INTRODUCTION

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The advent of molecular methodology has revolutionized our ability to investigate and understand microbial ecology, offering new and unique opportunities to explore the ecology of food-borne pathogens, including Listeria monocytogenes, throughout the food chain and in the food processing environment. Highly discriminatory molecular typing methods, including multilocus enzyme electrophoresis, pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA, ribotyping, and phage typing, have been successfully applied to investigations of contamination patterns in foods and in the food processing environment and are increasingly used for surveillance of human disease cases and for tracking of outbreak sources (2-4, 7, 12, 26, 34, 36, 37, 39). While each method provides discriminatory differentiation of L. monocytogenes subtypes, highly automated and standardized methods provide a simplified approach to molecular subtyping and data analysis. The RiboPrinter Microbial Characterization System (Qualicon, Inc., Wilmington, Del.) is one example of such an approach. This system is based on ribotyping, a subtyping method based upon scoring restriction polymorphisms in the rRNA operons of prokaryotes (8, 9, 25).

L. monocytogenes, an invasive food-borne pathogen capable of causing serious disease in immunocompromised individuals and pregnant women, is common to many natural and man-made environments (16). As this organism is ubiquitous and capable of growth at refrigeration temperatures, the zero-tolerance ruling issued by the U.S. Food and Drug Administration for L. monocytogenes in ready-to-eat foods presents a serious challenge to the food industry. Cold-smoked fish products, which are typically consumed without cooking, are among the ready-to-eat foods of particular concern due to the lack of a heat inactivation step during processing. The prevalence of L. monocytogenes in cold-smoked fish and cooked seafood products has been reported to range from 6 to 36% (6) up to 78% (15, 27). Many studies have also demonstrated a high prevalence of L. monocytogenes in a variety of food processing environments (3, 4, 29, 33, 34, 37) and in ready-to-eat foods that had been subjected to microbial destruction steps sufficient to eliminate L. monocytogenes present in raw materials (for a recent survey, see http://www.fsis.usda.gov/oa/topics/lm_action.htm), strongly suggesting that the processing environment represents a significant source of this organism in finished products.

We hypothesize that molecular studies on the ecology of L. monocytogenes strains present in food processing environments will provide information crucial for the development of better control and prevention strategies for this important food-borne pathogen. The smoked fish industry provides an ideal model for the development and evaluation of molecular detection and tracking systems, as L. monocytogenes is frequently isolated from cold-smoked fish and hazard analysis critical control point (HACCP) programs are mandatory for seafood processors (1). The primary objective of this study was to investigate the prevalence, ecology, and transmission of L. monocytogenes in different seafood processing facilities using molecular detection and subtyping methods. The application of a commercially available PCR-based screening system for L. monocytogenes (BAX for Screening/Listeria monocytogenes; Qualicon, Inc.) allowed us to evaluate its performance using a culture-based detection method as a standard of comparison.

	MATERIALS AND METHODS

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Samples. A total of 531 samples were collected from three New York State smoked fish processing facilities over five visits (30 April, 10 August, 26 August, 13 October, and 26 October) to each facility in 1998. Processors C and D were located 2 miles apart. Processor B was located approximately 42 miles from the other two facilities. Samples collected from the processing environment (n = 206) were taken from floor drains, sink drains, cooler floors, and equipment surfaces. Sterile swabs were moistened with 1.0% peptone water containing 0.85% NaCl prior to sampling dry surfaces. Samples of raw fish (n = 102), belonging predominantly to the family Salmonidae, were taken from the collar and belly flap area of fresh, frozen, or thawed whole gutted fish or fish fillets. In-process samples (n = 127) were taken from fish during the wet or dry brine step and from brine solutions. Finished product samples (n = 96) included paper-wrapped and vacuum-packaged cold-smoked fish. Environmental swabs were transported in 2.0 ml of 1.0% peptone water containing 0.85% NaCl. All other samples were transported in sterile Stomacher 400 closure bags (Seward Ltd., London, United Kingdom). Samples were transported to the laboratory and held on ice. Sample analysis was initiated within 24 h of collection.

Bacteriological analysis. The method for isolation of L. monocytogenes used in this study was a modification of the protocol recommended by the Food and Drug Administration for the isolation of L. monocytogenes from food (24). Twenty-five-gram portions of raw, in-process, and smoked fish, prepared using sterilized instruments, were homogenized in 225 ml of Listeria Enrichment Broth (LEB) (Difco Laboratories, Detroit, Mich.) using a Stomacher 400 laboratory blender (Seward Ltd.). Brine solutions, in 25-ml aliquots, were inoculated into 225 ml of LEB. Swabs and transport media were transferred aseptically to 8 ml of LEB. Following 24 and 48 h of incubation at 30°C, 0.1 ml of each enrichment culture was plated on Oxford medium containing the Oxford Antimicrobic Supplement (Difco Laboratories) and incubated at 30°C for 48 h. Esculin hydrolysis-positive colonies with morphology typical for Listeria spp. were streaked for isolation on brain heart infusion (Difco Laboratories) agar and incubated at 37°C for 24 h. Colonies were isolated preferentially from the Oxford plate inoculated after 24 h of sample enrichment to facilitate recovery of L. monocytogenes as opposed to other Listeria species (30, 35). Pure culture isolates used for hlyA PCR or BAX system analysis (for confirmation that this system correctly identified isolates from culture-positive, BAX system-negative samples) were grown in brain heart infusion broth at 37°C with shaking for 12 to 15 h.

Identification of L. monocytogenes. Four Listeria-like isolates obtained from each sample on Oxford plates were tested with a PCR assay targeting the listeriolysin O gene, hlyA, to specifically identify L. monocytogenes isolates. Primers -1 (CCT AAG ACG CCA ATC GAA AAG AAA) and -1 (TAG TTC TAC ATC ACC TGA GAC AGA) define an 858-bp fragment of the hlyA gene (10). Assays were performed using GeneAmp PCR core reagents (Perkin-Elmer Applied Biosystems, Foster City, Calif.). Each 25-µl reaction mixture contained 1× PCR buffer II, 1.5 mM MgCl₂, 125 µM each dATP, dCTP, dGTP, and dTTP, 0.5 µM each primer, 1 U of Amplitaq DNA polymerase, and 2 µl of a 1:10 dilution of a crude cell lysate prepared as described by Furrer et al. (18). Cycling was performed in the GeneAmp PCR system 2400 (Perkin-Elmer Applied Biosystems). Amplification cycling began with 2 min at 95°C, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. A final extension step of 72°C for 10 min was followed by a hold at 4°C. The PCR products were electrophoresed on 1.5% agarose gels at 120 V, stained with ethidium bromide, and photodocumented (41). A positive result was indicated by the presence of an approximately 858-bp band.

BAX for Screening/Listeria monocytogenes. Following 48 h of sample enrichment, 250 µl of the enrichment culture was removed and centrifuged at maximum speed in a bench-top microcentrifuge for 2 min. The supernatant was discarded and the pellet was resuspended in 250 µl of sterile deionized water. This prelysis wash step was incorporated to minimize the possibility of PCR inhibition by components of the enrichment culture (personal communication from Technical Assistance, Qualicon, Inc.). The lysate was prepared as outlined in the manufacturer's protocol. Temperature cycling for the DNA amplification step was performed in either the GeneAmp PCR system 2400 or PCR system 9600 (Perkin-Elmer Applied Biosystems). PCR products were loaded onto 1.5% agarose gels as outlined in the BAX for Screening protocol, electrophoresed at 120 V, stained with ethidium bromide, and photodocumented (41). A positive result was indicated by the presence of an approximately 400-bp band in the sample lane and an 800-bp band in the corresponding control lane. Aliquots of the resuspended enrichment culture pellets, stored at 80°C, were used to retest samples for which PCR-based screening and culture methods yielded discrepant results.

Ribotyping. One L. monocytogenes isolate from each culture-positive sample was subtyped. Automated ribotyping with normalized data was performed using the RiboPrinter (Qualicon, Inc.), as previously described (9, 25), in the Laboratory for Molecular Typing at Cornell University. This automated typing method involves EcoRI digestion of L. monocytogenes chromosomal DNA followed by Southern hybridization with an Escherichia coli rrnB rRNA operon probe. Images are acquired with a charge-coupled device and analyzed using custom software that normalizes fragment pattern data for band intensity and relative band size compared to a molecular size marker.

Statistical analyses. Culture- and PCR-based detection system results were compared by defining the culture-based system results as the standard of comparison ("gold standard") and then calculating the sensitivity (true positive rate), specificity (true negative rate), and accuracy (method agreement) of the PCR-based system using standard formulas (11). BAX system-negative, culture-positive results were interpreted as BAX system false-negative results. As characterization of as few as five Listeria-like isolates in a culture-based screen can result in an overall sample-negative result (24), BAX system-positive results were interpreted as false positive if hlyA screening of four Listeria-like isolates from the corresponding sample yielded negative results. If the presence of L. monocytogenes was subsequently confirmed by hlyA PCR screening of up to 16 additional isolates from a sample, the BAX results were interpreted as true positive for calculation of the revised specificity estimate.

	RESULTS

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Detection of L. monocytogenes in cold-smoked fish and in the smoked fish processing environment. A total of 531 environmental, raw material, in-process, and smoked fish samples were screened for the presence of L. monocytogenes using both the culture-based method and the BAX for Screening/Listeria monocytogenes system. The BAX PCR system detected L. monocytogenes in 17.8% of the 531 samples, compared to 16.0% positive-culture results. Sensitivity, specificity, and overall accuracy estimates for the PCR system are shown in Table 1. Thirteen samples that gave initial BAX system false-negative results were retested using the resuspended frozen enrichment culture pellets. Six samples gave positive results upon retesting, improving the sensitivity of the PCR system (Table 1). Twenty-nine samples gave BAX system false-positive results compared to the initial culture-based screening results. Subsequent hlyA PCR screening of up to 16 additional isolates per sample confirmed the presence of L. monocytogenes in 12 of these samples, thus improving the specificity of the PCR system (Table 1). While there was no apparent correlation between sample type and BAX system false-positive results, four of the seven BAX system false-negative samples were taken from finished products. The corrected BAX system sensitivity and specificity values reported in this study should be regarded as rough estimates, as only the discrepant results were further analyzed. Estimation of true corrected values would have required retesting of all samples.

Tracking L. monocytogenes in cold-smoked fish and in the smoked fish processing environment. L. monocytogenes strain characterization by ribotyping identified 15 ribotypes among 85 isolates analyzed. The distribution of ribotypes among processing facilities, grouped by sampling visit, is shown in Fig. 1 to 3.

View larger version (70K): [in this window] [in a new window]   FIG. 1.   Ribotypes of L. monocytogenes isolates from processor B environmental, raw material, in-process, and finished product samples grouped by sampling visit. (E), environmental sample; (R), raw materials; (IP), in-process sample; (F), finished product. *, ribotype isolated most frequently from processor B samples.

View larger version (62K): [in this window] [in a new window]   FIG. 2.   Ribotypes of L. monocytogenes isolates from processor C environmental, raw material, in-process, and finished product samples grouped by sampling visit. (E), environmental sample; (R), raw materials, (IP), in-process sample, (F), finished product. * and **, ribotypes isolated most frequently from processor C samples.

View larger version (97K): [in this window] [in a new window]   FIG. 3.   Ribotypes of L. monocytogenes isolates from processor D environmental, raw material, in-process, and finished product samples grouped by sampling visit. (E), environmental sample; (R), raw materials; (IP), in-process sample; (F), finished product. *, ribotype isolated most frequently from processor D samples.

	DISCUSSION

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L. monocytogenes is a food-borne bacterial pathogen that is ubiquitous in nature and shows the ability to persist in the food processing environment for prolonged time periods. Control of this organism is thus extremely challenging for the food industry. Development of a better understanding of the ecology of L. monocytogenes in food processing plants in combination with rapid, standardized detection and typing systems will provide critical tools and knowledge for the development and verification of improved control strategies.

In-plant L. monocytogenes contamination patterns. L. monocytogenes was detected in a variety of environmental, raw material, in-process, and cold-smoked fish samples (Table 2) collected from three smoked fish processing plants. We isolated L. monocytogenes from 11.5% of finished product samples, consistent with the prevalence range reported by previous surveys (6, 21, 27). As L. monocytogenes is a common contaminant of the food processing environment (6, 16), we were not surprised that this organism was isolated from over 20% of our environmental samples.

Potential sources of finished product contamination. Molecular subtyping data also provided useful information regarding potential sources of specific L. monocytogenes strains isolated from finished product samples. Our subtyping data, in conjunction with our prevalence results, indicate that both raw materials and the processing environment serve as potential sources of finished product contamination. For example, DUP-1039A was isolated from processor B raw and cold-smoked Chilean salmon but not from the processing environment (Fig. 1, visit 1), indicating that raw materials likely served as the source of finished product contamination at the time of sampling. While results from a study by Eklund et al. also implicated raw fish as an important source of L. monocytogenes in the smoked fish processing environment and in finished products (15), other studies employing molecular typing methods concluded that raw materials may not serve as a significant source of finished product contamination (4, 37). Additional comprehensive studies monitoring contamination of raw fish and finished products using molecular subtyping methods are needed to clarify the importance of raw materials as a source of L. monocytogenes in finished products.

Evaluation of a commercial PCR-based system for detection of L. monocytogenes. Conventional culture methods for the isolation and identification of L. monocytogenes from food and environmental samples require a minimum of 4 to 5 days to complete (6, 13, 16). These methods may lead to false-negative results if mixed populations of Listeria species are present in a given sample, as most selective plating media do not allow visual differentiation of L. monocytogenes from other Listeria spp. (6). Rapid sample screening methods, including immunochemical, nucleic acid probe-based, and PCR-based assays, may overcome some of these limitations (5, 13, 23, 40). An increasing number of commercial PCR-based assays are available for the specific detection of L. monocytogenes, including the BAX for Screening system and the Probelia PCR system (Sanofi Diagnostics Pasteur). These assays have the potential to provide rapid and reliable molecular methods for monitoring of L. monocytogenes in the food industry.

Conclusions. Our results indicate that molecular detection and subtyping methods facilitate a better understanding of the ecology of food-borne pathogens in the food processing environment. This work further demonstrates the utility of molecular subtyping, specifically ribotyping, for tracking the sources and spread of food-borne pathogens. Ribotyping was applied in our study because this subtyping method is highly discriminatory, automated, and reproducible from one laboratory to another, thus facilitating rapid identification and data sharing among research groups. Although a single typing method can often elucidate contamination patterns, the use of multiple typing methods (20), multiple enzymes for ribotyping (19), or other typing methods (e.g., PFGE) (20) may increase discriminatory power and provide additional information on contamination patterns. It is also important to remember that molecular typing methods rely on culture-based isolation of L. monocytogenes. As it has been shown that different enrichment procedures may favor the growth of different subtypes and that multiple subtypes may be present in a given sample (38), the employment of more than one enrichment method and subtyping of more than one isolate per sample may facilitate further elucidation of contamination patterns.