Evaluation of Culture- and PCR-Based Detection Methods for Escherichia coli O157:H7 in Inoculated Ground Beef†

Currently, several beef processors employ test-and-hold systems for increased quality control of ground beef. In such programs, each lot of product must be tested and found negative for Escherichia coli O157:H7 prior to release of the product into commerce. Optimization of three testing attributes (detection time, specificity, and sensitivity) is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low false-positive result rate so product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released and potentially cause disease. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. Ground beef samples were individually spiked with five genetically distinct strains of E. coli O157: H7 at concentrations of 17 and 1.7 CFU/65 g and then subjected to the various testing methodologies. There was no difference (P . 0.05) in the abilities of the PCR-based methods to detect E. coli O157:H7 inoculated in ground beef at 1.7 CFU/65 g. The culture-based systems detected more positive samples than did the PCR-based systems, but the detection times (21 to 48 h) were at least 9 h longer than those for the PCR-based methods (7.5 to 12 h). Ground beef samples were also spiked with potentially cross-reactive strains. The PCR-based systems that employed an immunomagnetic separation step prior to detection produced fewer false-positive results. Traditionally, detection of foodborne pathogens has involved sample collection, enrichment, and isolation of the target organism on selective and/or indicator media. Such culture-based approaches lack sensitivity and specificity and are time-consuming, taking from 48 to 96 h from sample collection to final results. The implementation of immunomagnetic separation (IMS) has greatly increased the sensitivity and specificity while decreasing the time of detection (4, 24, 33). Recently, PCR techniques, both conventional and real-time platforms, have been adopted for routine detection of foodborne pathogens (9, 20–22). The rapid PCR run times routinely reduce detection times by 24 h. Escherichia coli O157:H7 is a foodborne pathogen that has been associated with meat-, produce-, and water-related disease outbreaks (15, 17, 28). This pathogen, which is known for its low infective dose and its ability to cause severe disease and death, emerged as a foodborne threat in the 1980s and early 1990s (27, 31). Because early outbreaks were associated with ground beef, the U.S. Department of Agriculture Food Safety and Inspection Service produced several regulations aimed at eliminating this pathogen from red meat (32). At the same time, the public and private * Author for correspondence. Tel: 402-762-4227; Fax: 402-762-4149; E-mail: [email protected]. † Names are necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and the use of the name by the U.S. Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable. sectors of the beef processing industry were working to design, validate, and implement several antimicrobial interventions for use in combating E. coli O157:H7 contamination (7, 16, 26.) Unfortunately, no single intervention or combination of interventions have yet been identified that will eliminate E. coli O157:H7 on beef, and sporadic beefassociated outbreaks have continued to occur. The meat industry has recently employed test-and-hold systems to prevent release of product containing E. coli O157:H7 (6). In such test-and-hold programs, each lot of product is tested for E. coli O157:H7, and if the results are negative the product can be released into commerce. Optimization of testing methods with respect to detection time, specificity, and sensitivity is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low rate of false-positive results so wholesome product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released, potentially causing disease and defeating the purpose of the program. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for (i) their sample throughput capacity and time requirements for sample processing and detection, (ii) their specificity of discriminating between E. coli O157:H7 and other members of the background flora, and (iii) their sensitivity in detecting E. coli O157:H7 inoculated in ground beef at low concentrations. J. Food Prot., Vol. 68, No. 8 E. COLI O157:H7 DETECTION METHOD EVALUATION 1567 FIGURE 1. Flowchart outlines the methods by which samples were inoculated, incubated, and processed for detection of E. coli O157: H7. Bold headings indicate the official methods tested. Pathways not ending in a bold heading were used in the data correction process to identify samples that had been inoculated. MATERIALS AND METHODS Escherichia coli O157:H7 detection in ground beef. Five methods for the detection of E. coli O157:H7 in ground beef were evaluated for their sensitivity and specificity (Fig. 1). Batch samples (600 g of 80% lean ground beef) were inoculated with individual strains of E. coli O157:H7 and then divided into portions for use with each method to minimize variation in samples or processing conditions. For the PCR-based methods, Roman L. Hruska U.S. Meat Animal Research Center (MARC) personnel were trained to perform the various tests by representatives from the specific company that designed each test kit. All testing was carried out at MARC using ground beef purchased from local retailers. Sensitivity for E. coli O157:H7. Ground beef (80% lean) was spiked with approximately 17 or 1.7 CFU/65 g (equivalent to 100 or 10 CFU/375 g) for both PCR-based and culture-based procedures. Strains. For the sensitivity trials, five strains of E. coli O157: H7 (55AC1, 114AC1, 131AC1, 237AC1, and 299AB3) from the MARC Meats Research Unit (MRU) culture collection were used for individual inoculation of samples. These strains are genetically different from one another and represent major subtypes within three relatedness clusters identified by pulsed-field gel electrophoresis (PFGE) analysis of E. coli O157:H7 isolates recovered from beef processing plants (1, 3). Frozen stocks of the strains were prepared by inoculating tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, Md.) and growing the cultures overnight at 378C. The cultures were serially diluted in buffered peptone water (BPW; Difco, Becton Dickinson), sterile 50% (vol/vol) glycerol (Sigma, St. Louis, Mo.) was added to a final concentration of 15% (vol/vol), and cultures were then frozen at 2708C until use. Inoculation. For a final concentration of 17 or 1.7 E. coli O157:H7 CFU/65 g of ground beef, an appropriate volume was removed from a freshly thawed glycerol stock containing approximately 103 CFU/ml of the particular E. coli O157:H7 strain and added to 45 ml of BPW. The stock cultures were then plated (50 ml) in duplicate onto tryptic soy agar (Difco, Becton Dickinson) plates for enumeration of the inoculum. Samples (600 g) of refrigerated ground beef obtained from various local retailers were placed into 3,500-ml nonfilter stomacher bags (BA 6042, Seward, Worthington, UK). Inoculated BPW (45 ml) was then added to the bags, which were thoroughly hand massaged and then stomached at 190 rpm for 30 s. After the 600 g of ground beef was batch inoculated, 65or 375-g samples were removed and processed according to the various method protocols. Detection methods. At the time of this evaluation, all of the PCR-based methods tested here were premarket tests in their final stages of validation before entering the retail market. LightCycler E. coli (eae) Detection Kit. Probes, primers, and method for this kit were designed by Marshfield Clinic Laboratories, Food Safety Services (Marshfield, Wis.; marketed by Roche Applied Science, Indianapolis, Ind.). This method was J. Food Prot., Vol. 68, No. 8 1568 ARTHUR ET AL. evaluated using 375 g of ground beef (as specified by the manufacturer). We also ran the tests with 65-g samples to allow comparison with other procedures that specified 65-g samples. For the 65-g test, 70 g of spiked ground beef (65 g of ground beef and 5 ml of BPW from inoculum) was removed from the batch inoculation bag and placed in a 1,650-ml filter stomacher bag (B01318, Nasco Whirl-Pak, Fort Atkinson, Wis.), and 275 ml of BPW (preheated to 378C) was added to the bag. The sample was stomached at 230 rpm for 30 s and incubated at 378C with shaking at 125 rpm for 4 h. After 4 h, ca. 250 ml from each sample was poured off through the filter into a new 1,650-ml filter stomacher bag. The new sample bags were placed in Matrix/Pathatrix warming pots (Matrix Microsciences, Golden, Colo.), which had been preheated to 378C. The Pathatrix apparatus was inserted into the bag on the opposite side of the filter from that where the sample was poured. Fifty microliters of Matrix antiO157 immunomagnetic beads was added to the connector tubing, and the samples were recirculated for 1 h at 378C. The beads were then collected, washed, and resuspended in 100 ml of BPW. DNA was extracted from the 100-ml bead-cell suspension using the MagNA Pure LC Instrument (Roche Applied Science). Three microliters of the extracted DNA was used in the PCR performed on the LightCycler Instrument (Roche Applied Science). Following bead collection, the sample bags were incubated overnight at 378C. The following morning, all sample bags were processed by IMS and plating according to standard MRU IMS procedures. For the 375-g test, 403 g of spiked ground beef (375 g of ground beef and 28 ml of BPW from inoculum) was removed from the bag and placed in a 3,500-ml filter stomacher bag (123 010, Bagpage 3500, Interscience, Weymouth, Mass.), and 1 liter of BPW (preheated to 378C) was added to the bag. The sample was stomached at 190 rpm for 30 s and incubated at 378C with shaking at 125 rpm for 4 h. After 4 h, two ca. 250-ml portions from each sample were poured off through the filter into two 1,650-ml filter stomacher bags. The new sample bags were placed in Matrix/Pathatrix warming pots (preheated to 378C). The Pathatrix apparatus was inserted into each bag on the opposite side of the filter from that into which the sample was poured. Fifty microliters of Matrix anti-O157 immunomagnetic beads was added, and the samples were recirculated for 1 h at 378C. The beads were washed with BPW and then processed according to the LightCycler E. coli (eae) Detection Kit procedures. When this evaluation was performed the software for automated determination of positive or negative results was not available for this kit; therefore, the results were interpreted by the operator based on the assay protocol. Samples with positive amplification signals and melting curves with peaks of approximately 638C were considered positive. Assurance GDS for E. coli O157:H7, 6.5and 8-h methods. For the Assurance GDS test (BioControl Systems, Inc., Seattle, Wash.), 70 g of spiked ground beef (65 g ground beef and 5 ml BPW from inoculum) was removed from the batch inoculation bag and placed in a 1,650-ml filter stomacher bag (B01318, Nasco Whirl-Pak). EHEC8 medium (27.6 g; BioControl Systems) and 290 ml of sterile water (preheated to 428C) were added to the stomacher bag, the sample was stomached at 230 rpm for 30 s, and the remaining 290 ml of sterile water (preheated to 428C) was added for a final volume of 580 ml of medium. The bag was hand massaged and incubated at 428C for 6.5 h, and then 1 ml from each sample was removed to a deep-well block (each well containing 20 ml of sample preparation reagent). The sample bags were returned to incubation at 428C. The deep-well block was sealed and processed according to the Assurance GDS for E. coli O157:H7 protocol as written. Sample preparation reagent (20 ml) was mixed with each 1-ml sample in the deep-well block, and the block was vortexed at 900 rpm for 5 min. Samples were then processed by IMS using the PickPen eight-channel magnetic particle separation device. Recovered immunobeads were resuspended in 25 ml of resuspension buffer (BioControl Systems). Twenty microliters of the resuspension mixture was added to 5 ml of the polymerase buffer solution (BioControl Systems) and loaded onto a Rotor-Gene 3000 real-time PCR system (Corbett Research, Sydney, Australia). After 8 h of incubation, 1-ml aliquots were removed from all samples that did not give a positive result in the Assurance GDS assay after 6.5 h. These aliquots were processed for E. coli O157:H7 detection in the same manner as the 6.5-h sample aliquots. Following removal of the sample aliquots, all samples were returned to incubation at 428C. The following morning, all samples that gave negative results for the Assurance GDS assay were processed by IMS and plating according to standard MRU IMS procedures. BAX System PCR assay for screening E. coli O157:H7, MP method. For the BAX E. coli O157:H7 MP method (DuPont Qualicon, Wilmington, Del.), 70 g of spiked ground beef (65 g ground beef and 5 ml BPW from inoculum) were removed from the batch inoculation bag and placed in a 1,650-ml filter stomacher bag (B01318, Nasco Whirl-Pak), and 580 ml of BAX medium (preheated to 428C) was added to the bag. The sample was stomached at 230 rpm for 30 s and incubated at 428C for 8 h. After 8 h, 5 ml was removed from each sample enrichment and lysed according to the BAX protocol. After lysis, 50 ml of the cell lysate was added to the lyophilized PCR reagent pellet and loaded into the BAX System cycler/detector instrument. The results were obtained with the BAX system T1.70 software package. MRU standard method. After the aliquots for the BAX E. coli O157:H7 MP test were removed, the enrichments were returned to incubation at 428C for an additional 4 h (total of 12 h at 428C) and then held at 48C overnight. The following morning, all sample bags were processed by IMS and plating according to standard MRU IMS procedures. IMS was performed to recover E. coli O157:H7 based on a previously described method (2). One milliliter of enrichment was added to 20 ml of anti-O157 immunomagnetic beads (Dynal, Lake Success, N.Y.) in 1.5-ml microtubes and rotated for 30 min. The beads were held in place by a magnet while the supernatant was removed. The beads were then washed three consecutive times with 1 ml of phosphate-buffered saline plus 0.1% Tween 20 (PBS-Tween; Sigma) and resuspended in 100 ml of PBS-Tween. After IMS, E. coli O157:H7 beads were spread directly onto (i) ntRainbow medium, which is Rainbow agar (Biolog, Inc., Hayward, Calif.) supplemented with novobiocin (20 mg/liter; Sigma) and potassium tellurite (0.8 mg/liter; Sigma), and (ii) ctSMAC, which is sorbitol MacConkey agar (Becton Dickinson) supplemented with cefixime (0.05 mg/liter; Dynal) and potassium tellurite (2.5 mg/liter; Dynal). Pathatrix. The Pathatrix culture method (Matrix Microsciences) was tested in conjunction with the LightCycler E. coli (eae) PCR-based method and therefore the protocol used differed from that provided by the manufacturer. Four hundred three grams of spiked ground beef (375 g of ground beef and 28 g of BPW from inoculum) was removed from the batch inoculation bag and placed in a 3,500-ml filter stomacher bag (123 010; Bagpage 3500, Interscience), and 1 liter of BPW (preheated to 378C) was added to the bag. The sample was stomached at 190 rpm for 30 s and incubated at 378C with shaking at 125 rpm for 4 h. After J. Food Prot., Vol. 68, No. 8 E. COLI O157:H7 DETECTION METHOD EVALUATION 1569 TABLE 1. Sensitivity of various tests for E. coli O157:H7 inoculated at 17 CFU/65 g (100 CFU/375 g) Detection testa Sample size (g) Medium volume (ml) No. of samples No. (%) of positive samplesb Assurance GDS 6.5 h LightCycler E. coli (eae) Pathatrix MRU 65 375 375 65 585 1,000 1,000 585 57 57 57 57 57 (100) 57 (100) 57 (100) 57 (100) a The BAX E. coli O157:H7 MP test not included in these experiments because it was not available at the time of testing. b Percentage was determined by dividing the number of positive samples from the indicated test by the number of samples tested. 4 h, ca. 250 ml was poured off through the filter into a 1,650-ml filter stomacher bag, and the new sample bags were placed in Matrix/Pathatrix warming pots (preheated to 378C). The Pathatrix apparatus was inserted into each bag on the opposite side of the filter from where the sample was poured, and 50 ml of Matrix anti-O157 immunomagnetic beads was added to the connector tubing. The samples were recirculated for 1 h at 378C, and then the beads were washed and plated directly to ctSMAC and ntRainbow agar plates. Specificity for E. coli O157:H7. A subset of the MRU strain collection was screened in pure culture using only the detection portion (no IMS) of the PCR-based detection systems to identify those strains that would be recognized as E. coli O157:H7. For this experiment, pure cultures were grown overnight in TSB and then diluted in BPW to 104 CFU/ml. A portion of the diluted samples was used for detection. Once the cross-reacting strains were identified, frozen stocks of those strains were made as described for E. coli O157:H7. Detection experiments using the complete processes and spiked ground beef samples were conducted to determine whether the growth media or the immunomagnetic separation phase would prevent the false-positive detection of these strains. The experiments were performed using methods similar to those described for O157:H7. For these experiments, the inoculum was added at 17 CFU/65 g (100 CFU/375 g). Statistics. The various test results were compared in two-bytwo contingency tables using the chi-square test from SAS statistical software version 6.12 (SAS Institute, Inc., Cary, N.C.).