (Fitzpatrick 1999). Food is one of the most important sources of VTEC infection and some of those implicated are hamburger meat, bruised apples, unpasteurized apple juice and milk, potatoes, lettuce, unchlorinated water and mayonnaise. Cattle are considered to be one of the major sources of O157:H7, which is spread through faecal contamination of food. The bovine population is also an important reservoir as shedding occurs intermittently, the timing of which is difficult to predict. Thus, human beings may be infected at any time and all measures should be taken to reduce the risk to public health. The first major occurrences were in the USA in 1982 which involved hamburgers from fast food chains in Oregon and Michigan (Snyder 1998). In 1988, 30 students at a high school in Minnesota fell ill after consuming partially-cooked beef patties, and in late 1992 and early 1993, four deaths in four states (Washington, Idaho, California and Nevada) were recorded. These were once again attributed to hamburgers. In 1996, unpasteurized apple juice was implicated in an outbreak in California. Also in that year, an outbreak in Lanarkshire, Scotland claimed 20 lives at a nursing home. This was as a result of the victims consuming beef contaminated with the organism (Bell and Kyriakides 1998). In August 1997, Hudson Foods recalled 25 million pounds of ground beef after an E. coli outbreak was traced to its plant in Nebraska (Snyder 1998). In 1999, a serious outbreak affected New York state where 1000 people were affected with two deaths recorded (Charatan 1999). In this incident, the source was found to be a contaminated well at the Washington County Fair in upstate New York. Runoff from cow manure after torrential rain contaminated the well and water consumption through products such as ice, snow cones and lemonade was the likely exposure route (Charatan 1999). In 2000, the major outbreak was in Walkerton, Ontario where seven people died and 2300 became ill as a result of infected water supplies. In 2001, 100 cases of poisoning because of E. coli was reported at nursing homes in Ontario while in 2002 there were further concerns about contaminated water in southern Ontario. In general, the infected patients are usually vulnerable members of the community such as the very young or very old and immunocompromised individuals. The infective dose of E. coli O157:H7 is 50–100 organisms and the incubation period to the onset of diarrhoea can vary from 1 to 8 days (Singleton 1995). The satisfactory microbiological quality for E. coli O157 is <20 CFU g−1 with the acceptable range being 20 to <100 CFU g−1 (Gilbert et al. 2000). However, it is the opinion of the Advisory Committee for Food and Dairy Products (ACFDP) of the UK that ready-to-eat foods should be free from E. coli O157 and other VTEC organisms (Gilbert et al. 2000). An important perspective is the Public Health aspect associated with outbreaks of disease caused by E. coli O157:H7. The Centers for Disease Control in the USA has estimated that 76 million people suffer from food-borne illnesses annually with 325 000 being admitted to hospitals of whom more than 5000 die. It has been estimated that the yearly cost of these illnesses is US $5–6 billion in direct medical expenses and lost productivity. Escherichia coli O157:H7 causes 73 000 illnesses and 61 deaths per year in the USA (CDC 2003). In the UK, the Health Protection Agency (formerly, the Public Health Laboratory Service) has indicated that in 2001, there were 85 468 food poisoning notifications, which represent a sixfold increase from 1982. Of these, 768 were because of E. coli O157:H7 (Health Protection Agency 2003). Thus, it is clear that rapid and sensitive methods for this organism are required. Conventional methods have included plating and culturing, and the use of biochemical tests. With respect to E. coli O157:H7, detection has been carried out by the use of sorbitol MacConkey agar (SMAC), which consists of bile salts, a carbohydrate source, sorbitol and an indicator. Under normal laboratory conditions, O157:H7 does not ferment sorbitol. If O157:H7 is present, colourless colonies will appear while other Enterobacteriaceae will show up as pink colonies. Early work on the use of SMAC found that, for E. coli O157:H7, there was a high level of accuracy and sensitivity (March and Ratnam 1986). Enrichment broths containing peptone, vancomycin, cefixime, cefsulodin and potassium tellurite are also used to enhance the detection of the organism by providing nutrients which allow the specific organisms to produce more colonies. In recent years, modified agar methods have been described for different applications. Silk and Donnelly (1997) have reported that by using trypticase soya agar, acid-injured E. coli O157:H7 in autoclaved apple cider were detected at higher sensitivities than other media. The apple cider had a pH of 3·2 which will injure the bacteria and the method was developed to identify viable organisms after 72 h in the cider. The authors have indicated that special recovery steps are needed when analysing acidic foods, which may contain the bacterium. A universal pre-enrichment (UP) medium has been developed for the simultaneous recovery of E. coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella enterica serotype typhimurium (Thippareddi et al. 1995). It was found that the addition of oxyrase enhanced the growth of the organisms being investigated. Even injured bacteria were recovered from inoculated food samples such as turkey ham, mayonnaise and ground beef. This approach may prove useful where few organisms are implicated in causing disease. An interesting development has been the combined use of commercially available rainbow agar O157 and PCR. This was used to detect the organism in raw meat with the rainbow agar being selective and sensitive for the screening of E. coli O157:H7 from artificially and naturally contaminated meat samples in 24 h (Radu et al. 2000). Isolates suspected of containing the pathogen were amplified by PCR with this aspect adding a further 6–8 h to the analysis time. This method may be considered as time-consuming. Several biochemical and other tests (the IMViC tests) can be used to differentiate E. coli from other Enterobacteriaceae. These include the ability to produce indole from tryptophan (I), sufficient acid to reduce the medium pH below 4·4, the break point of the indicator methyl red (M), acetoin (V) and the ability to utilize citrate (C) (Adams and Moss 1995). Usually, the first preliminary test involves the use of the API system (bioMerieux, Paris, France), which can identify enterobacteria in 4 h based on the use of biochemical tests. Furthermore, the system can be used to detect other organisms such as Staphylococcus, Candida, Streptococcus and Campylobacter. Latex agglutination reactions are simple and easy to use, and are used to detect the presence of either antibody or antigen in a sample. These are attached to latex beads and if the corresponding antigen or antibody is present, the latex beads will agglutinate when mixed with the sample being investigated. The separation and detection of a specific microbial species from a mixed culture, such as food, by plating on selective media is usually inefficient without pre-enrichment steps (Safarik et al. 1995). One approach towards solving this problem is to use a specific magnetic separation of the target organism directly from the sample or pre-enrichment medium. Most of the particles used for these separations are super-paramagnetic, i.e. they only exhibit magnetic properties in the presence of an external magnetic field. They can be easily removed by a magnetic separator (Safarik et al. 1995). The most common magnetic carriers are Dynabeads® (Dynal, Oslo, Norway) which are polystyrene-based particles ranging from 2·8 to 4·5 μm. It is also possible to obtain coated Dynabeads® with covalently immobilized streptavidin or secondary antibodies against selective primary antibodies. In the direct IMS technique, immunomagnetic particles specific for the target organism are suspended in the mixed cell suspension. After incubation, the particles with bound target cells are separated from the suspension with a magnetic particle separator, the remaining suspension is removed and magnetic particles are washed several times (Safarik et al. 1995). The indirect approach involves the addition of primary antibodies to the bacterial suspension and binding of the target cell-surface structures. Magnetic particles with immobilized secondary antibodies are then added. After the interaction of the primary and secondary antibodies, the entire complex is removed from the suspension by a magnetic separator (Safarik et al. 1995). Immuno-detection has become a widely used approach for E. coli O157:H7 because it allows for sensitive and specific detection. In this section, we will describe some of the methods, which have been developed. Enzyme-linked approaches are very popular and some interesting methods have been reported in the literature. One of these involves the use of enzyme-linked immunomagnetic electrochemistry. This involves the sandwiching of bacterial analyte between antibody-coated magnetic beads and an alkaline phosphatase-conjugated antibody (Gehring et al. 1999). The beads were attached to the surface of magnetized graphite ink electrodes in a multiwell plate format. The substrate was then added and the electroactive product generated was measured by square-wave voltammetry. Detection of 4·7 × 103 cells ml−1 was possible in ca 80 min. A chemiluminescence enzyme immunoassay has been developed which uses different E. coli O157 serotypes (Kovacs and Rasky 2001). Tenfold dilutions of 24 h broth cultures of the test strains were performed and the detection limit was found to be 103–104 cells ml−1. Fratamico and Strobaugh (1998) compared enzyme-linked immunosorbent assay (ELISA) with the direct immunofluorescent filter technique (DIFT) and multiplex PCR for the detection of the bacterium in beef carcass wash water. They found that, following a 4 h enrichment culturing, ELISA could detect 100 colony forming units (CFU) ml−1 while DIFT detected 0·1 CFU ml−1 and multiplex PCR was 1 CFU ml−1. This gives excellent sensitivities but a major drawback is the lengthy enrichment procedure. Several other types of immunoassays have been developed for the detection of E. coli O157:H7 with a few interesting ones being chosen for study. Mansel Griffiths’ group has used immunomagnetic separation (IMS) and a fluorescently stained bacteriophage to detect the pathogen in broth (Goodridge et al. 1999a). In combination with flow cytometry, the fluorescent-bacteriophage assay (FBA) was able to detect ca 100 cells ml−1. These investigators have also used this approach to detect E. coli O157:H7 in inoculated ground beef and raw milk (Goodridge et al. 1999b). It was reported that 2·2 CFU g−1 of artificially contaminated ground beef were detected after a 6-h enrichment. In milk, the detection limit was 10–100 CFU ml−1, following a 10-h enrichment procedure. The FBA may be useful as a method for the preliminary detection of the organism in food but further research into the specificity and applications to other foods are required before it can be widely adopted. A solid phase fluorescent capillary immunoassay has also been developed (Czajka and Batt 1996). A soft-glass capillary tube served as a solid support for adsorption of heat-killed bacteria. Polyclonal anti-E. coli O157:H7 antibody, conjugated with biotin, was used with the bound antigen–antibody complex being detected by avidin labelled with a fluorescent cyanine dye. For ground beef, the detection limit was 1 CFU 10 g−1 after enrichment for 7 h while for apple cider it was 0·5 CFU ml−1 after a 7-h enrichment. In a similar approach, a time-resolved fluorescence immunoassay (TRFIA) using IMS was used to detect organisms in apple cider (Yu et al. 2002). The TRFIA used a polyclonal antibody bound to immunomagnetic beads as the capture antibody, with the same antibody labelled with europium as the detection antibody. The authors indicate that the limit of detection of the assay was 103 cells with 10–100 CFU ml−1 detected in 6 h. This appears comparable with the results obtained by the solid phase fluorescent immunoassay. In recent years, PCR has become very important as a technique for the detection of bacteria. The main reason for this is that the DNA from a single bacterial cell can be amplified in about 1 h, which is very rapid compared with the methods described previously. However, the method can also amplify dead cells and care must be taken in designing the experiments. Furthermore, this makes data interpretation complex and it is an issue that has to be addressed as it has long-range implications from a legal perspective. This is especially true in the EU where new legislation will be introduced shortly. Escherichia coli may be found in a number of unrelated environments and its ability to survive beyond a host, which will permit re-infection, is an important feature of its life cycle (Jaykus 2003; Winfield and Groisman 2003). This is of paramount importance and must be considered when designing and developing real-time detection methods. In this account, we will describe a few of the approaches involving this technique to detect E. coli O157:H7. A PCR assay targeting the 3′-end of the eae gene of E. coli O157:H7 was found to be specific, with sensitivity being 1 pg DNA or 103 CFU PCR per reaction (Uyttendaele et al. 1999). Furthermore, studies were carried out to determine the effect of the food matrix and sample preparation method on PCR detection of nonviable cells using heat-killed bacteria in ground beef. Sample preparation methods included centrifugation, buoyant density centrifugation (BDC), IMS, chelex extraction and swabbing. It was found that IMS was the only method which did not produce false-positive results, provided the number of cells were below 108 CFU g−1. Above this number, this method also produced false positives which is a severe limitation of this approach. Several variations of the standard PCR have recently appeared and these have assisted in producing more sensitive detection methods. Of these, multiplex PCR and real-time PCR are proving to be the most popular. The former allows several targets to be co-amplified in one PCR by combining or and PCR allows reactions to be characterized by the time when amplification of the PCR product is first detected by use of a 2000). et al. have described a multiplex PCR using of which amplified of eae and analysing E. coli strains O157:H7 and it was found that the assay could serotype O157:H7 from other In this bovine faeces was also A multiplex PCR was used to detect the pathogen in and water et al. 2001). and water samples were with E. coli O157:H7 and to PCR. The reported detection of 1 CFU ml−1 water and CFU g−1 An interesting aspect was that of the bacteria for days before addition to did not the detection of cell numbers as as 10 CFU g−1 were obtained in one a PCR has been developed to detect viable E. coli O157:H7. is an enzyme which is of DNA from in the and and several eae and as for They found that and were detected in all growth with the products of and being in isolated from viable However, the target was not amplified after The authors that the gene is the most target for viable bacteria. can detect of the This an those PCR which enrichment as it the time required for analysis while a high The BAX® system with automated detection was developed by and it allows for the rapid detection of bacteria in raw products and samples 2001). It uses DNA amplification followed by to determine the presence or of a specific target 2001). DNA and for a and an are combined into one an which the amplification and detection has been developed. This uses an of as the source and a tube to detect the fluorescent which is produced 2001). The assay are with from foods after enrichment. The does on the samples and then the to determine if the target is Several have appeared in the 4 which the BAX® PCR et al. food and for E. coli O157:H7, and reported that was more sensitive than culturing for some However, they reported that both and methods were to numbers of the bacteria from In it was claimed that the system was than methods with the former a detection of compared with for the method et al. 1998). The is for the of E. coli in various foods but it does not provide of the organisms One of the major in developing a rapid test for E. coli O157:H7 is that a number of steps are usually lengthy enrichment Thus, are more but there are several remaining to be These will be in this and in the along with the which have been to develop for this An was used to detect the bacterium in 10 and 25 ground beef samples and 2002). The uses a to direct which the the are and a of the into the A and the fluorescent A immunoassay was which allowed the detection of × 103 CFU g−1 for 25 samples and CFU g−1 for the sample. The authors reported that false positives were obtained with results being obtained 25 after sample group has used a in a to in the detection of DNA from E. coli et al. 2002). DNA was and to It was reported that detection of containing the was obtained in ca 20 by fluorescence and have described a combination to detect of E. coli O157:H7. PCR was used to amplify a to the pathogen and, in a was attached to the surface of the This was by using the the been to the surface of the The authors have that this approach may be for the organism in food, water and It has also been that this method may be into an integrated system for rapid PCR-based DNA analysis but this will more research before this may be Several on the use of surface have to appear in the literature. A using antibodies against E. coli O157:H7 was found to have a detection limit of × CFU ml−1 et al. However, does not well with other methods as described group has used of of with of DNA at the and a DNA to amplify DNA by PCR et al. 2000). A can as a in PCR. The PCR products were then by which allowed the of the O157:H7 strains from other bacteria. has reported on the use of to the binding reactions of immobilized E. coli O157:H7 with such as and A system was used to the of binding on the surface with and indicated that of binding while These studies were performed to allow a rapid of for carcass to or from of the surface Biosensors, 1997, by of and fluorescence with flow was used to detect E. coli O157:H7 in ground beef et al. 2001). The authors reported that this approach several advantages available it is able to of food or water in it can detect single organisms other methods may more than the method is automated and it is specific for the organisms being It is believed that this system can provide the sensitivity and specificity required for the detection of bacteria. a investigating the effect of E. coli O157:H7 cells labelled with an enhanced fluorescent was carried out and 2002). The cells were with and and in fluorescent were was also The indicate that used to the pathogen may not be for of viable and dead cells after with It was found that was for the detection of dead cells while antibodies labelled with is the of for the of and cell In this section, we will three approaches which are towards producing rapid and sensitive detection methods for E. coli O157:H7. These are the development of integrated systems the use of molecular beacons and the of However, it should be that of these methods is free from and the organism to provide a major in detection. allow of specific DNA or to be detected on a small or about 2001). This has allowed more rapid to but there are to the use of this are of and and to useful from the of data these are to appear which use this to detect E. coli O157:H7. and have reported on the use of PCR and acid microarrays to specific and sensitive detection. The was of to four targets and DNA was amplified by multiplex the being to the without any It has been claimed that the is times more sensitive than and was of 1 of The authors have further reported that by using a combination of immunomagnetic PCR and a CFU ml−1 of bacteria in carcass wash water were detected without the for In a this group has described the development of an flow cell and system for the automated IMS of E. coli O157:H7 from carcass et al. 2001). pre-enrichment was and high was used to enhance the magnetic the flow to of the immunomagnetic particles throughout the cells were isolated directly from carcass with a recovery at 103 CFU ml−1. Molecular beacons to DNA The essential feature of these is et al. 2000). The is of two one labelled with a and the other with a of the to the target causes the to with the result that the and are to each This to the of fluorescence et al. 2000). A of this approach involves the molecular beacons on solid These beacons exhibit similar properties to the et al. 2000). of detection of with molecular beacons of the The use of in the detection of E. coli O157:H7 is to have and a few have been and used a to detect the pathogen in milk PCR amplification of The was to to a of the gene and to when the became The

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