molecular studies on the ecology of listeria monocytogenes ... · made environments (16). as this...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.0010 DOI: 10.1128/AEM.67.1.198–205.2001

Jan. 2001, p. 198–205 Vol. 67, No. 1

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Molecular Studies on the Ecology of Listeria monocytogenes inthe Smoked Fish Processing Industry

DAWN M. NORTON,1 MEGHAN A. MCCAMEY,1 KENNETH L. GALL,2 JANET M. SCARLETT,3

KATHRYN J. BOOR,1 AND MARTIN WIEDMANN4*

Food Safety Laboratory, Department of Food Science,1 Department of Population Medicine and Diagnostic Sciences,3 andDepartment of Food Science,4 Cornell University, Ithaca, New York 14853, and Cornell University Cooperative

Extension, New York Sea Grant, State University of New York, Stony Brook, New York 11794-50022

Received 18 April 2000/Accepted 14 September 2000

We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprintingmethods, to study the ecology of Listeria monocytogenes in food processing environments. A total of 531 samples,including raw fish, fish during the cold-smoking process, finished product, and environmental samples, werecollected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) ofthe samples tested positive for L. monocytogenes using a commercial PCR system (BAX for Screening/Listeriamonocytogenes), including 57 (27.7%) environmental samples (n 5 206), 8 (7.8%) raw material samples (n 5102), 23 (18.1%) samples from fish in various stages of processing(n 5 127), and 7 (7.3%) finished productsamples (n 5 96). L. monocytogenes was isolated from 85 samples (16.0%) using culture methods. Used inconjunction with a 48-h enrichment in Listeria Enrichment Broth, the PCR system had a sensitivity of 91.8%and a specificity of 96.2%. To track the origin and spread of L. monocytogenes, isolates were fingerprinted byautomated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping dataestablished possible contamination patterns, implicating raw materials and the processing environment as poten-tial sources of finished product contamination. Analysis of the distribution of ribotypes revealed that eachprocessing facility had a unique contamination pattern and that specific ribotypes persisted in the environmentsof two facilities over time (P < 0.0006). We conclude that application of molecular approaches can providecritical information on the ecology of different L. monocytogenes strains in food processing environments. Thisinformation can be used to develop practical recommendations for improved control of this important food-borne pathogen in the food industry.

The advent of molecular methodology has revolutionizedour ability to investigate and understand microbial ecology,offering new and unique opportunities to explore the ecologyof food-borne pathogens, including Listeria monocytogenes,throughout the food chain and in the food processing environ-ment. Highly discriminatory molecular typing methods, includ-ing multilocus enzyme electrophoresis, pulsed-field gel electro-phoresis (PFGE), random amplification of polymorphic DNA,ribotyping, and phage typing, have been successfully applied toinvestigations of contamination patterns in foods and in thefood processing environment and are increasingly used forsurveillance of human disease cases and for tracking of out-break sources (2–4, 7, 12, 26, 34, 36, 37, 39). While eachmethod provides discriminatory differentiation of L. monocy-togenes subtypes, highly automated and standardized methodsprovide a simplified approach to molecular subtyping and dataanalysis. The RiboPrinter Microbial Characterization System(Qualicon, Inc., Wilmington, Del.) is one example of such anapproach. This system is based on ribotyping, a subtypingmethod based upon scoring restriction polymorphisms inthe rRNA operons of prokaryotes (8, 9, 25).

L. monocytogenes, an invasive food-borne pathogen capableof causing serious disease in immunocompromised individualsand pregnant women, is common to many natural and man-

made environments (16). As this organism is ubiquitous andcapable of growth at refrigeration temperatures, the zero-tol-erance ruling issued by the U.S. Food and Drug Administra-tion for L. monocytogenes in ready-to-eat foods presents aserious challenge to the food industry. Cold-smoked fish prod-ucts, which are typically consumed without cooking, are amongthe ready-to-eat foods of particular concern due to the lack ofa heat inactivation step during processing. The prevalence ofL. monocytogenes in cold-smoked fish and cooked seafoodproducts has been reported to range from 6 to 36% (6) up to78% (15, 27). Many studies have also demonstrated a highprevalence of L. monocytogenes in a variety of food processingenvironments (3, 4, 29, 33, 34, 37) and in ready-to-eat foodsthat had been subjected to microbial destruction steps suffi-cient 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 environ-ment represents a significant source of this organism in fin-ished products.

We hypothesize that molecular studies on the ecology ofL. monocytogenes strains present in food processing environ-ments will provide information crucial for the development ofbetter control and prevention strategies for this importantfood-borne pathogen. The smoked fish industry provides anideal model for the development and evaluation of moleculardetection and tracking systems, as L. monocytogenes is frequent-ly isolated from cold-smoked fish and hazard analysis criticalcontrol point (HACCP) programs are mandatory for seafood

* Corresponding author. Mailing address: Department of Food Sci-ence, 412 Stocking Hall, Cornell University, Ithaca, NY 14853. Phone:(607) 254-2838. Fax: (607) 254-4868. E-mail: [email protected].

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processors (1). The primary objective of this study was toinvestigate the prevalence, ecology, and transmission of L.monocytogenes in different seafood processing facilities usingmolecular detection and subtyping methods. The applica-tion of a commercially available PCR-based screening systemfor L. monocytogenes (BAX for Screening/Listeria monocyto-genes; Qualicon, Inc.) allowed us to evaluate its performanceusing a culture-based detection method as a standard of com-parison.

MATERIALS AND METHODS

Samples. A total of 531 samples were collected from three New York Statesmoked 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 werelocated 2 miles apart. Processor B was located approximately 42 miles from theother two facilities. Samples collected from the processing environment (n 5206) were taken from floor drains, sink drains, cooler floors, and equipmentsurfaces. Sterile swabs were moistened with 1.0% peptone water containing0.85% NaCl prior to sampling dry surfaces. Samples of raw fish (n 5 102),belonging predominantly to the family Salmonidae, were taken from the collarand belly flap area of fresh, frozen, or thawed whole gutted fish or fish fillets.In-process samples (n 5 127) were taken from fish during the wet or dry brinestep and from brine solutions. Finished product samples (n 5 96) includedpaper-wrapped and vacuum-packaged cold-smoked fish. Environmental swabswere transported in 2.0 ml of 1.0% peptone water containing 0.85% NaCl. Allother samples were transported in sterile Stomacher 400 closure bags (SewardLtd., London, United Kingdom). Samples were transported to the laboratory andheld on ice. Sample analysis was initiated within 24 h of collection.

Bacteriological analysis. The method for isolation of L. monocytogenes used inthis study was a modification of the protocol recommended by the Food andDrug Administration for the isolation of L. monocytogenes from food (24).Twenty-five-gram portions of raw, in-process, and smoked fish, prepared usingsterilized instruments, were homogenized in 225 ml of Listeria Enrichment Broth(LEB) (Difco Laboratories, Detroit, Mich.) using a Stomacher 400 laboratoryblender (Seward Ltd.). Brine solutions, in 25-ml aliquots, were inoculated into225 ml of LEB. Swabs and transport media were transferred aseptically to 8 mlof LEB. Following 24 and 48 h of incubation at 30°C, 0.1 ml of each enrichmentculture was plated on Oxford medium containing the Oxford Antimicrobic Sup-plement (Difco Laboratories) and incubated at 30°C for 48 h. Esculin hydrolysis-positive colonies with morphology typical for Listeria spp. were streaked forisolation on brain heart infusion (Difco Laboratories) agar and incubated at 37°Cfor 24 h. Colonies were isolated preferentially from the Oxford plate inoculatedafter 24 h of sample enrichment to facilitate recovery of L. monocytogenes asopposed to other Listeria species (30, 35). Pure culture isolates used for hlyAPCR or BAX system analysis (for confirmation that this system correctly iden-tified isolates from culture-positive, BAX system-negative samples) were grownin brain heart infusion broth at 37°C with shaking for 12 to 15 h.

Identification of L. monocytogenes. Four Listeria-like isolates obtained fromeach sample on Oxford plates were tested with a PCR assay targeting the listerio-lysin O gene, hlyA, to specifically identify L. monocytogenes isolates. Primers a-1(CCT AAG ACG CCA ATC GAA AAG AAA) and b-1 (TAG TTC TAC ATCACC TGA GAC AGA) define an 858-bp fragment of the hlyA gene (10). Assayswere performed using GeneAmp PCR core reagents (Perkin-Elmer AppliedBiosystems, Foster City, Calif.). Each 25-ml reaction mixture contained 13 PCRbuffer II, 1.5 mM MgCl2, 125 mM each dATP, dCTP, dGTP, and dTTP, 0.5 mMeach primer, 1 U of Amplitaq DNA polymerase, and 2 ml of a 1:10 dilution of acrude cell lysate prepared as described by Furrer et al. (18). Cycling was per-formed 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 for10 s, 60°C for 30 s, and 72°C for 30 s. A final extension step of 72°C for 10 minwas 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-bpband.

BAX for Screening/Listeria monocytogenes. Following 48 h of sample enrich-ment, 250 ml of the enrichment culture was removed and centrifuged at maxi-mum speed in a bench-top microcentrifuge for 2 min. The supernatant wasdiscarded and the pellet was resuspended in 250 ml of sterile deionized water.This prelysis wash step was incorporated to minimize the possibility of PCRinhibition by components of the enrichment culture (personal communication

from Technical Assistance, Qualicon, Inc.). The lysate was prepared as outlinedin the manufacturer’s protocol. Temperature cycling for the DNA amplificationstep was performed in either the GeneAmp PCR system 2400 or PCR system9600 (Perkin-Elmer Applied Biosystems). PCR products were loaded onto 1.5%agarose gels as outlined in the BAX for Screening protocol, electrophoresed at120 V, stained with ethidium bromide, and photodocumented (41). A positiveresult was indicated by the presence of an approximately 400-bp band in thesample lane and an 800-bp band in the corresponding control lane. Aliquots ofthe resuspended enrichment culture pellets, stored at 280°C, were used to retestsamples for which PCR-based screening and culture methods yielded discrepantresults.

Ribotyping. One L. monocytogenes isolate from each culture-positive samplewas subtyped. Automated ribotyping with normalized data was performed usingthe RiboPrinter (Qualicon, Inc.), as previously described (9, 25), in the Labora-tory for Molecular Typing at Cornell University. This automated typing methodinvolves EcoRI digestion of L. monocytogenes chromosomal DNA followed bySouthern hybridization with an Escherichia coli rrnB rRNA operon probe. Im-ages are acquired with a charge-coupled device and analyzed using customsoftware that normalizes fragment pattern data for band intensity and relativeband size compared to a molecular size marker.

Statistical analyses. Culture- and PCR-based detection system results werecompared by defining the culture-based system results as the standard of com-parison (“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-posi-tive results were interpreted as BAX system false-negative results. As character-ization of as few as five Listeria-like isolates in a culture-based screen can resultin an overall sample-negative result (24), BAX system-positive results wereinterpreted as false positive if hlyA screening of four Listeria-like isolates fromthe corresponding sample yielded negative results. If the presence of L. mono-cytogenes was subsequently confirmed by hlyA PCR screening of up to 16 addi-tional isolates from a sample, the BAX results were interpreted as true positivefor calculation of the revised specificity estimate.

The rates of recovery of specific ribotypes of L. monocytogenes among thethree processing plants and among the sample sources (environment, raw ma-terials, in-process product, and finished product) were compared using Pearson’schi-square test. Exact P values were calculated since expected cell values in somecells were less than five. No analyses were attempted for ribotypes isolated fewerthan four times overall due to the small sample size and infrequency of thoseobservations. P values of #0.05 were considered significant. All analyses wereperformed using the statistical software program StatXact-4 for Windows(CYTEL Software Corporation, Cambridge, Mass.).

RESULTS

Detection of L. monocytogenes in cold-smoked fish and in thesmoked fish processing environment. A total of 531 environ-mental, raw material, in-process, and smoked fish sampleswere screened for the presence of L. monocytogenes using boththe culture-based method and the BAX for Screening/Listeriamonocytogenes system. The BAX PCR system detected L.monocytogenes in 17.8% of the 531 samples, compared to16.0% positive-culture results. Sensitivity, specificity, and over-all accuracy estimates for the PCR system are shown in Table1. Thirteen samples that gave initial BAX system false-negativeresults were retested using the resuspended frozen enrichmentculture 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 comparedto the initial culture-based screening results. Subsequent hlyAPCR screening of up to 16 additional isolates per sample con-firmed the presence of L. monocytogenes in 12 of these sam-ples, thus improving the specificity of the PCR system (Table1). While there was no apparent correlation between sampletype and BAX system false-positive results, four of the sevenBAX system false-negative samples were taken from finishedproducts. The corrected BAX system sensitivity and specificityvalues reported in this study should be regarded as rough

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estimates, as only the discrepant results were further analyzed.Estimation of true corrected values would have required re-testing of all samples.

The BAX system detected L. monocytogenes in 27.7% of the206 environmental samples taken throughout the processingareas (Table 2) (the data reflect prevalence following reexam-ination of discrepant results). Positive samples included thosefrom floors and floor drains in raw material preparation areas,brining rooms, cold smokers, finished product processingareas, and food contact surfaces, including cutting tables andautomated finished product slicer blades. L. monocytogeneswas isolated from environmental samples with a lower fre-quency (21.4%) than was detected by the PCR-based system.Nine raw material samples (8.8%), including salmon, whitefish,and chub, were L. monocytogenes culture positive, compared toa slightly lower frequency using the PCR-based system. L.monocytogenes was detected in in-process samples with aslightly higher frequency (18.1%) by the PCR-based system.The prevalence of L. monocytogenes in finished products, in-cluding smoked sea bass, sablefish and cold-smoked salmon,was similar to that observed in raw materials. Additional Lis-teria species (L. monocytogenes hlyA PCR negative) were iso-lated from 54.1% of the 85 culture-positive samples.

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

Seven different ribotypes were isolated from samples col-lected at processor B (Fig. 1). L. monocytogenes DUP-1042C,isolated from 36.4% of positive samples, predominated in thesamples collected from this facility. This strain persisted inenvironmental samples collected over a period of 5 monthsand was also isolated from raw materials and in-process sam-ples. L. monocytogenes DUP-1045 was isolated from 31.8% ofpositive processor B samples and persisted in environmentalsamples collected over 2.5 months. This ribotype was isolatedonly from the environment of processor B, was not isolatedfrom processor C samples, and was isolated from only twoin-process samples collected from processor D (Fig. 3). Asshown in Table 3, the distribution of ribotypes DUP-1042Cand DUP-1045 varied significantly by processing facility (P 50.0003 and P 5 0.0006, respectively).

Eight different L. monocytogenes ribotypes were isolated from16 of the samples collected at processor C (Fig. 2). L. mono-cytogenes DUP-1044A and DUP-1046B were isolated with thehighest frequency (25.0% each) from positive samples. L. mono-cytogenes DUP-1044A persisted in samples collected from thisfacility over 4 months and was not isolated from processor Bsamples. The distribution of ribotype DUP-1044A, however,was not shown to vary significantly by processor (Table 3). Thedistribution of ribotype DUP-1046B, isolated only from sam-ples collected at processor C, was shown to vary significantlyamong processing facilities (P 5 0.0144) (Table 3).

Nine L. monocytogenes ribotypes were isolated from 47 ofthe samples collected at processor D (Fig. 3). L. monocytogenesDUP-1039C, isolated from 48.9% of positive processor D sam-ples, persisted in the processing environment over all samplingvisits (6 months). This strain was not isolated from raw mate-rial samples collected at processor D or from any samplescollected from processor B or C. The distribution of L. mono-cytogenes DUP-1039C was shown to vary significantly by pro-cessing facility (P 5 0.0000) (Table 3). While L. monocytogenesribotypes DUP-1044A and DUP-1062 also persisted in sam-ples collected from processor D over a 3-month period, thedistribution of these ribotypes did not vary significantly by pro-cessing facility (Table 3).

As preliminary analyses indicated that the type of samplecollected varied significantly (P 5 0.0001) according to pro-cessing facility, we suspected that the apparent difference inribotype distribution among processors could be attributed tothe prevalence of a given ribotype in a specific sample type.The distribution of each ribotype, however, was not shown tovary significantly by sample type (P . 0.05).

DISCUSSION

L. monocytogenes is a food-borne bacterial pathogen that isubiquitous in nature and shows the ability to persist in the foodprocessing environment for prolonged time periods. Control ofthis organism is thus extremely challenging for the food indus-try. Development of a better understanding of the ecology ofL. monocytogenes in food processing plants in combinationwith rapid, standardized detection and typing systems will pro-vide critical tools and knowledge for the development andverification of improved control strategies.

In-plant L. monocytogenes contamination patterns. L. mono-cytogenes was detected in a variety of environmental, raw ma-terial, in-process, and cold-smoked fish samples (Table 2) col-lected from three smoked fish processing plants. We isolatedL. monocytogenes from 11.5% of finished product samples,consistent with the prevalence range reported by previous sur-

TABLE 1. Sensitivity and specificity estimates for the BAXfor Screening/Listeria monocytogenes system using culture-based

detection results as the standard of comparison

BAXresult

No. of samples with the indicated culture result after:

Initial testinga Reexamination ofdiscrepant resultsb

Positive Negative Positive Negative

Positive 60 29 78 17Negative 13 429 7 429

Total 73 458 85 446

a Sensitivity, 82.2% (95% confidence interval, 71 to 91%); specificity, 93.7%(95% confidence interval, 92 to 96%); accuracy, 92.1%.

b Corrected sensitivity, 91.8% (95% confidence interval, 84 to 98%); correctedspecificity, 96.2% (95% confidence interval, 94 to 98%); corrected accuracy,95.5%.

TABLE 2. Detection of L. monocytogenes in samples from theprocessing environment, raw materials, fish during the

cold-smoking process, and cold-smoked fish

Sample sourceNo. (%) Total no.

of samplesBAX system positive Culture positive

Environment 57 (27.7) 44 (21.4) 206Raw materials 8 (7.8) 9 (8.8) 102In process 23 (18.1) 21 (16.5) 127Finished product 7 (7.3) 11 (11.5) 96

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veys (6, 21, 27). As L. monocytogenes is a common contaminantof the food processing environment (6, 16), we were not sur-prised that this organism was isolated from over 20% of ourenvironmental samples.

To specifically investigate L. monocytogenes contaminationpatterns and strain progressions in these plants, all isolateswere characterized by molecular subtyping. Our subtypingdata, in combination with statistical analysis, revealed a uniqueL. monocytogenes contamination pattern for each processingplant. Different ribotypes predominated in the samples col-lected from each processor, and specific ribotypes persisted insamples collected over time (Fig. 1 to 3). In support of aunique contamination pattern for each processor, the distribu-tions of L. monocytogenes ribotypes DUP-1039C, DUP-1042C,DUP-1045, and DUP-1046B were shown to vary significantlyby processing facility (Table 3) but not according to sampletype. The results for processor D provide the most strikingillustration, as L. monocytogenes DUP-1039C persisted in theprocessing environment over the 6-month sampling period andwas isolated from nearly half of the culture-positive samplescollected at this facility (Fig. 3). This ribotype was not isolatedfrom any samples collected from processors B and C. In con-trast, a different subtype (DUP-1042C) persisted in the envi-ronment of processor B over a period of 5 months (Fig. 1).

The persistence of L. monocytogenes DUP-1042C and DUP-1039C in the environments of processors B and D, respectively,for at least 5 months suggests that these strains are a part of theresident microflora and are not eliminated by current cleaningand sanitation programs. Previous reports have shown that L.

monocytogenes has the ability to adhere to surfaces and colo-nize the food processing environment (22, 28, 29, 31). Oncethese populations are established, they appear to become moreresistant to cleaning and sanitation measures (17, 28, 32). Theaffected processing areas, therefore, can serve as primarysources of finished product contamination. Our results showthat ribotyping provides an effective means for monitoring thepersistence of resident populations of L. monocytogenes in theprocessing environment and facilitates a better understandingof the ecology of this organism in food processing facilities.With this information available, the processor can develop andimplement specific programs to eliminate resident populationsand minimize the potential for L. monocytogenes colonization.

The predominance of specific L. monocytogenes subtypesover time is consistent with findings of previous investigationsof contamination patterns in a variety of food processing en-vironments, including those for smoked fish, poultry, meat, anddairy foods (3, 29, 33, 37). The persistence of specific strains ina variety of processing environments, along with the isolationof genetically diverse sporadic contaminants, suggests thatsome strains might have unique genetic characteristics confer-ring an enhanced ability to survive environmental stresses andcolonize the food processing environment. In support of thishypothesis, a recent study indicated that L. monocytogenesstrains differ in their ability to form biofilms (M. Chae andM. Schraft, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr.P-104, p. 531, 1999). In a previous study evaluating the rela-tionship between the resistance of specific strains to commer-cial sanitizers and their ability to persist in the processing

FIG. 1. Ribotypes of L. monocytogenes isolates from processor B environmental, raw material, in-process, and finished product samplesgrouped by sampling visit. (E), environmental sample; (R), raw materials; (IP), in-process sample; (F), finished product. p, ribotype isolated mostfrequently from processor B samples.



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environment, Earnshaw and Lawrence observed that while thesensitivity to sanitizers varied significantly among strains insuspension, there was not a significant difference betweenstrains which persisted in the poultry processing environmentover a 6-month period and sporadic isolates (14). Furtherstudies will likely elucidate characteristics that contribute tothe apparent enhanced ability of specific subtypes to colonizeprocessing environments and resist cleaning and sanitationmeasures.

Potential sources of finished product contamination. Mo-lecular subtyping data also provided useful information regard-ing potential sources of specific L. monocytogenes strains iso-lated from finished product samples. Our subtyping data, inconjunction with our prevalence results, indicate that both rawmaterials and the processing environment serve as potentialsources of finished product contamination. For example, DUP-1039A was isolated from processor B raw and cold-smokedChilean salmon but not from the processing environment (Fig.1, visit 1), indicating that raw materials likely served as thesource of finished product contamination at the time of sam-pling. While results from a study by Eklund et al. also impli-cated raw fish as an important source of L. monocytogenes inthe smoked fish processing environment and in finished prod-ucts (15), other studies employing molecular typing methodsconcluded that raw materials may not serve as a significantsource of finished product contamination (4, 37). Additionalcomprehensive studies monitoring contamination of raw fishand finished products using molecular subtyping methods are

needed to clarify the importance of raw materials as a sourceof L. monocytogenes in finished products.

Analysis of ribotyping data also provided strong evidence insupport of the processing environment as a significant sourceof finished product contamination. L. monocytogenes DUP-1046B, for example, was isolated from cold-smoked Novasalmon, cold-smoked sablefish, and two floor drain samplescollected during the second visit to processor C (Fig. 2). AsDUP-1046B was not isolated from any raw material samplescollected from this facility over the 6-month sampling period,the processing environment most likely served as the source ofthis subtype in finished product samples. L. monocytogenesDUP-1039C, which persisted in the processing environment ofprocessor D over the 6-month sampling period, was also iso-lated from in-process samples and from five of seven positivecold-smoked fish samples. DUP-1039C was not isolated fromraw material samples (Fig. 3), strongly suggesting that theenvironment serves as the primary source of finished productcontamination in this processing facility. Consistent with ourresults, previous studies employing discriminatory subtypingmethods have identified the processing environment as a sig-nificant source of L. monocytogenes isolated from samples dur-ing processing and from finished products (4, 37). Rørvik et al.observed the persistence of a single electrophoretic type in theprocessing environment of a smoked salmon processing plantand in finished products, strongly suggesting that the environ-ment served as a primary source of contamination (37). Simi-larly, analysis of PFGE subtyping data led Autio et al. to

FIG. 2. Ribotypes of L. monocytogenes isolates from processor C environmental, raw material, in-process, and finished product samplesgrouped by sampling visit. (E), environmental sample; (R), raw materials, (IP), in-process sample, (F), finished product. p and pp, ribotypes isolatedmost frequently from processor C samples.



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conclude that the contamination of cold-smoked rainbow troutwas most closely linked to sites associated with brining andslicing (4).

Evaluation of a commercial PCR-based system for detectionof L. monocytogenes. Conventional culture methods for the

isolation and identification of L. monocytogenes from food andenvironmental samples require a minimum of 4 to 5 days tocomplete (6, 13, 16). These methods may lead to false-negativeresults if mixed populations of Listeria species are present in agiven sample, as most selective plating media do not allow

FIG. 3. Ribotypes of L. monocytogenes isolates from processor D environmental, raw material, in-process, and finished product samplesgrouped by sampling visit. (E), environmental sample; (R), raw materials; (IP), in-process sample; (F), finished product. p, ribotype isolated mostfrequently from processor D samples.



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visual differentiation of L. monocytogenes from other Listeriaspp. (6). Rapid sample screening methods, including immuno-chemical, nucleic acid probe-based, and PCR-based assays,may overcome some of these limitations (5, 13, 23, 40). Anincreasing number of commercial PCR-based assays are avail-able for the specific detection of L. monocytogenes, includingthe BAX for Screening system and the Probelia PCR system(Sanofi Diagnostics Pasteur). These assays have the potentialto provide rapid and reliable molecular methods for monitor-ing of L. monocytogenes in the food industry.

We used the BAX for Screening/Listeria monocytogenes sys-tem for monitoring the presence of this organism in raw ma-terials, in-process fish, cold-smoked fish, and environmentalsamples. Our results indicate a sensitivity of 91.8% for theBAX system (Table 1). It appears that our PCR system false-negative results were likely due to low levels of L. monocyto-genes after sample enrichment or due to a high level of back-ground microflora. Fewer than 3 3 102 Listeria-like colonies(,3 3 103 CFU/ml), below the system detection limit of 1 3105 CFU/ml (40), were observed on the Oxford plates for six ofthe seven BAX system false-negative samples. The other sam-ple appeared to contain Listeria in a high background of amixed bacterial population. Further, pure-culture L. monocy-togenes isolates from all false-negative samples tested BAXsystem positive, suggesting that the false-negative results werenot due to the presence of nonreacting isolates. The employ-ment of a two-step sample enrichment, as recommended by themanufacturer of the BAX for Screening/Listeria monocyto-genes system, may increase the sensitivity of this system byfacilitating recovery and growth of this organism. We used asingle-step enrichment in LEB for this study based upon re-ports of comparable or enhanced recovery of L. monocytogenesfrom naturally contaminated seafood as compared to otherenrichment protocols (6).

Our results indicated a specificity of 96.2% for the BAXsystem (Table 1). We believe that the BAX system false-pos-itive results may be due to the specific detection of L. mono-cytogenes in a high background of other Listeria species. Asadditional Listeria spp. were observed in over half of ourL. monocytogenes-positive samples, it is possible that this or-ganism was overgrown by competing species during sampleenrichment (resulting in culture-negative screening results). Inagreement with our results, several researchers have observedthat Listeria innocua can outcompete L. monocytogenes if the

two species are grown together in commonly used enrichmentmedia, including LEB (30, 35). It is unlikely that nonspecificpriming contributed to the BAX system false-positive results,as this system has demonstrated 100% exclusivity for 60 Lis-teria spp. strains (non-L. monocytogenes) and 44 non-Listeriastrains (BAX for Screening/Listeria monocytogenes package in-sert, Qualicon, Inc., Wilmington, Del.). Further, a recent re-port indicated that the presence of other Listeria species didnot interfere with the detection of L. monocytogenes by theBAX system (40).

Conclusions. Our results indicate that molecular detectionand subtyping methods facilitate a better understanding of theecology of food-borne pathogens in the food processing envi-ronment. This work further demonstrates the utility of molec-ular subtyping, specifically ribotyping, for tracking the sourcesand spread of food-borne pathogens. Ribotyping was appliedin our study because this subtyping method is highly discrimi-natory, automated, and reproducible from one laboratory toanother, thus facilitating rapid identification and data sharingamong research groups. Although a single typing method canoften elucidate contamination patterns, the use of multipletyping methods (20), multiple enzymes for ribotyping (19),or other typing methods (e.g., PFGE) (20) may increase dis-criminatory power and provide additional information on con-tamination patterns. It is also important to remember thatmolecular typing methods rely on culture-based isolation ofL. monocytogenes. As it has been shown that different enrich-ment procedures may favor the growth of different subtypesand that multiple subtypes may be present in a given sample(38), the employment of more than one enrichment methodand subtyping of more than one isolate per sample may facil-itate further elucidation of contamination patterns.

A thorough understanding of strain persistence and progres-sion will be crucial for improved control strategies for L. mono-cytogenes. Based on our results, we propose that sanitationprotocols specifically targeting possible reservoirs of persistentstrains may efficiently reduce environmental contamination.Sanitation control procedures are of particular importance forproducts that do not receive a heat treatment sufficient to killfood-borne pathogens, including L. monocytogenes. Routineenvironmental monitoring programs featuring molecular de-tection and subtyping methods will help to provide the neces-sary information regarding the origins and spread of contam-inants to facilitate targeted control and sanitation strategies.

ACKNOWLEDGMENTS

We are indebted to the smoked fish processors who participated inthis study. We thank Qualicon, Inc., Cornell Research Support Spe-cialist Mary Bodis, and the members of the Food Safety Laboratory fortheir expert technical advice and assistance.

This paper is a result of research funded by the National Oceanicand Atmospheric Administration (award NA86RG0056 to the Re-search Foundation of State University of New York for New York SeaGrant).

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TABLE 3. Prevalence of L. monocytogenes ribotypes in eachprocessing facility over five sampling visits

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a Two-sided exact P value computed for Pearson’s chi-square test.b Total number of samples.



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