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Rapid and specific detection of Listeria monocytogenes in smoked salmon with BAX®-PCR
Food Control 16 (2005) 717–721 www.elsevier.com/locate/foodcont
Rapid and specific detection of Listeria monocytogenes in smoked salmon with BAXÒ-PCR B. Becker a
a,*
, S. Jordan b, W.H. Holzapfel
a
Institute of Hygiene and Toxicology, Federal Research Centre for Nutrition, D-76131 Karlsruhe, Germany b Du Pont Qualicon, Du Pont de Nemours GmbH, Bad Homburg, Germany
Abstract Members of the genus Listeria are ubiquitous, and are therefore also common to the food and the environment. Among them, only Listeria monocytogenes has a pathogenic potential, and can cause infectious diseases (listeriosis) in humans. Conventional microbiological testing methods are labour-intensive and time consuming (4–5 days), and often require a number of different culture media for final isolation and confirmatory tests. In order to overcome these limitations, numerous rapid methods have been developed in recent years. DNA-based methods such as the polymerase chain reaction (PCR) have increasingly been used for rapid and sensitive detection of L. monocytogenes. Among the various available PCR assays, we used the BAXÒ system (with two different detection procedures: gel detection and automated detection) to screen for L. monocytogenes in samples of vacuum packaged cold smoked salmon. A total of 27 samples were used for this study. The method was compared to the German standard microbiological detection method according to DIN 11290-1 and -2. Detection of Listeria and L. monocytogenes from salmon samples was performed using Palcam enrichment medium, followed by plating on both Palcam agar and ALOA agar. The BAXÒ assay gave identical results for 26 food samples compared to the standard method, including 15 positives. Only in one case the BAXÒ system gave a false-positive result, probably due to the amplification of DNA from nonviable cells of L. monocytogenes. In naturally contaminated food samples, the BAXÒ method gave good results after 24–48 h. Application of this rapid method is simple and time saving. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Listeria monocytogenes; PCR; BAXÒ system
1. Introduction Consumers around the world are more aware than ever about food safety issues and are seeking increased assurance about safety and quality of the foods they eat. The cost of illness, death, and business lost due to bacterial foodborne diseases is high. Some pathogenic bacteria that cause economically important foodborne
*
Corresponding author. Tel.: +49 721 6625 462; fax: +49 721 6625 453. E-mail address: [email protected] (B. Becker). 0956-7135/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2004.06.009
diseases in the United States include Salmonella (calculated at $4 billion in economic losses annually), S. aureus ($1500 million) and Listeria monocytogenes ($313 million) (Todd, 1989). In 2000, the Federal Institute of Health Protection of Consumers and Veterinary Medicine in Germany reported overall 200,000 cases of foodborne infections. Studies by this institute showed in some cases that illnesses caused by zoonotic pathogens had increased markedly compared to 1999. L. monocytogenes was repeatedly found in samples of meat and meat products, in raw milk, soft cheese and pasteurised dairy products and especially in fish and fish products. Several surveys reported the presence of the pathogen L. monocytogenes in cold smoked salmon (Ben Embarek, 1994;
718
B. Becker et al. / Food Control 16 (2005) 717–721
Dillon, Patel, & Ratnam, 1994; Norton et al., 2001). High counts of L. monocytogenes of more than 10,000 per gram of food were detected in some cases (Feldhusen, Jark, Etzel, Ballin, & Wilke, 2002). According to the latest scientific findings, a contamination level of higher than 100 Listeria per gram may cause illness. Contamination by levels exceeding 100 viable cells of L. monocytogenes per gram food are therefore considered to have the potential of causing serious, sometimes fatal, foodborne infections in young children, the elderly and people with weakened immune systems. It also can cause miscarriages or stillbirths even if a pregnant woman experiences no symptoms. In healthy people, L. monocytogenes can cause headache, high fever, nausea, abdominal pain and diarrhoea. Zero-tolerance for L. monocytogenes was introduced in the USA for commercial foods for consumption without further cooking. Other countries such as Switzerland, Austria, Italy, New Zealand and South Korea have adopted this regulation. Still, Listeria cannot be eliminated completely from food and food processing lines in the industry. Therefore, a tolerance of up to 100 L. monocytogenes per gram of food on the ‘‘sell-by’’ date would be more realistic. Considerations leading to this ‘‘tolerance’’ level probably have accepted this as ‘‘minimal infection dose’’ for people at risk. The mortality rate of 30% for listeriosis is the highest of any foodborne bacterial agent. This value is an average rate and will be higher for infants and people with immune deficiencies. Finding a quick and reliable method to detect and identify L. monocytogenes, has high priority for the early recognition of contaminated food products. The modern polymerase chain reaction (PCR) based assays provide a quick and reliable screening procedure for several foodborne pathogens (Baumgartner & Grand, 1995; Hoffman & Wiedmann, 2001; Lu¨cke & ten Bosch, 1998; Winters, Maloney, & Johnson, 1999). One of the latest test kit available for the rapid detection of L. monocytogenes is the BAXÒ system (Du Pont Qualicon, Germany). In April, 2002, the US Department of AgricultureÕs Food Safety and Inspection (FSIS) has adopted the BAXÒ system to screen meat and poultry samples for L. monocytogenes (FSIS adopts new screening method for Listeria monocytogenes, 2002). The scope of this study was to critically evaluate the use of this system for screening L. monocytogenes in cold smoked salmon.
2. Materials and methods A total of 27 samples of vacuum packaged smoked salmon (obtained from local supermarkets) were screened for L. monocytogenes using cultural Listeria detection according to DIN 11290-1 and -2 with a small modification, and the molecular based methods, automated BAXÒ and gel-based system.
2.1. Listeria—culture procedure A 25 g portion of each sample was blended in a stomacher in 225 ml of Palcam-Listeria-enrichment-broth (Merck, Germany) for 2 min and incubated at 37 °C for either 24 or 48 h. The enrichment method using Palcam broth is not recommended for the BAXÒ system, but since this medium is often used in Germany for routine isolation purposes it was included in this study. For determination of viable Listeria counts, a loopful from each enrichment was surface-plated, both on PalcamListeria-Agar (Merck, Germany) and the new selective ALOA agar (AES-Laboratoire, France). The plates were incubated for 24–48 h at 37 °C. Five suspect Listeria colonies from each plate were chosen and purified on Standard-I-Agar. Further biochemical characterisation was performed using API-Listeria (bioMe´rieux, Germany) as well as other tests like catalase, oxidase and CAMP reactions (with Staphylococcus aureus DSM 6732 and Rhodococcus equi DSM 20307). 2.2. PCR Subsequently to the enrichment procedure, the BAXÒ system protocol (Du Pont Qualicon, Germany) was followed. 2.3. BAXÒ system with gel detection The process consists of three major steps: DNA preparation One ml of enriched samples were centrifuged at 13,000g for 10 min. The pellet was washed twice in sterile demineralised water. Afterwards, 5 ll of the cell suspension was recovered in 200 ll lysis reagent (12.5 ll protease to 1 ml lysis buffer) and heated at 55 °C for 60 min, then 95 °C for 10 min. After cooling (about 5 min) 50 ll of lysed samples from lysis tubes were transferred into the PCR tubes. Amplification BAXÒ system tablets contain all necessary reagents for PCR amplification (buffer, primers, polymerase, dNTPs and positive control). The PCR tubes containing tablets and lysate were placed in the thermal cycler (Primus 25, MWG-Biotech, Germany), and PCR was carried out using the following condition: denaturation step at 94 °C for 2 min, followed by 38 amplification cycles. Each cycle consisted of 15 s at 94 °C and 3 min at 70 °C. Negative and positive controls were included in all experiments. Detection 15 ll of PCR products were loaded onto 2% agarose gels and electrophoresis was performed for 45 min at 180 V. The molecular weight marker supplied with the kit was also run on each gel. The gels were stained with
B. Becker et al. / Food Control 16 (2005) 717–721
ethidium bromide and photo-documentation was done at 312 nm. 2.4. BAXÒ system with automated detection Automated BAXÒ system kits can be used with an automated instrument (comprising cycler, computer system, monitor and printer) that eliminates the need for gel electrophoresis and photo-documentation. The process for the BAXÒ system consists of sample preparation followed by automated amplification and detection (in the same unit). Sample preparation, DNA preparation and lysis are identical with the BAXÒ system with gel detection (see point 2.3). The lysed samples were transferred to the PCR tubes (with PCR tablets) and then loaded into the BAXÒ cycler/detector. The appropriate program was run. Each tube contains a fluorescent dye, which binds with target DNA and emits a fluorescent signal in response to light. The full BAXÒ process program, which consists of cycling and detection, takes about 5 h to complete. Evaluation of the BAXÒ system with automated detection and the BAXÒ system with gel detection was performed by calculating their sensitivity, specificity and efficiency (Capita, Alonso-Calleja, Moreno, & Garcı´a-Ferna´ndez, 2001). Values were calculated as follows: sensitivity = a/(a + c); specificity = d/(b + d) and efficiency = (a + d)/(a + b + c + d). Data a, b, c and d represent true-positive, false-positive, false-negative and true-negative values, respectively.
719
have been described on which L. monocytogenes can be distinguished from other Listeria spp. (Johansson, 1998; Pinto et al., 2001; Restaino, Frampton, Irbe, Schabert, & Spitz, 1999; Vlaemynck, Lafarge, & Scotter, 2000). Its principle is based on hemolysis or on a chromogenic substrate. In the new selective agar medium, ALOA, the chromogenic compound X-glucoside is added as substrate for the detection of b-glucosidase, resulting in blue coloured Listeria colonies. The selectivity of medium is obtained by the addition of nalidixic acid, ceftazidime, polymyxin, and cycloheximide. Through the addition of a specific purified L -a-phosphatidylinositol, L. monocytogenes grow within 24 h as blue colonies surrounded by an opaque and regular halo (production of a phosphatidylinositol-specific phospholipase C). All positive samples that were detected with the reference method on Palcam medium have also been identified with ALOA agar. The picking of colonies for confirmation was much easier than on Palcam medium. The advantage of ALOA medium is the possibility of distinction between L. monocytogenes and other Listeria species. The detection of other Listeria spp. was sometimes difficult because L. monocytogenes often formed large opaque halos (Fig. 1). The results of the culture procedure very clearly demonstrate that control of L. monocytogenes in fish processing and supply systems is absolutely essential.
3. Results and discussion A total of 27 cold smoked salmon samples were screened for the presence of L. monocytogenes using both the culture-based method, and the BAXÒ system for screening of L. monocytogenes. 3.1. Culture procedure Using the cultural method, 70.4% of vacuum packaged cold smoked salmon were found to be contaminated with Listeria spp. L. monocytogenes was found in 15 (55.6%) of all samples tested, L. innocua in 11.1%, L. seeligeri, L. welshimeri and L. grayi in 7.4%. Five of the samples were contaminated by L. monocytogenes and other Listeria in combination. It is well known that the Palcam agar does not allow distinction between colonies of L. monocytogenes and other Listeria. The selection of five colonies for confirmation of L. monocytogenes from Palcam agar may not be sufficient if large numbers of other Listeria species are present in the food. For this reason, the enrichment broths were inoculated on ALOA agar. Many media
Fig. 1. Selective and differential isolation of L. monocytogenes on ALOA agar; nonpathogenic Listeria spp. appear as bluish colonies without a halo and L. monocytogenes as bluish colonies with opaque halo.
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B. Becker et al. / Food Control 16 (2005) 717–721
3.2. BAXÒ system with gel detection Traditional screening methods are based on bacterial behaviour. By comparison, the BAXÒ system relies on the actual genetic information of bacteria by detecting a specific DNA fragment found only in the target organism. Following enrichment procedures, the BAXÒ system yielded results within about eight hours. All necessary reagents for this method are already packaged inside the tubes as a single tablet. The PCR results were interpreted as follows: a sample was designated positive for L. monocytogenes if two bands appeared in the lane, one at the appropriate target level (around the 400 bp) and another at the control level (200 bp). A sample was also considered positive if a target band appeared without the control band. This situation can occur when the sample contains a high level of L. monocytogenes that competes with the positive control for amplification reagents. A sample was considered negative for L. monocytogenes if no target band appeared, but a control band was present. A band at the control level indicates that there has been no PCR inhibition. The gel detection system worked correctly, but we could not clearly detect six bands (indicated by the manufacturer) in the marker lane, as only five bands were sufficiently intensive. Fig. 2 shows the results of the BAXÒ system with agarose gel detection. The BAXÒ assay gave identical results
Fig. 2. 2% agarose gel electrophoresis of PCR BAXÒ-amplified products obtained from enrichment broth of cold smoked salmon. Lanes 1, 18, 19 and 36: molecular size markers. Lanes 2 and 20: L. monocytogenes DSM 20600 as positive control. Lanes 3 and 21: negative control. Lanes 4, 6, 11, 14, 15, 16, 23, 26, 30, 32, 33 and 34: L. monocytogenes-negative samples. Lanes 5, 7, 8, 9, 10, 12, 13, 17, 22, 24, 25, 27, 28, 29, 31 and 35: L. monocytogenes-positive samples.
for 26 of 27 food samples compared to the standard method, including 15 positives. Only in one case the BAXÒ system gave a false-positive result, probably due to the amplification of DNA from nonviable cells of L. monocytogenes. With the culture-based method, only L. innocua was found in this sample. By using a pure viable culture of this strain, a negative reaction was obtained with the BAXÒ system (Fig. 2). 3.3. BAXÒ system with automated detection The BAXÒ system can be used with an automated instrument that eliminates the need for gel electrophoresis and photo-documentation. Each PCR tablet of the BAXÒ system contains a fluorescent dye, which binds with DNA and emits a fluorescent signal in response to light. After amplification, the BAXÒ system begins a detection phase where the fluorescent signal is measured. During detection, the temperature of the samples is raised to the point where the DNA strands denature, releasing the dye and lowering the signal. This change in fluorescence can be plotted against temperature to generate a melting curve, which is interpreted by the BAXÒ system software. This software contains three choices of graphical views: processed, raw or alternate views of data associated with the selected wells. For easier reviewing of smaller changes of the melting curve, the alternate data graph is the best way of reporting (Fig. 3). The graph should show one peak for the positive control (under 80 °C) and, if L. monocytogenes is present, a second peak at a different temperature (higher than 83 °C). With 24 h enrichment prior to PCR analysis, 15 samples were positive for L. monocytogenes by using the BAXÒ system. After 48 h enrichment, the BAXÒ system was able to detect 16 positive samples. Therefore, a 48 h enrichment period seems to be impor-
Fig. 3. Individual well report of the BAXÒ system—alternate data graph: positive sample with a peak at 85 °C and negative sample with a peak under 80 °C.
B. Becker et al. / Food Control 16 (2005) 717–721
tant in order to avoid false-negative results, especially when the number of contaminating bacteria is low. One of the 12 culture-negative samples for L. monocytogenes gave a positive result with the BAXÒ system. This false-positive result is probably due, either to the amplification of DNA from nonviable cells of L. monocytogenes or to the specific detection of L. monocytogenes in a high background of other Listeria species. Several authors have observed that L. innocua can overgrow L. monocytogenes if they are grown together in commonly used enrichment media (MacDonald & Sutherland, 1994; Petran & Swanson, 1993). In this study, we detected also L. innocua (30 cfu/g) in the false-positive sample with the BAXÒ system. The enrichment method using the Palcam medium and an additional centrifugation step after enrichment, are not according to the BAXÒ protocol. Our study, however, has shown that Palcam enrichment medium, which is widely used in Germany, is just as suitable for enrichment as Fraser broth. The BAXÒ positive control reaction with each sample provided direct confirmation that no PCR inhibitors were present. L. monocytogenes present in cold smoked vacuum packaged salmon could be detected by BAXÒ-PCR with target DNA prepared by both BAXÒ protocols described above. Both BAXÒ procedures took 5–6 h after enrichment. Our results indicated a specificity of 92%, a sensitivity of 100% and an efficiency of 96.4% for the BAXÒ system. FSIS determined also that the BAXÒ system was as sensitive as the current method in detecting L. monocytogenes. With BAXÒ, fewer samples were falsely screened as positive. Data showed as well that the system reduced the reporting time for negative samples by one day when compared to the current method. Our results indicate that a simple enumeration on a specific isolation medium with reliable performance, and the rapid detection of pathogenic bacteria by PCR methods (BAXÒ system), may improve control of Listeria in food and environmental samples. The chromogenic ALOA agar and the BAXÒ system provided results comparable to the standard methods. All positive samples on Palcam agar were positive on ALOA agar and differentiation of L. monocytogenes from other Listeria was significantly better and easier to interpret. The introduction of the BAXÒ system in the detection procedure would reduce the time of analysis for L. monocytogenes in food considerably.
Acknowledgement We wish to thank Jennifer Murphy for excellent technical assistance.
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References Baumgartner, M., & Grand, A. (1995). Detection of verotoxinproducing Escherichia coli in minced beef and raw hamburgers: comparison of polymerase chain reaction (PCR) and immunomagnetic beads. Archiv fu¨r Lebensmittelhygiene, 46, 127–130. Ben Embarek, P. K. (1994). Presence, detection and growth of Listeria monocytogenes in seafoods: a review. International Journal of Food Microbiology, 23, 17–34. Capita, R., Alonso-Calleja, C., Moreno, B., & Garcı´a-Ferna´ndez, M. C. (2001). Occurrence of Listeria species in retail poultry meat and comparison of a cultural/immunoassay for their detection. International Journal of Food Microbiology, 65, 75–82. Dillon, R., Patel, T., & Ratnam, S. (1994). Occurrence of Listeria in hot and cold smoked seafood products. International Journal of Food Microbiology, 22, 73–77. Feldhusen, F., Jark, U., Etzel, V., Ballin, U., & Wilke, T. (2002). Listeria monocytogenes in Ra¨ucherlachs—Problemanalyse und Lo¨sungsansa¨tze. Archiv fu¨r Lebensmittelhygiene, 53, 8–12. FSIS adopts new screening method for Listeria monocytogenes (2002). Food Safety and Inspection Service, United States Department of Agriculture (FSIS), April 30, 2002. Available: http://www.fsis. usda.gov. Hoffman, A. D., & Wiedmann, M. (2001). Comparative evaluation of culture- and BAX polymerase chain reaction-based detection methods for Listeria spp. and Listeria monocytogenes in environmental and raw fish samples. Journal of Food Protection, 64(10), 1521–1526. Johansson, T. (1998). Enhanced detection and enumeration of Listeria monocytogenes from foodstuffs and food-processing environments. International Journal of Food Microbiology, 40, 77–85. Lu¨cke, F.-K., & Ten Bosch, C. (1998). Mo¨glichkeiten und Grenzen fu¨r den Einsatz der Polymerase-Kettenreaktion (PCR) zum Nachweis und zur Typisierung lebensmittelhygienisch relevanter Bakterien. Deutsche Lebensmittel-Rundschau, 6, 182–186. MacDonald, F., & Sutherland, A. D. (1994). Important differences between the generation times of Listeria monocytogenes and Listeria innocua in two Listeria enrichment broths. Journal of Dairy Research, 61, 433–436. Norton, D. M., McCamey, M. A., Gall, K. L., Scarlet, J. M., Boor, K. J., & Wiedmann, M. (2001). Molecular studies on the ecology of Listeria monocytogenes in the smoked fish processing industry. Applied and Environmental Microbiology, 67, 198–205. Petran, R. L., & Swanson, K. M. J. (1993). Simultaneous growth of Listeria monocytogenes and L. innocua. Journal of Food Protection, 56, 616–618. Pinto, M., Burri, S., Mena, C., Almeida, G., Carneiro, L., Teixeira, P., & Gibbs, P. A. (2001). Comparison of oxford agar, palcam and Listeria monocytogenes blood agar for the recovery of L. monocytogenes from foods and environmental samples. Food Control, 12, 511–514. Restaino, L., Frampton, E. W., Irbe, R. M., Schabert, G., & Spitz, H. (1999). Isolation and detection of Listeria monocytogenes using fluorogenic and chromogenic substrates for phosphatidylinositolspecific phospholipase. C. J. Food Protection, 62, 244–251. Todd, E. C. (1989). Costs of acute bacterial foodborne disease in Canada and the United States. International Journal of Food Microbiology, 9(4), 313–326. Vlaemynck, G., Lafarge, V., & Scotter, S. (2000). Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium. Journal of Applied Microbiology, 88(3), 430–441. Winters, D. K., Maloney, T. P., & Johnson, M. G. (1999). Rapid detection of Listeria monocytogenes by a PCR assay specific for an aminopeptidase. Molecular and Cellular Probes, 13, 127–131.
Rapid and specific detection of Listeria monocytogenes in smoked salmon with BAXÒ-PCR B. Becker a
a,*
, S. Jordan b, W.H. Holzapfel
a
Institute of Hygiene and Toxicology, Federal Research Centre for Nutrition, D-76131 Karlsruhe, Germany b Du Pont Qualicon, Du Pont de Nemours GmbH, Bad Homburg, Germany
Abstract Members of the genus Listeria are ubiquitous, and are therefore also common to the food and the environment. Among them, only Listeria monocytogenes has a pathogenic potential, and can cause infectious diseases (listeriosis) in humans. Conventional microbiological testing methods are labour-intensive and time consuming (4–5 days), and often require a number of different culture media for final isolation and confirmatory tests. In order to overcome these limitations, numerous rapid methods have been developed in recent years. DNA-based methods such as the polymerase chain reaction (PCR) have increasingly been used for rapid and sensitive detection of L. monocytogenes. Among the various available PCR assays, we used the BAXÒ system (with two different detection procedures: gel detection and automated detection) to screen for L. monocytogenes in samples of vacuum packaged cold smoked salmon. A total of 27 samples were used for this study. The method was compared to the German standard microbiological detection method according to DIN 11290-1 and -2. Detection of Listeria and L. monocytogenes from salmon samples was performed using Palcam enrichment medium, followed by plating on both Palcam agar and ALOA agar. The BAXÒ assay gave identical results for 26 food samples compared to the standard method, including 15 positives. Only in one case the BAXÒ system gave a false-positive result, probably due to the amplification of DNA from nonviable cells of L. monocytogenes. In naturally contaminated food samples, the BAXÒ method gave good results after 24–48 h. Application of this rapid method is simple and time saving. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Listeria monocytogenes; PCR; BAXÒ system
1. Introduction Consumers around the world are more aware than ever about food safety issues and are seeking increased assurance about safety and quality of the foods they eat. The cost of illness, death, and business lost due to bacterial foodborne diseases is high. Some pathogenic bacteria that cause economically important foodborne
*
Corresponding author. Tel.: +49 721 6625 462; fax: +49 721 6625 453. E-mail address: [email protected] (B. Becker). 0956-7135/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2004.06.009
diseases in the United States include Salmonella (calculated at $4 billion in economic losses annually), S. aureus ($1500 million) and Listeria monocytogenes ($313 million) (Todd, 1989). In 2000, the Federal Institute of Health Protection of Consumers and Veterinary Medicine in Germany reported overall 200,000 cases of foodborne infections. Studies by this institute showed in some cases that illnesses caused by zoonotic pathogens had increased markedly compared to 1999. L. monocytogenes was repeatedly found in samples of meat and meat products, in raw milk, soft cheese and pasteurised dairy products and especially in fish and fish products. Several surveys reported the presence of the pathogen L. monocytogenes in cold smoked salmon (Ben Embarek, 1994;
718
B. Becker et al. / Food Control 16 (2005) 717–721
Dillon, Patel, & Ratnam, 1994; Norton et al., 2001). High counts of L. monocytogenes of more than 10,000 per gram of food were detected in some cases (Feldhusen, Jark, Etzel, Ballin, & Wilke, 2002). According to the latest scientific findings, a contamination level of higher than 100 Listeria per gram may cause illness. Contamination by levels exceeding 100 viable cells of L. monocytogenes per gram food are therefore considered to have the potential of causing serious, sometimes fatal, foodborne infections in young children, the elderly and people with weakened immune systems. It also can cause miscarriages or stillbirths even if a pregnant woman experiences no symptoms. In healthy people, L. monocytogenes can cause headache, high fever, nausea, abdominal pain and diarrhoea. Zero-tolerance for L. monocytogenes was introduced in the USA for commercial foods for consumption without further cooking. Other countries such as Switzerland, Austria, Italy, New Zealand and South Korea have adopted this regulation. Still, Listeria cannot be eliminated completely from food and food processing lines in the industry. Therefore, a tolerance of up to 100 L. monocytogenes per gram of food on the ‘‘sell-by’’ date would be more realistic. Considerations leading to this ‘‘tolerance’’ level probably have accepted this as ‘‘minimal infection dose’’ for people at risk. The mortality rate of 30% for listeriosis is the highest of any foodborne bacterial agent. This value is an average rate and will be higher for infants and people with immune deficiencies. Finding a quick and reliable method to detect and identify L. monocytogenes, has high priority for the early recognition of contaminated food products. The modern polymerase chain reaction (PCR) based assays provide a quick and reliable screening procedure for several foodborne pathogens (Baumgartner & Grand, 1995; Hoffman & Wiedmann, 2001; Lu¨cke & ten Bosch, 1998; Winters, Maloney, & Johnson, 1999). One of the latest test kit available for the rapid detection of L. monocytogenes is the BAXÒ system (Du Pont Qualicon, Germany). In April, 2002, the US Department of AgricultureÕs Food Safety and Inspection (FSIS) has adopted the BAXÒ system to screen meat and poultry samples for L. monocytogenes (FSIS adopts new screening method for Listeria monocytogenes, 2002). The scope of this study was to critically evaluate the use of this system for screening L. monocytogenes in cold smoked salmon.
2. Materials and methods A total of 27 samples of vacuum packaged smoked salmon (obtained from local supermarkets) were screened for L. monocytogenes using cultural Listeria detection according to DIN 11290-1 and -2 with a small modification, and the molecular based methods, automated BAXÒ and gel-based system.
2.1. Listeria—culture procedure A 25 g portion of each sample was blended in a stomacher in 225 ml of Palcam-Listeria-enrichment-broth (Merck, Germany) for 2 min and incubated at 37 °C for either 24 or 48 h. The enrichment method using Palcam broth is not recommended for the BAXÒ system, but since this medium is often used in Germany for routine isolation purposes it was included in this study. For determination of viable Listeria counts, a loopful from each enrichment was surface-plated, both on PalcamListeria-Agar (Merck, Germany) and the new selective ALOA agar (AES-Laboratoire, France). The plates were incubated for 24–48 h at 37 °C. Five suspect Listeria colonies from each plate were chosen and purified on Standard-I-Agar. Further biochemical characterisation was performed using API-Listeria (bioMe´rieux, Germany) as well as other tests like catalase, oxidase and CAMP reactions (with Staphylococcus aureus DSM 6732 and Rhodococcus equi DSM 20307). 2.2. PCR Subsequently to the enrichment procedure, the BAXÒ system protocol (Du Pont Qualicon, Germany) was followed. 2.3. BAXÒ system with gel detection The process consists of three major steps: DNA preparation One ml of enriched samples were centrifuged at 13,000g for 10 min. The pellet was washed twice in sterile demineralised water. Afterwards, 5 ll of the cell suspension was recovered in 200 ll lysis reagent (12.5 ll protease to 1 ml lysis buffer) and heated at 55 °C for 60 min, then 95 °C for 10 min. After cooling (about 5 min) 50 ll of lysed samples from lysis tubes were transferred into the PCR tubes. Amplification BAXÒ system tablets contain all necessary reagents for PCR amplification (buffer, primers, polymerase, dNTPs and positive control). The PCR tubes containing tablets and lysate were placed in the thermal cycler (Primus 25, MWG-Biotech, Germany), and PCR was carried out using the following condition: denaturation step at 94 °C for 2 min, followed by 38 amplification cycles. Each cycle consisted of 15 s at 94 °C and 3 min at 70 °C. Negative and positive controls were included in all experiments. Detection 15 ll of PCR products were loaded onto 2% agarose gels and electrophoresis was performed for 45 min at 180 V. The molecular weight marker supplied with the kit was also run on each gel. The gels were stained with
B. Becker et al. / Food Control 16 (2005) 717–721
ethidium bromide and photo-documentation was done at 312 nm. 2.4. BAXÒ system with automated detection Automated BAXÒ system kits can be used with an automated instrument (comprising cycler, computer system, monitor and printer) that eliminates the need for gel electrophoresis and photo-documentation. The process for the BAXÒ system consists of sample preparation followed by automated amplification and detection (in the same unit). Sample preparation, DNA preparation and lysis are identical with the BAXÒ system with gel detection (see point 2.3). The lysed samples were transferred to the PCR tubes (with PCR tablets) and then loaded into the BAXÒ cycler/detector. The appropriate program was run. Each tube contains a fluorescent dye, which binds with target DNA and emits a fluorescent signal in response to light. The full BAXÒ process program, which consists of cycling and detection, takes about 5 h to complete. Evaluation of the BAXÒ system with automated detection and the BAXÒ system with gel detection was performed by calculating their sensitivity, specificity and efficiency (Capita, Alonso-Calleja, Moreno, & Garcı´a-Ferna´ndez, 2001). Values were calculated as follows: sensitivity = a/(a + c); specificity = d/(b + d) and efficiency = (a + d)/(a + b + c + d). Data a, b, c and d represent true-positive, false-positive, false-negative and true-negative values, respectively.
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have been described on which L. monocytogenes can be distinguished from other Listeria spp. (Johansson, 1998; Pinto et al., 2001; Restaino, Frampton, Irbe, Schabert, & Spitz, 1999; Vlaemynck, Lafarge, & Scotter, 2000). Its principle is based on hemolysis or on a chromogenic substrate. In the new selective agar medium, ALOA, the chromogenic compound X-glucoside is added as substrate for the detection of b-glucosidase, resulting in blue coloured Listeria colonies. The selectivity of medium is obtained by the addition of nalidixic acid, ceftazidime, polymyxin, and cycloheximide. Through the addition of a specific purified L -a-phosphatidylinositol, L. monocytogenes grow within 24 h as blue colonies surrounded by an opaque and regular halo (production of a phosphatidylinositol-specific phospholipase C). All positive samples that were detected with the reference method on Palcam medium have also been identified with ALOA agar. The picking of colonies for confirmation was much easier than on Palcam medium. The advantage of ALOA medium is the possibility of distinction between L. monocytogenes and other Listeria species. The detection of other Listeria spp. was sometimes difficult because L. monocytogenes often formed large opaque halos (Fig. 1). The results of the culture procedure very clearly demonstrate that control of L. monocytogenes in fish processing and supply systems is absolutely essential.
3. Results and discussion A total of 27 cold smoked salmon samples were screened for the presence of L. monocytogenes using both the culture-based method, and the BAXÒ system for screening of L. monocytogenes. 3.1. Culture procedure Using the cultural method, 70.4% of vacuum packaged cold smoked salmon were found to be contaminated with Listeria spp. L. monocytogenes was found in 15 (55.6%) of all samples tested, L. innocua in 11.1%, L. seeligeri, L. welshimeri and L. grayi in 7.4%. Five of the samples were contaminated by L. monocytogenes and other Listeria in combination. It is well known that the Palcam agar does not allow distinction between colonies of L. monocytogenes and other Listeria. The selection of five colonies for confirmation of L. monocytogenes from Palcam agar may not be sufficient if large numbers of other Listeria species are present in the food. For this reason, the enrichment broths were inoculated on ALOA agar. Many media
Fig. 1. Selective and differential isolation of L. monocytogenes on ALOA agar; nonpathogenic Listeria spp. appear as bluish colonies without a halo and L. monocytogenes as bluish colonies with opaque halo.
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3.2. BAXÒ system with gel detection Traditional screening methods are based on bacterial behaviour. By comparison, the BAXÒ system relies on the actual genetic information of bacteria by detecting a specific DNA fragment found only in the target organism. Following enrichment procedures, the BAXÒ system yielded results within about eight hours. All necessary reagents for this method are already packaged inside the tubes as a single tablet. The PCR results were interpreted as follows: a sample was designated positive for L. monocytogenes if two bands appeared in the lane, one at the appropriate target level (around the 400 bp) and another at the control level (200 bp). A sample was also considered positive if a target band appeared without the control band. This situation can occur when the sample contains a high level of L. monocytogenes that competes with the positive control for amplification reagents. A sample was considered negative for L. monocytogenes if no target band appeared, but a control band was present. A band at the control level indicates that there has been no PCR inhibition. The gel detection system worked correctly, but we could not clearly detect six bands (indicated by the manufacturer) in the marker lane, as only five bands were sufficiently intensive. Fig. 2 shows the results of the BAXÒ system with agarose gel detection. The BAXÒ assay gave identical results
Fig. 2. 2% agarose gel electrophoresis of PCR BAXÒ-amplified products obtained from enrichment broth of cold smoked salmon. Lanes 1, 18, 19 and 36: molecular size markers. Lanes 2 and 20: L. monocytogenes DSM 20600 as positive control. Lanes 3 and 21: negative control. Lanes 4, 6, 11, 14, 15, 16, 23, 26, 30, 32, 33 and 34: L. monocytogenes-negative samples. Lanes 5, 7, 8, 9, 10, 12, 13, 17, 22, 24, 25, 27, 28, 29, 31 and 35: L. monocytogenes-positive samples.
for 26 of 27 food samples compared to the standard method, including 15 positives. Only in one case the BAXÒ system gave a false-positive result, probably due to the amplification of DNA from nonviable cells of L. monocytogenes. With the culture-based method, only L. innocua was found in this sample. By using a pure viable culture of this strain, a negative reaction was obtained with the BAXÒ system (Fig. 2). 3.3. BAXÒ system with automated detection The BAXÒ system can be used with an automated instrument that eliminates the need for gel electrophoresis and photo-documentation. Each PCR tablet of the BAXÒ system contains a fluorescent dye, which binds with DNA and emits a fluorescent signal in response to light. After amplification, the BAXÒ system begins a detection phase where the fluorescent signal is measured. During detection, the temperature of the samples is raised to the point where the DNA strands denature, releasing the dye and lowering the signal. This change in fluorescence can be plotted against temperature to generate a melting curve, which is interpreted by the BAXÒ system software. This software contains three choices of graphical views: processed, raw or alternate views of data associated with the selected wells. For easier reviewing of smaller changes of the melting curve, the alternate data graph is the best way of reporting (Fig. 3). The graph should show one peak for the positive control (under 80 °C) and, if L. monocytogenes is present, a second peak at a different temperature (higher than 83 °C). With 24 h enrichment prior to PCR analysis, 15 samples were positive for L. monocytogenes by using the BAXÒ system. After 48 h enrichment, the BAXÒ system was able to detect 16 positive samples. Therefore, a 48 h enrichment period seems to be impor-
Fig. 3. Individual well report of the BAXÒ system—alternate data graph: positive sample with a peak at 85 °C and negative sample with a peak under 80 °C.
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tant in order to avoid false-negative results, especially when the number of contaminating bacteria is low. One of the 12 culture-negative samples for L. monocytogenes gave a positive result with the BAXÒ system. This false-positive result is probably due, either to the amplification of DNA from nonviable cells of L. monocytogenes or to the specific detection of L. monocytogenes in a high background of other Listeria species. Several authors have observed that L. innocua can overgrow L. monocytogenes if they are grown together in commonly used enrichment media (MacDonald & Sutherland, 1994; Petran & Swanson, 1993). In this study, we detected also L. innocua (30 cfu/g) in the false-positive sample with the BAXÒ system. The enrichment method using the Palcam medium and an additional centrifugation step after enrichment, are not according to the BAXÒ protocol. Our study, however, has shown that Palcam enrichment medium, which is widely used in Germany, is just as suitable for enrichment as Fraser broth. The BAXÒ positive control reaction with each sample provided direct confirmation that no PCR inhibitors were present. L. monocytogenes present in cold smoked vacuum packaged salmon could be detected by BAXÒ-PCR with target DNA prepared by both BAXÒ protocols described above. Both BAXÒ procedures took 5–6 h after enrichment. Our results indicated a specificity of 92%, a sensitivity of 100% and an efficiency of 96.4% for the BAXÒ system. FSIS determined also that the BAXÒ system was as sensitive as the current method in detecting L. monocytogenes. With BAXÒ, fewer samples were falsely screened as positive. Data showed as well that the system reduced the reporting time for negative samples by one day when compared to the current method. Our results indicate that a simple enumeration on a specific isolation medium with reliable performance, and the rapid detection of pathogenic bacteria by PCR methods (BAXÒ system), may improve control of Listeria in food and environmental samples. The chromogenic ALOA agar and the BAXÒ system provided results comparable to the standard methods. All positive samples on Palcam agar were positive on ALOA agar and differentiation of L. monocytogenes from other Listeria was significantly better and easier to interpret. The introduction of the BAXÒ system in the detection procedure would reduce the time of analysis for L. monocytogenes in food considerably.
Acknowledgement We wish to thank Jennifer Murphy for excellent technical assistance.
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