Journal of Applied Microbiology 2004, 96, 419–429
A REVIEW
Strategies for the detection of Escherichia coli O157:H7
in foods
A.K. Deisingh and M. Thompson
Department of Chemistry, University of Toronto, Toronto, Ontario, Canada
2003/0505: received 12 June 2003, revised 28 October 2003 and accepted 30 October 2003
1. Summary, 419
2. Introduction, 419
2.1 Epidemiology and occurrences, 420
3. Methods of detection, 420
3.1 Conventional methods, 420
3.1.1 Immunomagnetic separation, 421
3.2 Immunological detection, 421
3.2.1 Enzyme-linked immunosorbent assay, 421
3.2.2 Nonenzymatic immunoassays, 421
3.3 PCR-based detection methods, 422
3.3.1 The BAX
automated PCR system, 423
1. SUMMARY
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. Conven-
tional methods such as culturing and biochemical tests are
covered, followed by a discussion of immunological meth-
ods. 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.
Present address: Anil K. Deisingh, Caribbean Industrial Research Institute,
University of the West Indies Campus, St Augustine, Trinidad & Tobago, West
Indies.
Correspondence to: Anil K. Deisingh, Department of Chemistry, University of
Toronto, 80 St George Street, Toronto, Ontario, Canada M5S 3H6 (e-mail:
anildeisingh@aol.com).
ª 2004 The Society for Applied Microbiology
doi:10.1111/j.1365-2672.2003.02170.
3.4 Biosensors, 423
3.5 Fluorescence and microscopy, 424
4. Emerging technologies, 424
4.1 Microarrays, 424
4.2 Molecular beacons, 424
4.3 Integrated systems, 425
5. Concluding remarks, 425
6. References, 427
2. INTRODUCTION
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 syn-
drome (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
420 A . K . D E I S I N G H A N D M . T H O M P S O N
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).
2.1 Epidemiology and occurrences
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
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 419–429, doi:10.1111/j.1365-2672.2003.02170.x
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.
3 . M E THO D S O F D ET EC TI ON
3.1 Conventional methods
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 3Æ2 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-enrich-
 STRATEGIES FOR THE DETECTION OF E. COLI O157:H7 IN FOODS
ment (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 ampli-
fied 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 Enterobacteria-
ceae. These include the ability to produce indole from
tryptophan (I), sufficient acid to reduce the medium pH
below 4Æ4, 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 reac-
tions 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.
3.1.1 Immunomagnetic separation. 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 parti-
cles ranging from 2Æ8 to 4Æ5 lm. It is also possible to obtain
coated Dynabeads
with covalently immobilized streptavi-
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 419–429, doi:10.1111/j.1365-2672.2003.02170.x
421
din 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 suspen-
sion by a magnetic separator (Safarik et al. 1995).
3.2 Immunological detection
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.
3.2.1 Enzyme-linked immunosorbent assay. 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 elec-
trochemistry. 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 4Æ7 · 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
0Æ1 CFU ml)1 and multiplex PCR was 1 CFU ml)1. This
gives excellent sensitivities but a major drawback is the
lengthy enrichment procedure.
3.2.2 Nonenzymatic immunoassays. Several other
types of immunoassays have been developed for the
detection of E. coli O157:H7 with a few interesting ones
422 A . K . D E I S I N G H A N D M . T H O M P S O N
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 cyto-
metry, 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 2Æ2 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 anti-
body, 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 0Æ5 CFU ml)1 after a 7-h
enrichment. In a similar approach, a time-resolved fluores-
cence 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.
3.3 PCR-based detection methods
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 interpret-
ation 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-
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 419–429, doi:10.1111/j.1365-2672.2003.02170.x
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 multiplexing primer pairs (Newton and
Graham 1997). Real-time PCR allows reactions to be
characterized by the time when amplification of the PCR
product is first detected by use of a fluorogenic probe (Livak
2000).
Hu et al. (1999) have described a multiplex PCR using
five sets of primers which amplified regions of eae A, slt1,
slt2, fli C and rfb E genes. By analysing 82 E. coli strains
(both O157:H7 and non-O157:H7), it was found that the
assay could successfully distinguish serotype O157:H7 from
other serotypes. In this instance, bovine faeces was also
studied. A multiplex PCR was used to detect the pathogen
in soil and water (Campbell et al. 2001). Soil and water
samples were spiked with E. coli O157:H7 and enriched
prior to PCR. The researchers reported detection limits of
1 CFU ml)1 drinking water and 2 CFU g)1 soil. An
interesting aspect was that starvation of the bacteria for
35 days before addition to soil did not affect the detection of
initial cell numbers as low as 10 CFU g)1 soil. Results were
obtained in one working day.
Finally, a reverse transcriptase PCR (RT-PCR) has been
developed to detect viable E. coli O157:H7. RT is an enzyme
which is capable of synthesizing single-stranded DNA from
RNA in the 5¢–3¢ direction (Newton and Graham 1997).
Yaron and Matthews (2002) studied several genes (rfb E, fli
C, slt1, slt2, mob A, eae A, hly and 16S rRNA) as indicators
for viability. They found that rfb E, slt1, hly and 16S rRNA
amplicons were detected in all growth phases with the
products of 16S rRNA, mob A, rfb E and slt1 being readily
visualized in RNA isolated from viable cells. However, the
16S rRNA target was not amplified after heat treatment.
 STRATEGIES FOR THE DETECTION OF E. COLI O157:H7 IN FOODS
The authors suggest that the rfb E gene is the most suitable
target for detecting viable bacteria. Without pre-enrichment,
RT-PCR can detect 107 CFUs of the organism. This
represents an advance over those PCR approaches, which
require enrichment as it reduces the time required for
analysis while retaining a high sensitivity.
3.3.1 The BAX
automated PCR system. The BAX

system with automated detection was developed by Du Pont
Qualicon (Wilmington, DE, USA) and it allows for the rapid
detection of bacteria in raw ingredients, finished products
and environmental samples (Qualicon 2001). It uses DNA
amplification followed by gel electrophoresis to determine
the presence or absence of a specific target (Fritschel 2001).
Primers, DNA polymerase and deoxynucleotides for PCR, a
positive control and an intercalating dye are combined into
one tablet. Additionally, an instrument, which integrates the
amplification and detection steps, has been developed. This
uses an array of 96 blue light-emitting diodes as the
excitation source and a photomultiplier tube to detect the
fluorescent signal, which is produced (Fritschel 2001). The
assay tablets are hydrated with lysates from foods after
overnight enrichment. The instrument does thermal cycling
on the samples and then analyses the melting curve to
determine if the target is present. Several papers have
appeared in the last 4 years which evaluated the BAX
PCR
system. Shearer et al. (2001) studied 15 food samples,
including cabbage, radish, mushroom, tomato and mango,
for E. coli O157:H7, and reported that BAX was more
sensitive than culturing for some samples. However, they
reported that both BAX and culture methods were unable to
recover low numbers of the bacteria from alfalfa sprouts. In
another study, it was claimed that the BAX system was better
than conventional methods with the former having a
detection rate of 96Æ5% compared with 39% for the best
culture method (Johnson et al. 1998). The BAX is ideal for
the identification of E. coli O 157:H7 in various foods but it
does not provide quantification of the organisms present.
3.4 Biosensors
One of the major problems in developing a rapid test for
E. coli O157:H7 is that a number of steps are usually involved,
including lengthy enrichment procedures. Thus, biosensors
are becoming more commonplace but there are still several
issues remaining to be solved. These will be discussed in this
section and in the Conclusion along with the attempts, which
have been made to develop biosensors for this pathogen.
An evanescent-wave fibre optic biosensor was used to
detect the bacterium in 10 and 25 g ground beef samples
(Demarco and Lim 2002). The biosensor uses a 635-nm
laser diode to direct light onto optical fibre probes, which
generates the evanescent wave. Fluorescent molecules
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 419–429, doi:10.1111/j.1365-2672.2003.02170.x
423
within the evanescent field are excited and a part of the
emission re-couples into the fibre probe. A photodiode
detects and quantifies the fluorescent signal. A sandwich
immunoassay was utilized which allowed the detection
of 9Æ0 · 103 CFU g)1 for 25 g samples and 5Æ2 ·
102 CFU g)1 for the 10-g sample. The authors reported
that no false positives were obtained with results being
obtained 25 min after sample processing. Krull’s group has
used a fibre optic biosensor operated in a total internal
reflection format to assist in the detection of genomic DNA
from coliforms, including E. coli (Almadidy et al. 2002).
Genomic DNA was extracted and sonicated to prepare
300-mer fragments. It was reported that detection of
fragments containing the lac Z sequence was obtained in
ca 20 s by fluorescence measurements.
Deisingh and Thompson (2001) have described a PCR-
acoustic wave sensor combination to detect sequences of
E. coli O157:H7. PCR was used to amplify a 509 base
sequence unique to the pathogen and, in real time, a
biotinylated probe was attached to the surface of the sensor.
This was achieved by using the biotin–neutravidin interac-
tion, the latter having been pre-adsorbed to the surface of
the sensor. The authors have suggested that this approach
may be suitable for detecting the organism in food, water
and clinical samples. It has also been proposed that this
method may be incorporated into an integrated system for
rapid PCR-based DNA analysis but this will require much
more research before this aim may be achieved.
Several papers reporting on the use of surface plasmon
resonance (SPR) have begun to appear in the literature. A
BIACore biosensor (Fig. 1) using antibodies against E. coli
O157:H7 was found to have a detection limit of
5 · 107 CFU ml)1 (Fratamico et al. 1997). However, this,
does not compare well with other biosensor methods as
described above. Karube’s group has used chimeric oligo-
nucleotides consisting of 21 bases of RNA with six bases of
DNA at the 3¢-hydroxyl terminus and a recombinant
thermostable DNA polymerase to amplify DNA fragments
by PCR (Miyachi et al. 2000). A chimeric RNA–DNA
primer can serve as a primer in conventional PCR. The PCR
products were then examined by SPR, which allowed the
differentiation of the O157:H7 strains from other bacteria.
Medina (2002) has reported on the use of SPR to investigate
the binding reactions of immobilized E. coli O157:H7 with
extracellular components such as collagen, fibronectin,
laminin and glucoaminoglycans. A model system was used
to evaluate the inhibition of collagen–laminin binding on the
sensor surface with polysulphated polysaccharides (heparan
sulphate and carrageenans). She indicated that carrageenans
inhibited 71–99% of binding while heparan sulphate
inhibited 39–41%. These studies were performed to allow
a rapid assessment of compounds for carcass treatment to
inhibit or detach pathogens from meat.
424 A . K . D E I S I N G H A N D M . T H O M P S O N
Fig. 1 Schematic of the BIACORE surface plasmon resonance
spectrometer (reprinted from: G. Ramsay, Commercial Biosensors,
1997, by permission of John Wiley and Sons Inc.)
3.5 Fluorescence and microscopy
Laser-induced fluorescence coupled with flow cytometry
was used to detect E. coli O157:H7 in ground beef (Johnson
et al. 2001). The authors reported that this approach offers
several advantages over currently available techniques
including: (i) it is able to examine large quantities of food
or water in real time, (ii) it can detect single organisms
whereas other methods may require more than 104 microbes,
(iii) the method is automated and (iv) it is specific for the
organisms being studied. It is believed that this system can
provide the sensitivity and specificity required for the
detection of pathogenic bacteria.
Recently, a study investigating the effect of treating E. coli
O157:H7 cells labelled with an enhanced green fluorescent
protein (EGFP) plasmid was carried out (Burnett and
Beuchat 2002). The cells were treated with chlorine,
hydrogen peroxide and acetic acid, and changes in fluores-
cent intensity were observed. Confocal scanning laser
microscopy was also performed. The researchers indicate
that EGFP used to label the pathogen may not be suitable for
microscopic differentiation of viable and dead cells after
treatment with sanitizers. It was found that SYTOX Green
(Molecular Probes, Eugene, OR, USA) was preferable for the
detection of dead cells while antibodies labelled with Alexa
Fluor 594 (Molecular Probes) is the substance of choice for
the determination of total (dead and viable) cell counts.
4. EMERGING TECHNOLOGIES
In this section, we will consider three approaches which are
having great impact towards producing rapid and sensitive
detection methods for E. coli O157:H7. These are the
development of integrated systems (lab-on-a-chip), the use
of molecular beacons and the production of microarrays.
However, it should be noted, that none of these methods is
free from problems and the organism continues to provide a
major challenge in detection.
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 419–429, doi:10.1111/j.1365-2672.2003.02170.x
4.1 Microarrays
Microarrays allow thousands of specific DNA or RNA
sequences to be detected simultaneously on a small glass or
silica slide about 1–2 cm2 (Aitman 2001). This has allowed
more rapid analyses to take place but there are drawbacks to
the use of this technology. Microarray instruments are
expensive, of limited availability and require specialist
knowledge and training to extract useful information from
the huge amount of data generated. Notwithstanding these
problems, papers are starting to appear which use this
technology to detect E. coli O157:H7.
Call and co-workers (2001) have reported on the use of
multiplexed PCR and nucleic acid microarrays to attempt
specific and sensitive detection. The array was composed of
25–30-mer oligonucleotide probes complementary to four
targets (intimin, slt-1, slt-2 and haemolysin A). Target DNA
was amplified by multiplex PCR, the amplicons being
hybridized to the array without any purification. It has been
claimed that the array is 32 times more sensitive than gel
electrophoresis and was capable of detecting 1 fg of genomic
DNA. The authors have further reported that by using a
combination of immunomagnetic capture, PCR and a
microarray, 55 CFU ml)1 of bacteria in chicken carcass
wash water were detected without the need for pre-
enrichment.
In a later paper, this group has described the development
of an electromagnetic flow cell and fluidics system for the
automated IMS of E. coli O157:H7 from poultry carcass rinse
(Chandler et al. 2001). No pre-enrichment was necessary and
high porous nickel foam was used to enhance the magnetic
field gradient within the flow path to ensure immobilization
of the immunomagnetic particles throughout the fluid.
Bacterial cells were isolated directly from poultry carcass
rinse with a 39% recovery efficiency at 103 CFU ml)1.
4.2 Molecular beacons
Molecular beacons (MBs) fluoresce upon hybridization to
their complementary DNA target. The essential feature of
these probes is their stem-loop structure (Brown et al.
2000). The stem is constructed of two short oligodeoxynu-
cleotide arms, one terminally labelled with a fluorophore and
the other with a quencher (Fig. 2). Hybridization of the loop
to the target causes the stem to open with the result that the
fluorophore and quencher are no longer close to each other.
This leads to the emission of fluorescence (Brown et al.
2000). A modification of this approach involves supporting
the molecular beacons on solid glass particles. These
beacons exhibit similar properties to the soluble versions
(Brown et al. 2000).
The use of MBs in the detection of E. coli O157:H7 is now
beginning to have success and a few papers have been
 STRATEGIES FOR THE DETECTION OF E. COLI O157:H7 IN FOODS
Fig. 2 Principle of detection of hybrids
with molecular beacons (reprinted from:
M.L.M. Anderson, Nucleic Acid Hybridiza-
tion, Bios Scientific, 1999. By permission of Molecular beacon
the publisher) (nonfluorescent)
published recently. McKillip and Drake (2000) used a
beacon to detect the pathogen in skimmed milk during PCR
amplification of DNA. The probe was designed to hybridize
to a region of the slt2 gene and to fluoresce when the stem-
loop structure became open upon hybridization. The use of
an molecular beacon-PCR (MB-PCR) approach led to faster
results than with gel electrophoresis and it allows for real-
time monitoring of PCR reactions. Also, there is no need for
gel electrophoresis or Southern blotting to be performed,
thereby considerably reducing analysis times. It was possible
to detect 103 CFU ml)1. In another report, MBs were
designed to recognize a 26-bp region of the rfb E gene
(Fortin et al. 2001). The authors indicate that there was
specific detection, with closely related strains (such as O55)
not causing any problems. Without enrichment, it was
possible to detect 102 CFU ml)1 in raw milk and apple
juice. When enrichment was carried out for 6 h, the
detection limit dramatically improved to 1 CFU ml)1.
Molecular beacons can provide rapid, sensitive and semi-
automated detection of E. coli O157:H7 and it is expected
that many more papers will appear where this approach is
used.
4.3 Integrated systems
Integrated systems (lab-on-a-chip) are growing in popularity
as, if successful, they can decrease analysis times and
increase efficiency of detection. To date, no published report
has been seen with respect to E. coli O157:H7 but this will
surely change very soon. Thus, we will briefly survey the
field as this area holds a lot of promise.
Woolley et al. (1996) described the integration of PCR
and capillary electrophoresis in a microfabricated DNA
analysis device (Fig. 3). This approach combines thermal
cycling with high-speed DNA separation by the CE chips.
This system provided an assay of genomic Salmonella DNA
in about 45 min.
Andreas Manz’s group has used a micromachined chem-
ical amplifier to perform PCR in continuous flow at high
speed (Kopp et al. 1998). The authors report that input and
output of DNA is continuous and amplification is inde-
pendent of input concentration. In this report, Neisseria
gonorrhoeae was investigated and a 20-cycle PCR was
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 419–429, doi:10.1111/j.1365-2672.2003.02170.x
425
Target Dabcyle
Fluorophore
Hybrid
(Fluorescent)
completed in 90 s to 18Æ7 min, depending on the flow rate
used.
Finally, researchers at the Lawrence Livermore National
Laboratory have described the use of the Advanced Nucleic
Acid Analyzer (ANAA) for the detection of pathogens such
as Erwinia herbicola, Bacillus subtilis and B. anthracis
(Belgrader et al. 1998). The instrument was composed of
ten silicon reaction chambers with thin -film resistive heaters
and solid-state optics. They reported that detection times
were as short as 16 min and that 102–104 organisms ml)1
could be detected. This instrument allows for rapid analysis,
low power consumption, real-time monitoring and for
ruggedness due to a lack of moving parts.
5. CONCLUDING REMARKS
In this review, we have considered the various strategies
which have been used to detect E. coli O157:H7. This
pathogen is causing more and more outbreaks of disease
every year and so a rapid and reliable detection method is
needed. In the United States, almost 75000 cases of infection
occur annually (Perna et al. 2001). As Perna et al. 2001 state
in the introduction to their paper, the severity of disease,
the lack of effective treatment and the potential for large-
scale outbreaks from contaminated food supplies have
propelled intensive research on the pathogenesis and
detection of E. coli O157:H7. To this list, we can also add
the range of serotypes, low infectious dose and several
transmission modes which all make detection methods even
more difficult (Ge et al. 2002). Complicating the issue even
further is that there can be very high levels of competitor
organisms, which cannot be easily distinguished phenotyp-
ically (Johnson et al. 1998). Sampling methods must be
carefully considered especially when dealing with animal
samples such as faeces, hides and carcasses as different
methods have varying effectiveness (Ransom et al. 2002).
Furthermore, viable versus nonviable culture states of
bacteria must be an integral part of experimental design.
This is a significant challenge to the successful application of
molecular-based methods. However, a partial solution may
be obtained by the use of mRNA as a potential target,
especially if it could be linked to real-time PCR methodo-
logies.
426 A . K . D E I S I N G H A N D M . T H O M P S O N
Many approaches have been utilized and although there
have been successes in developing sensitive and specific
detection methods, many problems remain. A summary of
all the approaches is given in Table I. Conventional methods
are labour-intensive and time-consuming, and results can
take several days to be obtained. However, new methods are
generally compared with the gold standard of plating and
culturing as this is the method, which tends to guarantee the
presence or absence of the bacterium. Still, cultural methods
give variable results for E. coli O157:H7. Although immu-
nological approaches have provided sensitive analyses,
several steps are required to achieve these results. This
means results can take up to 1–2 days to be obtained,
although the actual analysis times may be reported as a few
hours. It has to be recognized that, in many cases,
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 419–429, doi:10.1111/j.1365-2672.2003.02170.x
Fig. 3 Schematic of the integrated PCR-CE
microdevice. (a) Laser-excited confocal fluor-
escence detection apparatus and an integrated
PCR-CE microdevice. (b) Expanded view of
the microfabricated PCR chamber.
(c) Expanded cross-sectional view of the
junction between the PCR and CE devices
(reprinted with permission from:
A.T. Woolley, D. Hadley, P. Landre, A.J. de
Mello, R.A. Mathies and M.A. Northrup,
Analytical Chemistry, 68, 4081–4086.
Copyright 1996, American Chemical Society)
enrichment procedures are necessary and these may require
up to 8 h.
Newer methods, providing advances towards solving
some of the issues, are also plagued by different problems.
Some fluorescence methods, for example, can lead to cross-
signalling which may result in overestimation of the
number of dead cells (Burnett and Beuchat 2002). PCR
has been one of the methods which has made detection
much easier and it has continued to grow strongly. The
process, however, can be inhibited by the food matrix and
by the large amounts of nontarget DNA derived from the
food source (Bhaduri and Cottrell 2001). In these complex
environments, the potential exists for inhibiting substances
to be co-purified resulting in reaction failure and a false-
negative result. It should also be noted that extraction of
 STRATEGIES FOR THE DETECTION OF E. COLI O157:H7 IN FOODS 427

Table 1 Summary of methods used to detect Escherichia coli O157:H7

Method Approx. detection time Detection limit Selected references

Plating/culturing 1 day to 1 week Low CFUs Silk and Donnelly (1997)

Biochemical tests 1 day to several days Low CFUs Adams and Moss (1995)
ELISA 12 h to 2 days 10–100 CFU ml)1 Gehring et al. (1999)
Fluorescent bacteriophage assay 10 h 10–100 CFU ml)1 Goodridge et al. (1999a)
Chemiluminescence enzyme immunoassay 6–8 h 103–104 cells ml)1 Kovacs and Rasky (2001)
Capillary immunoassay 7h 0Æ5–1 CFU ml)1 Czajka and Batt (1996)
Time-resolved fluorescence immunoassay 6h 10–100 CFU ml)1 Yu et al. (2002)
PCR 2–24 h depending 102–105 CFU ml)1 Uyttendaele et al. (1999)
on enrichment
Multiplex PCR 24 h 1–2 CFU ml)1 Hu et al. (1999)
RT- PCR 6–12 h 107 CFU ml)1 Yaron and Matthews (2002)
Laser-induced fluorescence Few hours Single organism Johnson et al. (2001)
Fibre optic biosensor ca 30 min 5Æ2 · 102 CFU g)1 Demarco and Lim (2002)
SPR biosensor 1h 5 · 107 CFU ml)1 Fratamico et al. (1997)
Microarrays <1 h 55 CFU ml)1 Call et al. (2001)
Molecular beacon 1–6 h depending 1–103 CFU ml)1 Fortin et al. (2001)
on enrichment
Integrated systems (lab-on-a-chip) 16–45 min 102–104 organisms ml)1 Belgrader et al. (1998)

DNA is a time-consuming process. As discussed in section
2Æ3, there may be the possibility of false-positives with PCR
in instances where more than 108 CFUs are involved.
Finally, there is still the need for IMS and/or enrichment
procedures, which also increase analysis time. Regardless of
these drawbacks, PCR (and, especially, the newer approa-
ches such as 5¢-nuclease, multiplex and reverse transcrip-
tase assays) has made detection procedures much easier for
all the researchers involved.
Biosensors and microarrays can provide real-time meas-
urements and rapid analysis times. In both cases, less
preparation time is required when compared with some of
the other methods described. Usually, no enrichment
procedures are necessary. However, both methods are in
early stages of development and much speculation is
involved. For biosensors, commercial developments have
been slow as a result of the intense competition from other
methods, and the intrinsic difficulties in rendering the
technology sensitive and reliable enough (Deisingh and
Thompson 2002). On a bright note, biosensors have
produced many interesting developments and they can be
expected to lead to greater successes soon. However,
microarrays generate lots of data, which require very skilled
workers to analyse over many hours. It is also vital to have
proper experimental design. Lucchini et al. (2001) have
summarized the issue succinctly when they stated that
microarray experiments should not be entered into lightly.
The roles of molecular beacons and integrated systems are
still to be evaluated but it is expected that both can produce
major developments in the detection of this pathogen.
Molecular beacons can be very useful in the development of
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 419–429, doi:10.1111/j.1365-2672.2003.02170.x
biosensors and lab-on-a-chip systems. They exhibit a higher
degree of specificity and have better signal to noise ratios
than conventional nucleic acid probes (Park et al. 2000).
They have also been used in an initial integrated system
where a biotinylated beacon was bound to the surface of a
silica chip. An evanescent wave was generated from a prism
placed next to the chip and fluorescent signals were recorded
(Liu and Tan 1999).
Although E. coli O157:H7 is still a great threat to human
and animal welfare, many attempts are being made to
develop new detection methods. Several of these are
promising in terms of speed, reliability, specificity and
sensitivity. These will eventually provide decreased turn-
around times in clinical laboratories. However, unless issues
related to viability and template purification are addressed, it
is difficult to foresee if the food industry and diagnostic
laboratories will have sufficient confidence to adopt these
new procedures.
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