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Appl Environ Microbiol, May 1998, p. 1640-1643, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Influence of Cold Stress on the Preliminary Enrichment Time
Needed for Detection of Enterohemorrhagic Escherichia
coli in Ground Beef by PCR
M.
Uyttendaele,1
C.
Grangette,2
F.
Rogerie,2
S.
Pasteau,2
J.
Debevere,1 and
M.
Lange2,*
Department of Food Technology and Nutrition,
Faculty of Agricultural and Applied Biological Sciences, University
of Ghent, 9000 Ghent, Belgium,1 and
Service de Recherche et Développement, Institut
Pasteur de Lille, 59019 Lille Cedex, France2
Received 30 September 1997/Accepted 23 February 1998
 |
ABSTRACT |
The influence of cold stress at 4 and 0°C on the detection time
as assessed by impedance technology (Bactometer;
Biomérieux, Marcy l'Etoile, France) of different
enterohemorrhagic Escherichia coli (EHEC) strains was
determined. Although there is some variation in susceptibility among
EHEC strains, prolonged exposure of EHEC to cold stress, i.e., 4 and 5 days at 4 and 0°C, respectively, in general significantly increased
their detection time. This reflects an increase of the lag-phase time
caused by cold stress. Two EHEC strains were selected to determine the
minimum preliminary enrichment time that would ensure a positive PCR
detection of low numbers of verotoxin-producing E. coli
(VTEC; 2 to 2 × 105 CFU/25 g) inoculated into ground
beef (25 g) and stored at 4 or 
20°C for 8 and 14 days,
respectively. Incubation times of 6 and 9 h of 1 to 10 CFU/g and 1 to 10 CFU/25 g, respectively, were sufficient for PCR detection of VTEC
in ground beef when analysis was performed immediately after
inoculation (no cold stress). When cells are exposed to cold stress (4 or 20°C) a 24-h enrichment period is recommended. Restriction of
enrichment time to 9 h under these circumstances decreases the
sensitivity of PCR detection to 80 CFU/g. Hence, to obtain maximum
sensitivity, PCR detection of VTEC in naturally contaminated ground
beef should be performed after 24 h of enrichment.
| |
INTRODUCTION |
Escherichia coli O157:H7
is an emerging cause of foodborne illness. Since 1982, when E. coli O157:H7 was first recognized as a pathogen, many food-related
outbreaks have been reported in the United States, Canada, the
United Kingdom, and many other parts of the world, the increase being
due in part to increased awareness of this pathogen (11).
The most common symptom of E. coli O157:H7 infection is
hemorrhagic colitis. The infective dose may be very low, i.e., <100
cells (9).
The mechanism of pathogenicity of E. coli O157:H7 has
not been fully elucidated, but important virulence factors have
been identified. All clinical isolates produce one or two types of verotoxins (VT1 and VT2). Other serotypes of verotoxin-producing E. coli (VTEC) have also been associated with human sporadic
infections or small to large outbreaks of hemorrhagic colitis, e.g.,
O26:H11, O111:H
, O104:H21, etc. They are designated
enterohemorrhagic E. coli (EHEC) (14).
There are no universal biochemical or physiological characteristics for
non-O157 VTEC, thus preventing the use of selective agars to isolate
these serogroups. Detection of part of the VT gene by using DNA probes
and PCR is the method usually used to screen for VTEC (1, 4, 5,
13, 15, 16, 18). These molecular methods are predominantly used
with DNA obtained from suspected colonies isolated by using traditional
coliform media.
Many cases and outbreaks of E. coli O157:H7 infection have
been linked to the consumption of contaminated beef. In order to test
for the presence of VTEC in beef products, the PCR protocol can be
adapted for the detection of VTEC directly from the homogenized sample
without prior isolation of the bacterium. To obtain a sensitive PCR-based assay for direct detection of VTEC in contaminated beef, the
PCR protocol should be preceded by a suitable sample preparation method
that will eliminate components that can interfere with the PCR
reaction. Begum and Jackson (2) used a 10- to 1,000-fold dilution of ground beef to reduce the concentration of components that
inhibit the PCR. The detection limit for ground beef with a decreased
fat content was 6,744 CFU/ml, while in nondefatted ground beef the mean
detection limit was 50,000 CFU/ml. The detection limit of PCR for VTEC
in ground-beef samples, however, can be significantly decreased by
including a 4- to 12-h enrichment phase (2, 20). In
agreement with the classical microbiological methodology
(19), a preliminary 6-h enrichment phase is suggested in PCR
screening studies to detect VTEC in artificially contaminated foods
(7).
In naturally contaminated foods, however, cells are subjected
to stress. VTEC in ground beef is exposed to cold stress because the
product is usually either refrigerated or frozen. Bacterial cells
subjected to stress show an increased lag-phase time (6). The enrichment time needed for a positive PCR detection of
cold-stressed VTEC cells will be adversely affected, and restriction of
the enrichment time to 6 h could lead to an underestimation of the contamination of the beef products. This study was undertaken to
determine the relationship between VTEC cells subjected to stress and
their corresponding minimum enrichment time for a positive PCR
detection.
| |
MATERIALS AND METHODS |
Susceptibility of EHEC to cold stress.
Fifteen EHEC strains
(Table 1) were tested by impedance
technology to determine their susceptibilities to cold stress. An overnight culture (18 to 20 h) incubated at 37°C in buffered
peptone water (BPW; Axcell, St. Genis l'Argentien, France) was diluted to 104 CFU/ml of BPW, and dilutions were kept at 4 and
0°C (melting ice).
After 1, 4, 7, and 14 days of storage at 4°C and after 1, 2, 5, and 7 days of storage at 0°C, samples were taken in duplicate
and analyzed
by impedance technology (Bactometer; Biomérieux,
Marcy l'Etoile,
France) as described by the manufacturer. Briefly,
0.1 ml was added to
0.9 ml of general purpose medium (Biomérieux),
which was then
dispensed into the wells of a Bactometer module.
The module was then
placed into the Bactometer incubator (30°C)
and monitored with the
Bactometer software for 24 h; the detection
time could then be
read. At the same time, to check for any die-off
during the cold
stress, the number of EHEC cells in BPW was determined
by enumeration
on tryptic soy agar (Difco, Detroit, Mich.). Data
were subjected to
statistical analysis (one-sample
t test) by
using SPSS for
Windows, release 7.5 (SPSS, Inc.).
Determination of the length of the enrichment phase for detection
of cold-stressed EHEC in ground beef by PCR.
Samples (25 g) of
ground beef were inoculated with either E. coli O26:H11
(strain Co26; VT1, eae) or E. coli
O157:H (strain Co7; VT2, eae) at different
inoculation levels: 2, 20, 200, 2 × 103, 2 × 104, and 2 × 105 CFU/25 g. An
uninoculated sample served as a control. The ground-beef samples were
analyzed for VTEC by PCR immediately after inoculation, after storage
for 4 and 8 days at 4°C, and after storage for 4, 8, and 14 days at
20°C. Then 25-g samples were prepared for each of the chosen time
intervals.
For analysis the following protocol was used. The ground-beef samples
were 10 times diluted in EC broth (Merck AG, Darmstadt,
Germany) and
homogenized with a Colworth stomacher. Depending
upon the inoculation
level and the subjected stress, samples were
taken for PCR analysis
during enrichment at 37°C at one or more
of the following time
intervals: 0, 3, 6, 9, or 24 h. A 4-ml sample
of the enrichment
phase was withdrawn and centrifuged for 2 min
at 700 rpm to eliminate
residual sediments. Then 1 ml of the supernatant
was removed and
centrifuged for 3 min at 13,000 rpm. DNA was extracted
from the
bacterial pellet as described below.
DNA extraction.
DNA was purified by a simple method that is
a modification of the silica-binding method of Boom et al.
(3). Briefly, purified DNA was obtained from the pelleted
cells by lysis in a guanidinium thiocyanate (GuSCN) buffer and silica
extraction with the Wizard DNA Clean-Up Resin (Promega,
Charbonnières, France). The bacterial pellet was suspended in 450 µl of GuSCN lysis buffer (5.25 mM GuSCN in 0.1M Tris-HCl [pH 6.4]
containing 20 mM EDTA and 1.3% [wt/vol] Triton X-100) and incubated
for 15 min at 55°C. After a cooldown, 300 µl of Wizard DNA Clean-Up
Resin (silica suspension) was added, and the mixture was incubated for
5 min at room temperature. The mixture was then filtered with a Wizard
Minicolumn connected to a vacuum manifold. The retained silica was
washed with 2 ml of 80% isopropanol. The Wizard Minicolumn was dried
by centrifugation in a microcentrifuge tube for 2 min at 12,000 rpm.
The DNA was then eluted by the addition of 50 µl of prewarmed
(70°C) extra-pure water to the Wizard Minicolumn and incubation at
70°C for 5 min. The eluted DNA was collected by centrifugation of the
Wizard Microcolumn in a microcentrifuge tube for 2 min at 12,000 rpm
and stored at 20°C until amplification by PCR.
PCR detection of VT genes.
PCR detection of VT genes, was
performed with degenerate primers ES149 and ES151 as described by Read
et al. (18). This primer set amplifies a conserved sequence
of the VT1 and VT2 genes, generating a 323-bp fragment. Ten microliters
of DNA was added to a reaction mixture containing 1× PCR buffer
(supplied with the enzyme); 200 µM dATP, dCTP, dGTP, and dUTP
(Pharmacia); 50 pmol of each primer (Eurogentec); 2 mM
MgCl2 (Perkin-Elmer); albumin (200 µg/ml; Boehringer); 1 U of Taq DNA polymerase (AmpliTaq; Perkin-Elmer); and 5 µl
(10 pg/µl) of internal control for a final volume of 50 µl. The
internal control was a Bluescript plasmid vector carrying an insert of
a gene different in sequence from and longer than the VT gene and
bordered by the PCR primers ES151 and ES149. Amplification of the
internal control resulted in a 609-bp fragment and confirmed that the
PCR was functioning well. The reaction mixtures were processed through
40 cycles in a DNA thermal cycler (Perkin-Elmer). After an initial
denaturation step of 5 min at 94°C, the cycle program consisted of
denaturation at 94°C for 30 s, annealing of primers at 49°C
for 30 s, and primer extension by DNA polymerase at 72°C for
30 s. To ensure complete strand extension, the reaction mixture
was incubated for 7 min at 72°C after the last cycle. The amplified
product was visualized by ethidium bromide staining after standard
submarine gel electrophoresis of 15 µl of the final reaction mixture
on 1.5% agarose. A "no template" control, in which sterile
distilled water was substituted for the prepared sample, and a positive
control were included with each amplification run.
| |
RESULTS |
Susceptibility of EHEC to cold stress.
The data presented in
Table 2 show that prolonged storage of
EHEC strains at 4°C (4, 7, and 14 days) significantly increases their
detection time (95% confidence intervals not overlapping). In general,
the detection time at day 14 of cold stress at 4°C was 3.55 h
(T14-T0 [Table 2]) longer than for the non-cold-stressed cells (day
0). There is, however, some variation between EHEC strains in their
susceptibility to cold stress. Based on the limits of the 95%
confidence interval for T14-T0 (the detection time at day 14 minus the
detection time at day 0), three groups can be distinguished: (i) those
uneffected by cold stress (T14-T0 < 2.712), i.e., Co17, Co26,
Co29, Co11, and Co12; (ii) those that are susceptible to cold stress
(2.712 < T14-T0 < 4.382), i.e., Co8, Co9, Co14, and Co19;
and (iii) those that are extremely susceptible to cold stress
(T14-T0 < 4.382), i.e., Co7, Co15, Co16, Co18, Co27, and Co6.
Strains (Co7, Co15, Co16, Co18, Co26, Co27, and Co29) of a subset were
evaluated for their susceptibilities to cold stress at 0°C (Table
3). After 1 and 2 days of storage at 0°C, the detection time was not significantly different from that of
the non-cold-stressed cells. After prolonged storage at 0°C (5 or 7 days), the detection time significantly increased. In general, the
detection time increased by 3.086 h after storage for 7 days at 0°C.
One strain, Co7, seemed to be extremely sensitive when subjected to
cold stress.
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TABLE 2.
Influence of cold stress at 4°C on detection time of
15 EHEC strains as assessed by impedance technology
|
|
Two strains (Co7 and Co26) were selected for the study to determine the
effect of cold stress on the enrichment time needed
for positive PCR
detection of EHEC strains.
Determination of the length of the enrichment phase for detection
of cold-stressed EHEC in ground beef by PCR.
This experiment was
performed to determine the minimum preliminary enrichment time needed
to ensure a positive detection by PCR (i.e., amplification of a
conserved region of the VT genes) of low numbers of VTEC (2 to 2 × 105 CFU/25 g) inoculated into ground-beef samples (25 g)
and stored at 4 or 20°C. The detection limit of the applied PCR,
previously established on a bacterial dilution, was 2 CFU per PCR tube.
One milliliter of culture was taken for DNA extraction at different times of enrichment. In agreement with the above-described protocol, DNA was finally eluted in 50 µl of extra-pure water, and 10 µl was
taken for PCR amplification. This results, if a 100% recovery of DNA
is obtained, in a minimum detection limit of 10 CFU/ml of enrichment
medium. A 100% effective homogenization would correspond to a
theoretical detection limit of 2,500 CFU/25 g of contaminated meat
diluted 10-fold in the enrichment medium.
In the control group of ground beef, which was not submitted to cold
stress but was homogenized and incubated immediately
after inoculation,
a PCR detection limit of 2 × 10
4 CFU/25 g was
obtained at 0 h (Table
4). If we
take into account
some deviations due to suboptimal homogenization and
differences
in DNA extraction efficiency, this finding is in agreement
with
the calculated detection limit mentioned above. This means that
for the PCR to detect low numbers of VTEC cells, enrichment of
the
target bacteria is necessary.
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TABLE 4.
Effect of cold stress at 4°C on the enrichment time
needed for detection of EHEC in ground beef as assessed by PCR
detection of the VT genes
|
|
Incubation for 6 and 9 h at 37°C in EC broth is sufficient to
detect, respectively, 1 to 10 CFU/g and 1 to 10 CFU/25 g. In
view
of a limit for direct detection of about 50 CFU/ml, an initial
lag phase of 1 to 2 h, and a generation time of 0.49 h at
37°C
(
12), it was expected that a 6-h enrichment would be
the minimum
needed for a positive PCR detection of these low inoculum
levels,
while a 9-h enrichment would be largely sufficient.
An increase of the minimum enrichment time of 3 h is noticed when
the pathogen is subjected to cold stress due to storage
of the ground
beef at 4°C for 4 days. Thus, in the present experiment
the
pathogen was detected by PCR after 9 and 24 h of enrichment
for
inoculum levels of 1 to 10 CFU/g and 1 to 10 CFU/25 g, respectively.
Exposure to 4 days of cold stress at 4°C only increased the detection
time by 0.6 h as assessed by impedance technology (Table
2).
Longer additional enrichment times were expected for PCR
detection
than for detection with the Bactometer because for the
impedance
experiment pure cultures of EHEC in optimal brain heart
infusion
medium were used, whereas for PCR detection EHEC strains were
inoculated into ground beef, where they are subjected to additional
environmental stresses (e.g., competitive flora, nonoptimal pH,
etc.)
apart from the cold stress.
Prolongation of storage of contaminated ground beef at 4°C for
another 4 days resulted in an additional increase of the preliminary
incubation time by 3 h to achieve a positive PCR detection. Both
of the inoculum sizes (1 to 10 CFU/g and 1 to 10 CFU/25 g) were
only
positive by PCR after 24 h of incubation. Shorter incubation
times
(12 to 15 h) would probably have been sufficient to recover
cold-stressed cells, but these times were not tested since, from
a
practical point of view, these incubation times are not likely
to be
used.
Similar results were obtained when ground beef was stored at
20°C
(Table
5). After 4 and 8 days of storage
at
20°C, a prolongation
of the enrichment time was noticed that was
similar to that which
occurred with storage at 4°C, although in some
cases an additional
3-h delay was obtained after freezing but not after
refrigeration.
Continued storage of the contaminated ground-beef
samples at
20°C
for up to 14 days after inoculation did not affect
the enrichment
time for PCR detection any further.
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TABLE 5.
Effect of cold stress at 20°C on the enrichment time
needed for detection of ETEC in ground beef by PCR detection of the
VT genes
|
|
Even after a 24-h enrichment period, inoculum levels of less than 10 CFU/25 g were not detected after more than 1 week of
storage at
20°C for EHEC strain Co26. This may have been due
to a die-off of
the bacterial cells during the freezing process.
Apart from these
storage conditions, there were four discrepancies
between the
results of the replicates with the two EHEC strains
in which the
enrichment time was prolonged for an additional 3
h each time for
the Co26 strain.
| |
DISCUSSION |
The physiological state and prior history of the cell affect the
lag phase of the bacterial cell. It was reported that the preincubation
temperature influences the lag-phase duration of foodborne pathogenic
microorganisms. The growth rate is not affected by preincubation
conditions (6). In the present study, prolonged exposure of
EHEC cells to cold stress for 4 and 5 days at 4 and 0°C,
respectively, significantly affected their detection time by impedance
technology. This reflects an increase of the lag-phase time caused by
cold stress.
Microbial populations occurring in processed foods have incurred
sublethal structural and metabolic injury. Hence, resuscitation treatments are required for their reproducible recovery. In food microbiology, it is well established that it is necessary to rely on
overnight preenrichment. The intrinsic sensitivity of the PCR procedure
allows detection of very low numbers of bacteria. In addition, DNA is
always present in the bacterial cell, even if it is sublethally
injured. Hence, sensu stricto, bacterial growth (and thus enrichment)
is no longer necessary. Direct application of PCR for detection of VTEC
in foods, however, is restricted because of the physical enclosure of
the target cell in the food and because of inhibitory food components.
Most of the time, PCR detection is performed after a short or long
enrichment period. Primary culture dilutes out interfering substances
and the culture step results in increased sensitivity. Paton et al.
(17) used an overnight incubation at 37°C and Heuvelink et
al. (10) even used a secondary culture before applying the
PCR protocol to detect VTEC in, respectively, naturally contaminated
dry fermented sausage and ground-meat samples.
Attempts have been made to decrease the enrichment time. Gannon et al.
(7) were able to detect 10 and 1 VTEC organisms/g of ground
beef after 4 and 6 h of enrichment, respectively, although the PCR
signal became much more intense when a 24-h enrichment period was used.
These detection limits were obtained with extracted DNA. If boiled
cultures were used, the detection limit after 6 h of enrichment
increased to 100 VTEC organisms/g. In a limited experimental setup,
Whitham et al. (20) found minimum preenrichment times of
12, 8, and 4 to 8 h would detect 0.5, 50, and 5,000 CFU/g of
ground beef. In their study the template DNA was prepared by boiling.
Neither of these studies took into account the factor of environmental
stress affecting the lag phase of the bacterial cell.
Incubation times of 6 and 9 h for detecting 1 to 10 CFU/g and 1 to
10 CFU/25 g, respectively, were determined to be sufficient for
PCR detection of VTEC in ground beef when the analysis was performed
immediately after inoculation. When cells were exposed to cold stress
(4 or 20°C), a 24-h enrichment period is recommended. Given that
all ground-beef samples should be stored cold, it is assumed that all
samples may have some degree of cold damage unless analyzed
immediately. In view of the results presented here, a 24-h incubation
period for naturally contaminated ground-beef samples is recommended
for the reliable detection by PCR of low numbers of EHEC organisms.
The combination of immunomagnetic separation performed after 6 h
of enrichment together with PCR detection may reduce the total analysis
time without a loss of sensitivity. However, although immunomagnetic
separation is available for E. coli O157:H7, the technique
is difficult to develop for VTEC strains, which represent an undefined
group of E. coli serotypes. For stool samples it may be
possible to decrease the enrichment time from 24 to 6 h because in
this case the VTEC cells are not stressed and they are present in
higher numbers (>10 CFU/g), while at the same time smaller volumes are
taken for analysis (1 g instead of 25 g).
| |
ACKNOWLEDGMENT |
Mieke Uyttendaele is a research assistant of the Fund for
Scientific Research, Flanders, Belgium (F.W.O.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Service de
Recherche et Développement, Institut Pasteur de Lille, rue de
Professeur Calmette 1
B.P.245, 59019 Lille Cedex, France. Phone: 33 (03) 20 87 72 08. Fax: 33 (03) 20 87 72 06. E-mail:
Marc.Lange{at}pasteur-lille.fr.
| |
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Appl Environ Microbiol, May 1998, p. 1640-1643, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
