In ternational Journal of Food Microbiology, 13 ( 1991 ) 97 - 104
97
© 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50 FOOD 00401
Listeria monocytogenes in foods in Norway Liv Marit R~rvik and Magne Yndestad Department of Food Hygiene, The Norwegian College of Veterinary Medicine, Oslo, Norway (Received II June 1990; revision received 15 October 1990; accepted 14 January 1991)
Threc-hundred-and-eighty-two samples of different retail food items in Norway (imported soft cheese, raw chicken, minced meat, fermented sausages, vacuum-packed processed meat products, smoked salmon, peeled shrimps, raw minced fish) and 78 carcass samples (sheep, pig, cattle), were screened for Listeria monocytogenes. Of the 460 samples investigated, 78 were found to contain L, monocytogenes. Five of these contained > 103 cfu/g, four > 102 cfu/g, while the remainder were shown to contain /_. monocytogenes only after enrichment. L. monocytogenes was isolated most frequently from raw chicken, sporadically from soft cheese, shrimps, processed meat products and smoked salmon, and not at all from carcasses and fermented sausages. Key words: Listeria monocytogenes; Foods
Introduction Listeria monocytogenes has for many years been recognised as an ubiquitous bacterium that may cause serious illness in animals and, occasionally, also in humans. During the last decade several serious, confirmed foodborne outbreaks, and some outbreaks in which foodborne infection was suspected but not proven (Schlech et al., 1983; James et al., 1985; BiUe and Glauser, 1988), have led to growing concern regarding the role of foods in transmission of listeriosis in humans. In addition to the outbreaks, background levels of sporadic cases of listeriosis have been increasing in several countries during recent years (Aasen, 1989; McLauchlin et al., 1988; Goulet and Brohier, 1989). Some sporadic cases have also been associated with the consumption of food containing L. monocytogenes (Azadian et al., 1989; Kaczmarski and Jones, 1989), It is therefore now generally assumed that listeriosis in man is mainly a food-transmitted illness. As the infective dose and the possible role of co-factors in human listeriosis have not yet been elucidated, and as most cases are not linked to special food items, it is important to find out which foods are the most common vehicles for L. monocytogenes. Correspondence address: L.M. R~rvik, Department of Food Hygiene, The Norwegian College of Veterinary Medicine, P.O. Box 8146 Dep., 0033 Osio 1, Norway.
98 The purpose of this study was to obtain an indication of the extent to which the Norwegian consumer is exposed to L. monocvtogenes-contaminated food.
Materials and Methods
Sample collection Samples of imported, soft cheese were collected from retail stores in Oslo. About 90% of the samples were cut from large cheeses at the stores, while the remainder were unopened, pre-packed portions. Carcasses of sheep, pig and cattle from the main slaughterhouse in the area of Oslo were rubbed with three sterile cotton swabs on each of five different areas (anal excision area, inside and outside the abdomen, the back, and the top of the neck). The swabs from each carcass were placed in bottles containing 100 ml of peptone water, and transported to the laboratory within 2 h of sampling. After vigorous shaking of the bottles, 10 ml of the peptone water from each sample were used for further analysis. Ready-for-sale chicken were collected at two poultry processing plants immediately after slaughter, 75 chickens from one and 15 from the other. They represented 12 poultry farms. The carcasses were packed separately and transported to the laboratory under refrigeration. On arrival each chicken was put into a plastic bag and rinsed thoroughly in 100 ml of peptone water, a volume of 10 ml of the peptone water then being used for further analysis. The fermented sausage and minced meat samples were obtained from retail stores and butcher's shops in Oslo at various times. About 90% of the fermented sausages were sliced at the stores, the rest was vacuum-packed at the processing plants. The minced meat samples were packed at the stores. The samples of sliced, vacuum-packed processed meat products originated from eight different processing plants and represented batches produced on different days. The samples of smoked salmon, shrimps and minced fish were collected at different processing plants on the Norwegian coast and transported under refrigeration, by plane, to our laboratory, and immediately stored at 4 ° C. The smoked salmon were ready-to-eat vacuum-packed pieces. The shrimp samples consisted of peeled shrimps in brine, ready-for-sale. Minced fish samples (coalfish and cod) were obtained from blocks at the processing plants. Enrichment procedures The investigation was carried out during a period of 1.5 years in the course of which a change was made in the enrichment procedure employed at the laboratory. Consequently, two different methods were used in this survey. Method 1. Listeria Enrichment Broth (LEB, M E R C K 10985), a selective broth with potassium thiocyanate ( M E R C K 5125) added was employed for both primary and secondary enrichment. A 10-g sample was homogenized with 90 ml LEB in a stomacher, or in the case of the chicken and swab samples, 10 ml of the peptone water and 90 ml LEB were mixed well. The primary enrichment broth was
99 incubated at 37 ° C. After 24 h, 0.5 ml of the primary broth was transferred to a tube containing 10 ml of LEB and this secondary broth incubated at 37 °C for 24 h. Method 1 was used for the examination of soft cheese, minced meat, fermented sausages, 12 samples of vacuum-packed processed meat products, and 60 of the chicken samples. Method 2. A modification of the USDA enrichment procedure (Skovgaard and Morgen, 1988) was employed. This method was used for the carcasses, 60 of the chicken, 23 of the vacuum-packed processed meat samples, the smoked salmon, the shrimps, and the minced fish samples.
Isolation procedure When enrichment method 1 was used LEB was spread on Listeria Selective Agar (LSA, MERCK 10986) and blood agar (BA, 7% young cattle citrate blood) with wire loops directly after homogenisation, from the 24- and 48-h primary enrichment cultures, and from the 24-h secondary enrichment tube. The plates were incubated at 37 * C, BA for 24 h and LSA for 24-48 h. Isolation after enrichment method 2 was carried out according to the method described by Skovgaard and Morgen (1988), LSA and Modified McBride Agar (MMA, OXOID CM819) being used as selective media. In addition, for the samples of vacuum-packed processed meat products, 0.1 ml of the primary enrichment broth was spread directly on LSA before enrichment for enumeration. The selective agar plates were examined in accordance with Henry's Oblique Light System (Henry, 1933). Presumptive L monocytogenes colonies on LSA, MMA and BA were streaked secondarily on BA for purification and confirmation. Confirmation Listeria-like, bluish grey colonies, whether fl-haemolytic or not on BA (secondary cultures), were identified by the following examinations: microscopy, catalase activity, motility at 20-25°C as umbrella-like growth on 'motility agar' and ' tumbling motility' as seen under phase microscopy after cultivation on meat extract broth (laboratory's own recipe). Further identification included tests for fermentation of rhamnose, xylose, methyl a-D-mannopyranoside, and the CAMP test performed with Staphylococcus aureus and Rhodococcus equi. Several selected strains were tested for mouse pathogenicity. Two white laboratory mice each weighing about 20 g were used for each strain, 0.3 and 0.5 ml of 24-h meat extract broth cultures incubated at 37°C being inoculated intraperitonally. Mortality in the mice was then registered. All strains were serotyped with Bacto-Listeria-O-antisera Type 1 and Type 4 (DIFCO 2300-50-2 and 2301-50-1) according to the manufacturer's procedure.
Results The results are listed in Table 1. Seven of the ten cheese samples containing L.
monocytogenes were cut in the same store, two were cut in different shops, while one
100 TABLE I Isolation of Listeria monocytogenes (Lm) in foods in Norway Types of food
Number of samples
Number of samples containing/..m (%)
Serotypes
Soft cheese Carcasses Sheep Cattle Pigs Chicken Minced meat Fermented sausages Processed meat Smoked salmon Shrimps Minced fish
90
10 (11%)
1, 4. NT a
40 13 25 90 40 70 35 33 16 8
0 0 0 55 (61%) 2 (5%) 0 4 (11%) 3 (9%) 3 (18%) 1 (12%)
1, NT 1 1 1.4 1.4 1
a Not typable with Bacto-Listeria-O-antisera Type 1 and Type 4.
was an unopened prepacked portion. Serotyping of the isolated stratus showed that nine were serotype 1 and one was serotype 4. The seven isolates from the samples cut at the same store were all serotype 1. For four of the positive samples, L. monocytogenes was isolated directly from the 1 : 1 0 homogenate, which indicates that the content of L. monocytogenes was > 203 cfu//g. All the other isolates were found after enrichment. All the 55 samples of chicken yielding L. monocytogenes were collected from the same processing plant, and comprised 73% of all the samples taken at this plant, All nine flocks sampled at this plant included positive samples. The chickens from the other plant in which no 1.. monocytogenes was found, came from three flocks. L. monocytogenes serotype 1 was isolated from 13 chicken samples, 34 samples contained strains which were untypable, while from eight samples both serotype 1 and untypable strains were isolated. No significant differences in percent positive results between the two isolation techniques were found. In only one of 37 positive samples detected by method 1 was L. monocytogenes isolated directly from the peptone water, i.e. present at levels of > 10 3 cfu/ml. The two positive samples of minced meat, both containing serotype 1, were bought at different supermarkets. The four samples of vacuum-packed processed meat products containing L. monocytogenes originated from four different processing plants, with levels between 1 and 3 × 10: cfu/g. All the strains were serotype 1. The three L. monocytogenes strains isolated from smoked salmon were serotype 1 (one strain) and 4 (two strains). The three shrimp samples containing L monocytogenes were obtained from different factories. One strain was serotype 1 and two were serotype 4. The strain isolated from minced fish (coalfish) was serotype 1.
101
Discussion
Soft cheese The L monocytogenes contamination rate in imported soft cheese was relatively high. Other authors have reported from 3.6 to 10% contamination in their studies (Terplan et al., 1986; Pini and Gilbert, 1988; Breer and Schopfer, 1989). In our study, cheese contained the highest levels of L. monocytogenes, though the amounts were not high when compared with some of those found in other countries, where up to 105-106 cfu/g have been revealed (Beckers et al., 1987; Pini and Gilbert, 1988). The fact that seven of the positive samples were purchased from the same store and were all one serotype, together with the generally low levels found might indicate that contamination of the cheese occurred mostly during handling in the stores. This is supported by the fact that official import control statistics for cheese for the same period shows a contamination rate for/_, monocytogenes of only 1.5%. The Norwegian policy when cheese is found to contain L. monocytogenes demands rejection of the lot in question independent of the contamination level regarding both imported and home produced cheese. This study included imported cheese only, as a previous survey carried out by the Norwegian Control Institute for Milk and Dairy Products on Norwegian-produced cheese, failed to detect L monocytogenes in the 850 samples examined (Graendsen, 1988, personal communication). Carcasses It is known that slaughter animals may be carriers of L monocytogenes (Larsen, 1969; Kampelmacher and van Noorle Jansen, 1980), and it was therefore expected that some carcasses contaminated with L monocytogenes would be found. However, it must be borne in mind that all the carcasses examined were from only one slaughterhouse, where good hygienic standards were maintained. There might well be differences in contamination levels between abattoirs, related to differences in hygienic standards.
Chicken The 61% incidence of L monocytogenes in chicken is high, compared with findings in surveys in other countries where L. monocytogenes contamination rates of 23% (Bailey et al., 1989), 32% (McClain and Lee, 1988), 47% (Skovgaard and Morgen, 1988) and 60% (Pini and Gilbert, 1988) have been reported. The abscence of serotype 4 from the chicken samples correspond with the results from Bailey et al. (1989) with only serotype 1/2a and 1/2b isolates, while Pini and Gilbert (1988) isolated serotypes 1/2, 3a, 3b, 3c, 4a and 4b. In this study, however, 67% of the chicken isolates were untypable with the typing system used, therefore further investigations are planned. This study showed a great difference between the two processing plants as regards contamination rate. Samples from one were all negative, whereas samples from the other revealed that all the flocks represented were more or less con-
102 taminated. Though this might partly result from differences in general hygiene standards and slaughtering procedures at the two plants, it might also have been due to the small number of samples taken from the plant in which no evidence of L. rnonocvtogenes contamination was found. Despite relatively low levels found, the high incidence of L. monocvtogenes implies a considerable contamination potential. Handling and preparation of raw chicken might allow cross-contamination of other foodstuffs, as well as of kitchen or processing environment, if sufficient precautions are not taken. Furthermore, inadequately heated chicken might pose a threat with regard to listeriosis, sporadic cases having already been associated with the consumption of undercooked chicken (Kerr et al., 1988: Schwartz et al., 1988; Kaczmarski and Jones, 1989). In Norway, most poultry is distributed and sold as raw-frozen or grilled. There is some uncertainty about to what degree the amount of L. monocytogenes is decreased during freezing. Cooked poultry meat offers good growth conditions for L. monocytogenes (Glass and Doyle, 1989), and because of the risk both of inadequate heating and recontamination after cooking, a short storage time for grilled, prepacked chicken should be recommended in order to reduce the possibility of multiplication of the organism. Meat products Both the contamination incidence and the contamination levels in the minced meat samples were low, L. monocytogenes being found only after enrichment. Different authors have reported contamination rates of up to 77% in minced beef samples, with L. monocytogenes usually present at levels of < 103 c f u / g (Karches and Teufel, 1988; Breer and Schopfer, 1989; Farber et al.. 1989). Minced meat probably does not favour growth of L. monoc.vtogenes during storage (Shelef and Monte, 1988). It has a short keeping life and is usually cooked before consumption. This probably means that minced meat does not constitute a major source of L monocytogenes for the Norwegian consumer. In this survey we failed to detect L. monocytogenes in fermented sausages. An incidence of up to 20% has been reported in other countries (Breer and Schopfer, 1989: Farber et al., 1989). The fermented sausages included in this study were products of the salami type containing 4 to 5% ( w / w ) NaCI and 120 mg N a N O J k g , with a pH of about 5 and a water activity below 0.90, They were mostly produced with a starter culture. Preliminary studies in Norway have shown that L. monocytogenes is present in raw materials and in the environment of meat-processing plants (Nesbakken, 1990, personal communication). However, the processing conditions seem to be infavourable for the multiplication or even survival of the bacteria. According to Johnson et ai. (1988) L. monocytogenes will not be recovered from hard salami after processing if the initial count is less then 103 cfu/g. In contrast, Trtissel (1989) found that L. monocytogenes and L. innocua in salami survived processing even when present in only low concentrations. The detection of L. monocytogenes in vacuum-packed, processed meat products requires considerable attention. Even when initial contamination levels are low, the bacteria may still be able to multiply abundantly during a keeping period of several
103
weeks (Glass and Doyle, 1989), which is usual for these products. Precautions must be taken to avoid contamination during processing. Adequate cooking must be assured, and storage at temperatures lower than those commonly used for refrigeration ( < 4 ° C ) , as well as reduction of the recommended shelf life should be considered as measures to prevent transmission of L. monocytogenes. Smoked salmon, shrimps and minced fish The 1- monocytogenes contamination incidences of respectively 9 and 18% found in smoked salmon and shrimps are high. Several surveys on imported and domestic seafood in the U.S.A. have indicated an incidence of about 5 to 6% L. monocytogenes contamination (Kvenberg, 1988). There is some uncertainty regarding the possibilities for multiplication of the bacteria during storage in these products. The brine pickle used for peeled shrimps in Norway, however, usually has a pH of about 6, and might allow the multiplication of L. monocvtogenes. It was not surprising to find low levels of L. monocytogenes contamination in raw, minced fish. This food item is normally subjected to a heat treatment sufficient to destroy L. monocytogenes before consumption, and the detection of L. monocytogenes is therefore of lesser importance.
Conclusions According to this study, the most frequent source of L. monocytogenes in foods in Norway is raw chicken. Ready-to-eat foods such as cheese, vacuum-packed processed meat products, peeled shrimps and smoked salmon also seem to be regularly contaminated. The contamination of ready-to-eat food is of great concern, especially with regard to food items with a long storage period at refrigeration temperature. Even when initial numbers are small, as in most of the examined samples, some foods are good substrates for the growth of L. monocytogenes, and may permit considerable multiplication of the bacterium during the storage period. Because of its ubiquitous character, the sporadic occurrence of low levels of L. monocytogenes probably has to be tolerated in raw foods, even though cross-contamination and undercooking represent possible hazards to health.
Acknowledgements The authors wish to thank Turid Bergsmyr and Brit Heidenreich for technical assistance. This investigation was supported by the Norwegian Agriculture Research Council and the Norwegian Food Control Authority. References Aasen, S. (1989) Listeria-infection in Norway 1975-89. ISSN 0801-3497, 17, (46). Azadian, B.S., Finnerty, G.T. and Pearson, A.D. (1989) Cheese-borne meningitis in an immunocompetent patient. Lancet i, 322-323.
104 Bailey, J.S., Fletcher, D.L. and Cox. N.A. (1989) Recove~ and serotype distribution of Lasterta monocvtogenes from broiler chickens in the Southeastern United States. J. Food Protect. 52, 148-150. Beckers, H.J.. Soentoro, P.S.S. and Delfgou-van Asch, E.H.M. (1987) The occurrence of LJsterla monocvtogenes in soft cheeses and raw milk and its resistance to heat. Int. J. Food Microbiol. 4. 249-256. Bilte, J. and Glauser, M.P. (1988) Zur Listeriose-situation in der Schweiz. Bull. Bundesamt. Gesundheitsw. 3, 28. Breer, C. and Schopfer, K. (1989) Listerien in Nahrungsmatteln. Schweiz. reed. Wschr. 119. 306-311. Farber, J.M., Sanders, G.W. and Johnston, M.A. (1989) A survey of various foods for the presence of Listeria species. J. Food Protect. 52, 456-458. Glass, K.A. and Doyle, M.P. (1989) Fate of Listeria monocytogenes in processed meat products during refrigerated storage. Appl. Environ. Microbiol. 55, 1565-1569. Goulet, V. and Brohier, S. (1989) La listeriose en France en 1986. Path. Biol. 37, 206-211. Henry, B.S. (1933) Dissociation in the genus Brucella. J. Infect. Dis. 52, 374-402. James, S.M., Fannin, S.L., Agee, B.A., Hall, B., Parker, E., Vogt, J., Run, G., Williams, J., Lieb, L., Salminen, C., Prendergast, T., Werner, S.B. and Chin, J. (1985) Listeriosis outbreak associated with Mexican style cheese - California. Morbid. Mortal. Weekly Rep. 34, 357-359. Johnson, J.L.+ Doyle, M.P., Cassens, R.G. and Schoeni, J.L. (1988) Fate of Listerta monocytogenes in tissues of experimentally infected cattle and in hard salami. Appl. Environ. Microbiol. 54, 497-501. Kaczrnarski, E.B. and Jones, D.M. (1989) Listeriosis and ready-cooked chicken. Lancet i, 549. Kampelmacher, E.H. and van Noorle Jansen, L.M. (1980) Listeriosis in humans and animals in The Netherlands (1958-1977). Zbl. Bakt. Hyg., I. Abt. Orig. A 246, 211-227. Karches, H. and Teufel, P. (1988) La'steria monocytogenes. Vorkommen in Hackfleisch und Verhalten in frischer Zwiebelmettwurst. Fleischwirtschaft 68, 1388-1392. Kerr, K.G., Dealler, S.F. and Lacey, R.W. (1988) Materno-foetal tisteriosis from cook-chill and refrigerated food. Lancet ii, 1133. Kvenberg, J.E. (1988) Outbreaks of listeriosis/La'steria-contaminated foods. Microbiol. Sci. 5, 355-357. Larsen, H.E. (1969) Epidemiology. In: l.,arsen, H.E., Lasteria monocytogenes. Studies on Isolation Techniques and Epidemiology. PhD Thesis, University of Copenhagen, pp. 177-216. McClain, D. and Lee, W.H. (1988) Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 71,660-663. McLauchlin, J., Saunders, N.A., Ridley, A.M. and Taylor, A.G. (1988) Listeriosis and foodborne transmxssion. Lancet ii, 177-178. Pini, P.N. and Gilbert, R.J. (1988) The occurrence in the U.K. of Lasteria species in raw chickens and soft cheeses. Int. J. Food Microbiol. 6, 317-326. Schwartz, B., Broome, C.V., Brown, G.R., Hightower, A.W., Ciesielski, C.A., Gaventa, S., Gellin, B.G. and Mascola, L. (1988) Association of sporadic fisteriosis with consumption of uncooked hot dogs and undercooked chicken. Lancet ii, 779-780. Schlech, W.F., Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Wort, A.J., Hightower, A . W . , Johnson, S.E., King, S.H., Nicholls, E.S and Broome, C.V. (1983) Epidemic listeriosis - evidence for transmission by food. N. Engl. J. Med. 312, 203-206. Shelef. L.A. and Monte, L.A. (1988) Survival of Listeria monocytogenes in ground beef or liver during storage at 4 ° and 25"C. J. Food Protect. 51, 828. Skovgaard. N. and Morgen, C.A. (1988) Detection of Listeria spp. in faeces from animals, in feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 6. 229-242. Terplan. G., Schoen, R., Springmeyer, W., Degle, I. and Becker, H. (1986) Vorkommen, Verhalten und Bedeutung von Listerien in Milch und Milchprodukten. Arch. Lebensmittelhyg. 37, 129-156. Tr~ssel. M. (1989) Zum Vorkommen von Listerien bei der Produktion von Biindnerfleisch, Salami und Mettwurst. Schweiz. Arch. Tierheilk. 131,409-421.
97
© 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50 FOOD 00401
Listeria monocytogenes in foods in Norway Liv Marit R~rvik and Magne Yndestad Department of Food Hygiene, The Norwegian College of Veterinary Medicine, Oslo, Norway (Received II June 1990; revision received 15 October 1990; accepted 14 January 1991)
Threc-hundred-and-eighty-two samples of different retail food items in Norway (imported soft cheese, raw chicken, minced meat, fermented sausages, vacuum-packed processed meat products, smoked salmon, peeled shrimps, raw minced fish) and 78 carcass samples (sheep, pig, cattle), were screened for Listeria monocytogenes. Of the 460 samples investigated, 78 were found to contain L, monocytogenes. Five of these contained > 103 cfu/g, four > 102 cfu/g, while the remainder were shown to contain /_. monocytogenes only after enrichment. L. monocytogenes was isolated most frequently from raw chicken, sporadically from soft cheese, shrimps, processed meat products and smoked salmon, and not at all from carcasses and fermented sausages. Key words: Listeria monocytogenes; Foods
Introduction Listeria monocytogenes has for many years been recognised as an ubiquitous bacterium that may cause serious illness in animals and, occasionally, also in humans. During the last decade several serious, confirmed foodborne outbreaks, and some outbreaks in which foodborne infection was suspected but not proven (Schlech et al., 1983; James et al., 1985; BiUe and Glauser, 1988), have led to growing concern regarding the role of foods in transmission of listeriosis in humans. In addition to the outbreaks, background levels of sporadic cases of listeriosis have been increasing in several countries during recent years (Aasen, 1989; McLauchlin et al., 1988; Goulet and Brohier, 1989). Some sporadic cases have also been associated with the consumption of food containing L. monocytogenes (Azadian et al., 1989; Kaczmarski and Jones, 1989), It is therefore now generally assumed that listeriosis in man is mainly a food-transmitted illness. As the infective dose and the possible role of co-factors in human listeriosis have not yet been elucidated, and as most cases are not linked to special food items, it is important to find out which foods are the most common vehicles for L. monocytogenes. Correspondence address: L.M. R~rvik, Department of Food Hygiene, The Norwegian College of Veterinary Medicine, P.O. Box 8146 Dep., 0033 Osio 1, Norway.
98 The purpose of this study was to obtain an indication of the extent to which the Norwegian consumer is exposed to L. monocvtogenes-contaminated food.
Materials and Methods
Sample collection Samples of imported, soft cheese were collected from retail stores in Oslo. About 90% of the samples were cut from large cheeses at the stores, while the remainder were unopened, pre-packed portions. Carcasses of sheep, pig and cattle from the main slaughterhouse in the area of Oslo were rubbed with three sterile cotton swabs on each of five different areas (anal excision area, inside and outside the abdomen, the back, and the top of the neck). The swabs from each carcass were placed in bottles containing 100 ml of peptone water, and transported to the laboratory within 2 h of sampling. After vigorous shaking of the bottles, 10 ml of the peptone water from each sample were used for further analysis. Ready-for-sale chicken were collected at two poultry processing plants immediately after slaughter, 75 chickens from one and 15 from the other. They represented 12 poultry farms. The carcasses were packed separately and transported to the laboratory under refrigeration. On arrival each chicken was put into a plastic bag and rinsed thoroughly in 100 ml of peptone water, a volume of 10 ml of the peptone water then being used for further analysis. The fermented sausage and minced meat samples were obtained from retail stores and butcher's shops in Oslo at various times. About 90% of the fermented sausages were sliced at the stores, the rest was vacuum-packed at the processing plants. The minced meat samples were packed at the stores. The samples of sliced, vacuum-packed processed meat products originated from eight different processing plants and represented batches produced on different days. The samples of smoked salmon, shrimps and minced fish were collected at different processing plants on the Norwegian coast and transported under refrigeration, by plane, to our laboratory, and immediately stored at 4 ° C. The smoked salmon were ready-to-eat vacuum-packed pieces. The shrimp samples consisted of peeled shrimps in brine, ready-for-sale. Minced fish samples (coalfish and cod) were obtained from blocks at the processing plants. Enrichment procedures The investigation was carried out during a period of 1.5 years in the course of which a change was made in the enrichment procedure employed at the laboratory. Consequently, two different methods were used in this survey. Method 1. Listeria Enrichment Broth (LEB, M E R C K 10985), a selective broth with potassium thiocyanate ( M E R C K 5125) added was employed for both primary and secondary enrichment. A 10-g sample was homogenized with 90 ml LEB in a stomacher, or in the case of the chicken and swab samples, 10 ml of the peptone water and 90 ml LEB were mixed well. The primary enrichment broth was
99 incubated at 37 ° C. After 24 h, 0.5 ml of the primary broth was transferred to a tube containing 10 ml of LEB and this secondary broth incubated at 37 °C for 24 h. Method 1 was used for the examination of soft cheese, minced meat, fermented sausages, 12 samples of vacuum-packed processed meat products, and 60 of the chicken samples. Method 2. A modification of the USDA enrichment procedure (Skovgaard and Morgen, 1988) was employed. This method was used for the carcasses, 60 of the chicken, 23 of the vacuum-packed processed meat samples, the smoked salmon, the shrimps, and the minced fish samples.
Isolation procedure When enrichment method 1 was used LEB was spread on Listeria Selective Agar (LSA, MERCK 10986) and blood agar (BA, 7% young cattle citrate blood) with wire loops directly after homogenisation, from the 24- and 48-h primary enrichment cultures, and from the 24-h secondary enrichment tube. The plates were incubated at 37 * C, BA for 24 h and LSA for 24-48 h. Isolation after enrichment method 2 was carried out according to the method described by Skovgaard and Morgen (1988), LSA and Modified McBride Agar (MMA, OXOID CM819) being used as selective media. In addition, for the samples of vacuum-packed processed meat products, 0.1 ml of the primary enrichment broth was spread directly on LSA before enrichment for enumeration. The selective agar plates were examined in accordance with Henry's Oblique Light System (Henry, 1933). Presumptive L monocytogenes colonies on LSA, MMA and BA were streaked secondarily on BA for purification and confirmation. Confirmation Listeria-like, bluish grey colonies, whether fl-haemolytic or not on BA (secondary cultures), were identified by the following examinations: microscopy, catalase activity, motility at 20-25°C as umbrella-like growth on 'motility agar' and ' tumbling motility' as seen under phase microscopy after cultivation on meat extract broth (laboratory's own recipe). Further identification included tests for fermentation of rhamnose, xylose, methyl a-D-mannopyranoside, and the CAMP test performed with Staphylococcus aureus and Rhodococcus equi. Several selected strains were tested for mouse pathogenicity. Two white laboratory mice each weighing about 20 g were used for each strain, 0.3 and 0.5 ml of 24-h meat extract broth cultures incubated at 37°C being inoculated intraperitonally. Mortality in the mice was then registered. All strains were serotyped with Bacto-Listeria-O-antisera Type 1 and Type 4 (DIFCO 2300-50-2 and 2301-50-1) according to the manufacturer's procedure.
Results The results are listed in Table 1. Seven of the ten cheese samples containing L.
monocytogenes were cut in the same store, two were cut in different shops, while one
100 TABLE I Isolation of Listeria monocytogenes (Lm) in foods in Norway Types of food
Number of samples
Number of samples containing/..m (%)
Serotypes
Soft cheese Carcasses Sheep Cattle Pigs Chicken Minced meat Fermented sausages Processed meat Smoked salmon Shrimps Minced fish
90
10 (11%)
1, 4. NT a
40 13 25 90 40 70 35 33 16 8
0 0 0 55 (61%) 2 (5%) 0 4 (11%) 3 (9%) 3 (18%) 1 (12%)
1, NT 1 1 1.4 1.4 1
a Not typable with Bacto-Listeria-O-antisera Type 1 and Type 4.
was an unopened prepacked portion. Serotyping of the isolated stratus showed that nine were serotype 1 and one was serotype 4. The seven isolates from the samples cut at the same store were all serotype 1. For four of the positive samples, L. monocytogenes was isolated directly from the 1 : 1 0 homogenate, which indicates that the content of L. monocytogenes was > 203 cfu//g. All the other isolates were found after enrichment. All the 55 samples of chicken yielding L. monocytogenes were collected from the same processing plant, and comprised 73% of all the samples taken at this plant, All nine flocks sampled at this plant included positive samples. The chickens from the other plant in which no 1.. monocytogenes was found, came from three flocks. L. monocytogenes serotype 1 was isolated from 13 chicken samples, 34 samples contained strains which were untypable, while from eight samples both serotype 1 and untypable strains were isolated. No significant differences in percent positive results between the two isolation techniques were found. In only one of 37 positive samples detected by method 1 was L. monocytogenes isolated directly from the peptone water, i.e. present at levels of > 10 3 cfu/ml. The two positive samples of minced meat, both containing serotype 1, were bought at different supermarkets. The four samples of vacuum-packed processed meat products containing L. monocytogenes originated from four different processing plants, with levels between 1 and 3 × 10: cfu/g. All the strains were serotype 1. The three L. monocytogenes strains isolated from smoked salmon were serotype 1 (one strain) and 4 (two strains). The three shrimp samples containing L monocytogenes were obtained from different factories. One strain was serotype 1 and two were serotype 4. The strain isolated from minced fish (coalfish) was serotype 1.
101
Discussion
Soft cheese The L monocytogenes contamination rate in imported soft cheese was relatively high. Other authors have reported from 3.6 to 10% contamination in their studies (Terplan et al., 1986; Pini and Gilbert, 1988; Breer and Schopfer, 1989). In our study, cheese contained the highest levels of L. monocytogenes, though the amounts were not high when compared with some of those found in other countries, where up to 105-106 cfu/g have been revealed (Beckers et al., 1987; Pini and Gilbert, 1988). The fact that seven of the positive samples were purchased from the same store and were all one serotype, together with the generally low levels found might indicate that contamination of the cheese occurred mostly during handling in the stores. This is supported by the fact that official import control statistics for cheese for the same period shows a contamination rate for/_, monocytogenes of only 1.5%. The Norwegian policy when cheese is found to contain L. monocytogenes demands rejection of the lot in question independent of the contamination level regarding both imported and home produced cheese. This study included imported cheese only, as a previous survey carried out by the Norwegian Control Institute for Milk and Dairy Products on Norwegian-produced cheese, failed to detect L monocytogenes in the 850 samples examined (Graendsen, 1988, personal communication). Carcasses It is known that slaughter animals may be carriers of L monocytogenes (Larsen, 1969; Kampelmacher and van Noorle Jansen, 1980), and it was therefore expected that some carcasses contaminated with L monocytogenes would be found. However, it must be borne in mind that all the carcasses examined were from only one slaughterhouse, where good hygienic standards were maintained. There might well be differences in contamination levels between abattoirs, related to differences in hygienic standards.
Chicken The 61% incidence of L monocytogenes in chicken is high, compared with findings in surveys in other countries where L. monocytogenes contamination rates of 23% (Bailey et al., 1989), 32% (McClain and Lee, 1988), 47% (Skovgaard and Morgen, 1988) and 60% (Pini and Gilbert, 1988) have been reported. The abscence of serotype 4 from the chicken samples correspond with the results from Bailey et al. (1989) with only serotype 1/2a and 1/2b isolates, while Pini and Gilbert (1988) isolated serotypes 1/2, 3a, 3b, 3c, 4a and 4b. In this study, however, 67% of the chicken isolates were untypable with the typing system used, therefore further investigations are planned. This study showed a great difference between the two processing plants as regards contamination rate. Samples from one were all negative, whereas samples from the other revealed that all the flocks represented were more or less con-
102 taminated. Though this might partly result from differences in general hygiene standards and slaughtering procedures at the two plants, it might also have been due to the small number of samples taken from the plant in which no evidence of L. rnonocvtogenes contamination was found. Despite relatively low levels found, the high incidence of L. monocvtogenes implies a considerable contamination potential. Handling and preparation of raw chicken might allow cross-contamination of other foodstuffs, as well as of kitchen or processing environment, if sufficient precautions are not taken. Furthermore, inadequately heated chicken might pose a threat with regard to listeriosis, sporadic cases having already been associated with the consumption of undercooked chicken (Kerr et al., 1988: Schwartz et al., 1988; Kaczmarski and Jones, 1989). In Norway, most poultry is distributed and sold as raw-frozen or grilled. There is some uncertainty about to what degree the amount of L. monocytogenes is decreased during freezing. Cooked poultry meat offers good growth conditions for L. monocytogenes (Glass and Doyle, 1989), and because of the risk both of inadequate heating and recontamination after cooking, a short storage time for grilled, prepacked chicken should be recommended in order to reduce the possibility of multiplication of the organism. Meat products Both the contamination incidence and the contamination levels in the minced meat samples were low, L. monocytogenes being found only after enrichment. Different authors have reported contamination rates of up to 77% in minced beef samples, with L. monocytogenes usually present at levels of < 103 c f u / g (Karches and Teufel, 1988; Breer and Schopfer, 1989; Farber et al.. 1989). Minced meat probably does not favour growth of L. monoc.vtogenes during storage (Shelef and Monte, 1988). It has a short keeping life and is usually cooked before consumption. This probably means that minced meat does not constitute a major source of L monocytogenes for the Norwegian consumer. In this survey we failed to detect L. monocytogenes in fermented sausages. An incidence of up to 20% has been reported in other countries (Breer and Schopfer, 1989: Farber et al., 1989). The fermented sausages included in this study were products of the salami type containing 4 to 5% ( w / w ) NaCI and 120 mg N a N O J k g , with a pH of about 5 and a water activity below 0.90, They were mostly produced with a starter culture. Preliminary studies in Norway have shown that L. monocytogenes is present in raw materials and in the environment of meat-processing plants (Nesbakken, 1990, personal communication). However, the processing conditions seem to be infavourable for the multiplication or even survival of the bacteria. According to Johnson et ai. (1988) L. monocytogenes will not be recovered from hard salami after processing if the initial count is less then 103 cfu/g. In contrast, Trtissel (1989) found that L. monocytogenes and L. innocua in salami survived processing even when present in only low concentrations. The detection of L. monocytogenes in vacuum-packed, processed meat products requires considerable attention. Even when initial contamination levels are low, the bacteria may still be able to multiply abundantly during a keeping period of several
103
weeks (Glass and Doyle, 1989), which is usual for these products. Precautions must be taken to avoid contamination during processing. Adequate cooking must be assured, and storage at temperatures lower than those commonly used for refrigeration ( < 4 ° C ) , as well as reduction of the recommended shelf life should be considered as measures to prevent transmission of L. monocytogenes. Smoked salmon, shrimps and minced fish The 1- monocytogenes contamination incidences of respectively 9 and 18% found in smoked salmon and shrimps are high. Several surveys on imported and domestic seafood in the U.S.A. have indicated an incidence of about 5 to 6% L. monocytogenes contamination (Kvenberg, 1988). There is some uncertainty regarding the possibilities for multiplication of the bacteria during storage in these products. The brine pickle used for peeled shrimps in Norway, however, usually has a pH of about 6, and might allow the multiplication of L. monocvtogenes. It was not surprising to find low levels of L. monocytogenes contamination in raw, minced fish. This food item is normally subjected to a heat treatment sufficient to destroy L. monocytogenes before consumption, and the detection of L. monocytogenes is therefore of lesser importance.
Conclusions According to this study, the most frequent source of L. monocytogenes in foods in Norway is raw chicken. Ready-to-eat foods such as cheese, vacuum-packed processed meat products, peeled shrimps and smoked salmon also seem to be regularly contaminated. The contamination of ready-to-eat food is of great concern, especially with regard to food items with a long storage period at refrigeration temperature. Even when initial numbers are small, as in most of the examined samples, some foods are good substrates for the growth of L. monocytogenes, and may permit considerable multiplication of the bacterium during the storage period. Because of its ubiquitous character, the sporadic occurrence of low levels of L. monocytogenes probably has to be tolerated in raw foods, even though cross-contamination and undercooking represent possible hazards to health.
Acknowledgements The authors wish to thank Turid Bergsmyr and Brit Heidenreich for technical assistance. This investigation was supported by the Norwegian Agriculture Research Council and the Norwegian Food Control Authority. References Aasen, S. (1989) Listeria-infection in Norway 1975-89. ISSN 0801-3497, 17, (46). Azadian, B.S., Finnerty, G.T. and Pearson, A.D. (1989) Cheese-borne meningitis in an immunocompetent patient. Lancet i, 322-323.
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