Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood

International Journal of Food Microbiology 42 (1998) 127–131

Short Communication

Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood Lasse Vigel Jørgensen*, Hans Henrik Huss Danish Institute for Fisheries Research, Department of Seafood Research, Technical University of Denmark, Building 221, DK-2800 Lyngby, Denmark Received 17 September 1997; received in revised form 2 February 1998; accepted 21 April 1998

Abstract Listeria monocytogenes contamination of seafood varies with product category. The highest prevalence was found in cold-smoked fish (34–60%), while the lowest was found in heat-treated and cured seafood (4–12%). The prevalence of L. monocytogenes differed greatly in cold-smoked salmon between production sites, ranging from , 1.4% (nil out of 70 samples) to 100%. The prevalence at the individual production sites was reproducible at repeated sampling. The results indicate that it is possible to produce cold-smoked salmon with a low prevalence of L. monocytogenes. The organism showed moderate growth in naturally contaminated cold-smoked, and ‘gravad’, fish while the growth appeared faster in hot smoked fish. Thus L. monocytogenes is not under control in these products. Finally, the prevalence and growth of L. monocytogenes in naturally contaminated cold-smoked salmon are discussed in relation to controlling this risk.  1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Cold-smoked salmon; Detection; Growth; Incidence; Level of contamination; Listeria monocytogenes; Prevalence; Seafood

1. Introduction Ready-to-eat fish products are often contaminated with L. monocytogenes, as reviewed by Ben Embarek (1994). However, published results on the levels and growth of L. monocytogenes in naturally contaminated products such as cold-smoked salmon

*Corresponding author. Tel.: 1 45 4588 3322; fax: 1 45 4588 4774; e-mail: [email protected]

are scarce, and either show no growth or growth in only a few samples (Guyer and Jemmi, 1990; Cortesi et al., 1997). In some countries, food agencies have established a zero-tolerance in ready-to-eat food products such as ‘gravad’ or cold-smoked fish, whereas other countries have established an upper limit of 100 L. monocytogenes per gram. The objective of this study was to determine the prevalence and growth of L. monocytogenes in ready-to-eat seafood from Danish production sites. In addition, the prevalence of L. monocytogenes in cold-smoked

0168-1605 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 98 )00071-3

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salmon from individual production sites was investigated.

3. Results and discussion

3.1. Prevalence and growth in cold-smoked and ‘ gravad’ fish 2. Materials and methods

2.1. Fish samples and production sites Fish samples in retail packs were collected from the production sites. Each lot, in most cases consisting of 10 samples, was collected from the production site and kept below 58C until arrival at the laboratory. From each lot five samples were analysed 4–12 days from the production date; this corresponds to the initial analysis. The remaining five samples were stored (5618C) until the end of the commercial shelf-life stated by the processors; this corresponds to the final analysis. After 1–15 months additional lots were collected from selected sites producing cold-smoked salmon with a particularly low or high prevalence of L. monocytogenes.

2.2. Bacteriological analysis Listeria monocytogenes was determined in seafood by three different methods. (i) Qualitative determination in 25 g with a two-step enrichment (308C, 24 h) in modified University of Vermont broth (CM863, SR142, SR143, Oxoid, UK) followed by plating on Oxford and Palcam selective agars (378C, 48 h) (CM856, CM877, SR140, SR150E, Oxoid, UK). (ii) Semi-quantitative determination with 0.1, 0.01 and 0.001 g of seafood using the enrichment procedure described above (i). This gave values of , 10, 10–100, 100–1000 and . 1000 L. monocytogenes per gram. (iii) Direct enumeration by spread plating on Oxford and Palcam agar (see above). The seafood was homogenised in physiological saline (0.85% NaCl) with 0.1% peptone in a Stomacher. Presumptive Listeria colonies (5–10 per plate) from Oxford and Palcam agars were identified as L. monocytogenes by b-hemolysis, positive Gram and catalase tests, motility by microscopy, and the ability to ferment rhamnose and methyl-mannoside but not xylose (Seeliger and Jones, 1986). In addition, isolates were verified as L. monocytogenes using the Accuprobe TM L. monocytogenes Culture Identification test (Gen-Probe Inc., San Diego, USA).

The prevalence of L. monocytogenes in coldsmoked salmon and halibut was 34–43% and 45– 60%, respectively (Table 1). This is somewhat higher than most published surveys on cold-smoked fish where , 1 to 31% was observed, except for Hudson et al. (1992), who found a prevalence of 75% (Ben Embarek, 1994; Dillon et al., 1994; Jin et al., 1994; McLauchlin and Nichols, 1994; Garland, 1995; Loncarevic et al., 1996). Some growth of L. monocytogenes takes place in cold-smoked fish during storage at 58C. Thus more positive samples were found in the second examination (end of shelflife), and the general level of contamination was higher, as shown by the increase in samples containing . 100 L. monocytogenes per gram, from 0 to 20% in cold-smoked halibut and from 1 to 8% in cold-smoked salmon. On the other hand, no growth was observed in the majority of samples. These results are in agreement with those of Cortesi et al. (1997), who found growth of L. monocytogenes in some samples of cold-smoked salmon while most samples showed no growth when stored at 28C and 108C for 60 and 40 days, respectively. Guyer and Jemmi (1990) observed no growth of L. monocytogenes in naturally contaminated cold-smoked salmon during 10 days of storage at 48C. L. monocytogenes grows slowly in naturally contaminated cold-smoked fish and in fact much slower than demonstrated in the challenge test and as determined by several predictive models (Dalgaard and Jørgensen, 1998). A wide variation in the prevalence of L. monocytogenes in cold-smoked salmon was observed at the different production sites. Some sites had a prevalence of , 1.4% while others had close to 100% in their products (Table 2). Most interestingly, the prevalence of L. monocytogenes in cold-smoked salmon from different sites remained unchanged when additional lots where sampled 1–15 months after the initial sampling. Thus production sites A, B, C, D and E repeatedly produced cold-smoked salmon with a low prevalence of L. monocytogenes, whereas sites I and J produced products with a prevalence close to 100% (Table 2). The results indicate that it is possible to produce cold-smoked salmon with a low prevalence of L. monocytogenes.

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Table 1 Contamination level of L. monocytogenes in naturally contaminated ready-to-eat fish products stored at 58C Product

Storage time between initial and final analyses days at 5618C

Cold-smoked salmon Initial Final Final Cold-smoked halibut Initial Final Gravad fish Initial Final Heat-treated seafood b Initial Final Cured seafood c

No. of positive samples in 25 g (%)

, 10 / g a

10–100 / g

100–1000 / g

. 1000 / g

14–20 21–50

64(34) 46(40) 32(43)

53(28) 11(10) 17(23)

9(5) 23(20) 11(15)

2(1) 10(9) 2(3)

0 2(2) 2(3)

190 115 75

14–28

9(45) 12(60)

9(45) 6(30)

0 2(10)

0 3(15)

0 1(5)

20 20

12–28

28(33) 23(25)

15(18) 5(5)

11(13) 10(11)

2(2) 5(5)

0 3(3)

85 91

4(5) 6(12) 8(4)

4(5) 2(4) 8(4)

0 3(6) 0

0 1(2) 0

74 50 191

16–23

Level of contamination, No. of positive samples (%)

0 0 0

Total No. of samples

a

Most probable number per gram. ´ and fish cake. Heat-treated seafood includes hot-smoked fish, fish patees c Cured seafood includes lightly preserved products such as brined shrimps and surimi, oil marinated shrimps, caviar and marinated herring, the latter being a semi-preserved product. b

Table 2 Prevalence of L. monocytogenes in cold-smoked salmon found at repeated sampling from 10 production sites Production site

A B C D E F G H I J

No. of lots

7 2 2 4 3 2 3 2 5 4

a

No. of positive samples in lots Initial (%6SD)

Final (%6SD)

0 0 0 1(5610) 1(7612) 3(30614) 6(40620) 5(50614) 21(84622) 10(50625)

0 0 0 0 1(7612) 1(1067) 7(47623) 7(70642) 25(10060) 20(10060)

a

Number of lots produced with intervals of 1–15 months, each lot consisting of 10 individual samples.

The prevalence of L. monocytogenes in ‘gravad’ salmon and halibut was 25–33% (Table 1), which is similar to the results obtained from other countries where 21–27% was observed in ‘gravad’ salmon, ocean perch and rainbow trout (Hartemink and Georgsson, 1991; Jemmi, 1990; Loncarevic et al., 1996). The prevalence ranged from , 10% in products from five production sites to 90% from two sites (results not shown). When stored at 58C L. monocytogenes was able to grow in ‘gravad’ salmon and

halibut. Thus the contamination shifted towards a higher level at the end of the shelf-life; this is seen in the increase of samples having . 100 L. monocytogenes per gram from 2 to 8% at the end of the shelf-life (Table 1).

3.2. Prevalence and growth in heat-treated and cured seafood In heat-treated seafood, L. monocytogenes was present in 5–12% of the samples (Table 1). The positive samples originated from two production lots of vacuum-packed hot-smoked mackerel and trout packed together with ‘gravad’, and cold-smoked fish. The shelf-life indicated by the processors was 28 and 18 days at 58C, respectively. At the end of the shelf-life L. monocytogenes reached a level of 100– 1000 L. monocytogenes per gram and in one sample contained . 1000 L. monocytogenes per gram. The conditions for growth of L. monocytogenes in hotsmoked fish appear to be better as compared to cold-smoked fish. Consequently, when ‘gravad’, cold-smoked and hot-smoked fish are marketed in the same pack the shelf-life must be determined on the basis of the more rapid growth in the peat-treated product. The difference in growth of L. monocytogenes in these naturally contaminated products may

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result from the elimination of the associate microflora in the heat-treated samples. The prevalence of L. monocytogenes was 4% in cured seafood (brined shrimps and surimi, caviar and marinated herring) but no growth was observed during storage at 58C, indicating that these products are of low-risk with respect to L. monocytogenes (Table 1). Growth is most likely inhibited by the preserving parameters: salt ( $ 3.5% in water phase) reduced pH ( # 5.5) and often up to 2000 ppm sorbates or benzoates.

1998) and with lactate (Pelroy et al., 1994). Although the complete absence of this organism will be difficult if not impossible to obtain, the contamination should be reduced to an absolute minimum. To do this successfully there is a need for research to increase our knowledge of the contamination route of L. monocytogenes in salmon slaughterhouses and in cold-smoked salmon processing sites (Jemmi and Keusch, 1994; Rørvik et al., 1995).

Acknowledgements

3.3. Detection of L. monocytogenes in ready-to-eat seafood The contamination level was found to be higher in naturally contaminated seafood using the semi-quantitative enrichment procedure as compared to direct enumeration. This was the case for 53 of 225 (Oxford) and 70 of 232 (Palcam) samples (results not shown). Other researchers have reported similar differences between these methods (Hayes et al., 1992; Pinner et al., 1992).

3.4. Control of L. monocytogenes in cold-smoked salmon The control of L. monocytogenes has been the subject of much discussion. The minimal infection dose is not known, but it is generally accepted in many countries and endorsed by the International Commission on Microbiological Specifications that foods containing , 100 L. monocytogenes per gram are safe (ICMSF, 1994). This is supported by studies on the epidemiology of listeriosis suggesting that low levels of L. monocytogenes in foods are too frequent to be responsible for listeriosis even in susceptible humans, whereas exposure to higher doses ( $ 10 3 CFU g 21 ) of L. monocytogenes might result in foodborne listeriosis (Pinner et al., 1992; Hitchins, 1996; McLauchlin, 1996). Our results show that ready-to-eat fish products are commonly contaminated with L. monocytogenes and that growth occurs during normal storage conditions (no temperature abuse). Additional measures are needed to control the growth of L. monocytogenes in these products and promising results have been obtained with a combination of nisin and modified atmosphere packaging (Nilsson et al.,

The Danish Ministry of Food, Agriculture and Fisheries funded this work. The authors thank L. Korsholm Tokkesdal, M. Juhl Riisager and J. Hasløv for their technical assistance and Thyra Bjergskov for making it possible to collect the samples.

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