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.
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).
TABLE 1.
E. coli strains and their characteristics
| E. coli straina | VT typeb | Serotype | Presence of eae genec |
|---|---|---|---|
| E-ES-Co6/ATCC43888* | O157:H7 | + | |
| E-ES-Co7 | VT2 | O157:H− | + |
| E-ES-Co8 | VT1-VT2 | O157:H7 | + |
| E-ES-Co9 | VT1 | O157:H− | + |
| E-ES-Co11 | VT2 | O157:H7 | − |
| E-ES-Co12 | O157:H− | − | |
| E-ES-Co14 | VT1-VT2 | O157:H− | + |
| E-ES-Co15 | VT2 | O157:H− | + |
| E-ES-Co16 | VT2 | O157:H− | + |
| E-ES-Co17 | VT2 | O157:H7 | + |
| E-ES-Co18 | VT2 | O157:H7 | + |
| E-ES-Co19 | VT2 | O157:H7 | + |
| E-ES-Co26/H19* | VT1 | O26:H11 | + |
| E-ES-Co27/90.2380* | VT2 | O157 | + |
| E-ES-Co29/87.1215* | VT1-VT2 | O157 | + |
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.
TABLE 2.
Influence of cold stress at 4°C on detection time of 15 EHEC strains as assessed by impedance technology
| Duration of cold stress (days) | Mean detection timea (h) | 95% Confidence interval (h) |
|---|---|---|
| 0 | 6.992 | 6.898–7.087 |
| 1 | 6.580 | 6.422–6.738 |
| 4 | 7.600 | 7.386–7.814 |
| 7 | 8.990 | 8.705–9.257 |
| 14 | 10.535 | 9.872–11.197 |
| T14-T0b | 3.547 | 2.712–4.382 |
Mean detection time of all strains was determined by impedance technology (Bactometer).
Detection time at day 14 − detection time at day 0.
TABLE 3.
Influence of cold stress at 0°C on detection time of 7 EHEC strains as assessed by impedance technology
| Duration of cold stress (days) | Mean detection timea (h) | 95% Confidence interval (h) |
|---|---|---|
| 0 | 8.531 | 8.122–8.939 |
| 1 | 8.393 | 8.217–8.569 |
| 2 | 8.500 | 8.394–8.606 |
| 5 | 10.092 | 9.411–10.772 |
| 7 | 11.629 | 11.011–12.247 |
| T7-T0b | 3.086 | 2.170–4.002 |
Mean detection time of all strains as determined by impedance technology (Bactometer).
Detection time at day 7 − detection time at day 0.
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 × 104 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.
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
| Duration of cold stress (days) | Enrichment time (h) | EHEC detection with inoculum (CFU/25 g)a of:
|
|||||
|---|---|---|---|---|---|---|---|
| 2 | 2 × 10 | 2 × 102 | 2 × 103 | 2 × 104 | 2 × 105 | ||
| None | 0 | ND | ND | − | − | + | + |
| 3 | − | − | − | + | + | + | |
| 6 | − | − | + | + | + | + | |
| 9 | + | + | + | ND | ND | ND | |
| 24 | + | + | ND | ND | ND | ND | |
| 4 | 0 | ND | ND | − | − | − | − |
| 3 | − | − | − | −/+ | + | + | |
| 6 | − | − | − | + | + | + | |
| 9 | − | + | + | + | ND | ND | |
| 24 | + | + | + | ND | ND | ND | |
| 8 | 0 | ND | ND | ND | − | − | − |
| 3 | − | − | − | − | −/+ | + | |
| 6 | − | − | − | − | + | + | |
| 9 | − | − | − | −/+ | ND | ND | |
| 24 | + | + | + | + | ND | ND | |
ND, not determined; −, negative PCR detection for E. coli strains Co26 and Co7; +, positive PCR detection for E. coli strains Co26 and Co7; −/+, negative PCR detection for E. coli strain Co26, positive PCR detection for E. coli strain Co7.
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.
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
| Duration of cold stress (days) | Enrichment time (h) | ETEC detection with inoculum (CFU/25 g)a of:
|
|||||
|---|---|---|---|---|---|---|---|
| 2 | 2 × 10 | 2 × 102 | 2 × 103 | 2 × 104 | 2 × 105 | ||
| None | 0 | ND | ND | − | − | + | + |
| 3 | − | − | − | + | + | + | |
| 6 | − | − | + | + | + | + | |
| 9 | + | + | + | ND | ND | ND | |
| 24 | + | + | ND | ND | ND | ND | |
| 4 | 0 | ND | ND | − | − | − | − |
| 3 | − | − | − | − | + | + | |
| 6 | − | − | − | + | + | + | |
| 9 | − | − | −/+ | + | ND | ND | |
| 24 | + | + | + | ND | ND | ND | |
| 8 | 0 | ND | ND | ND | − | − | − |
| 3 | − | − | − | − | − | − | |
| 6 | − | − | − | − | + | + | |
| 9 | − | − | − | + | + | + | |
| 24 | −/+ | −/+ | + | + | ND | ND | |
| 14 | 0 | ND | ND | ND | − | − | − |
| 3 | − | − | − | − | − | − | |
| 6 | − | − | − | − | + | + | |
| 9 | − | − | − | + | + | + | |
| 24 | −/+ | −/+ | + | + | ND | ND | |
ND, not determined; −, negative PCR detection for E. coli strains Co26 and Co7; +, positive PCR detection for E. coli strains Co26 and Co7; +/−, negative PCR detection for E. coli strain Co26, positive PCR detection for E. coli strain Co7.
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.).
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