• Decrease font size
  • Return font size to normal
  • Increase font size
U.S. Department of Health and Human Services

Food

  • Print
  • Share
  • E-mail

Processing Parameters Needed to Control Pathogens in Cold Smoked Fish Chapter II. Potential Hazards in Cold-Smoked Fish: Listeria monocytogenes

(Table of Contents)

 

Scope

This section outlines what is currently known about the prevalence and behavior of Listeria monocytogenes during the cold-smoking process, including its survival in the processing environment and its presence and growth in the final product. Control methods in both the final product and the environment are discussed. While the section provides some of the data relevant for a science-based risk assessment, it does not give complete information on all four components: hazard identification, hazard characterization, exposure assessment, and risk characterization. Recent texts by Farber and others (1996), Farber and Peterkin (2000), Buchanan and others (1997), and Notermans and others (1998) cover these issues. Finally, based on the scientific data, approaches to minimize the presence and growth of L. monocytogenes in cold-smoked fish are considered. Lists of conclusions and research areas that need future attention are also included.

1. Introduction 

Listeria monocytogenes is a gram-positive, foodborne pathogen. It is widely distributed in the environment and occurs naturally in many raw foods. Listeria monocytogenes is psychrotrophic and halotolerant (Seeliger and Jones 1986) and can, under otherwise optimal conditions, grow in the range of 1 to 45 °C (34 to 113 °F) and between 0 and 10% NaCl. As a consequence it may grow in many food products with extended shelf lives (Barakat and Harris 1999; Rørvik and others 1991). Products that do not receive a heat treatment by the consumer, including ready-to-eat (RTE) products such as cheeses, meat, and fish delicatessen products, may contain high levels of L. monocytogenes when eaten, and many of these types of foods have been associated with listeriosis (McLauchlin 1997). In general, populations in foods are low (0-103 cfu / g with 90-99% being below 102 cfu / g and less than 1% being between 103 and 104 cfu / g) (Teufel and Benzulla 1993; Jørgensen and Huss 1998; Farber and Peterkin 2000); however, higher concentrations (105 - 107 cfu/g) have been reported (Farber and Peterkin 1991; Teufel and Bendzulla 1993).

 

Listeria monocytogenes may be divided into 13 different serotypes, all of which may cause listeriosis. The vast majority of cases, however, is caused by serotypes 1/2a, 1/2b and 4b (Farber and Peterkin 2000). The ingestion of high numbers of L. monocytogenes is a significant threat to health for people in risk groups such as immunocompromised, elderly, fetuses, and newborn babies. In these groups, the mortality from listeriosis is high, typically 20-30% (McLauchlin 1997). Because L. monocytogenes can readily be isolated from smoked fish and because inoculation trials have demonstrated significant growth in such products, the risk of getting listeriosis from consuming these types of products must be taken seriously.

Cold-smoked fish is an RTE product and such products have been linked to sporadic cases of listeriosis. Epidemiological evidence suggests that listeriosis has been associated with smoked mussels (Brett and others 1998), "gravad" trout (Ericsson and others 1997), and smoked trout (Miettinen, Siitonen and others 1999). In the latter case (Miettinen, Siitonen and others 1999), the outbreak was not the classical invasive listeriosis, but cold-smoked trout was associated with febrile gastroenteritis in five healthy people. There are, to date, no reports in the literature linking listeriosis cases/outbreaks in the United States to cold-smoked fish. Although cold-smoked fish, as discussed below, potentially is a high risk product with respect to listeriosis, case-control studies have linked listeriosis to a number of other products such as uncooked hotdogs, undercooked chicken, paté and cold cuts, and cheeses (Elliot and Kvenberg 2000; Rocourt and others 2000).

The virulence of L. monocytogenes varies with the strain. Although no single test can predict the pathogenicity of a strain, a combination of tests is used to indicate virulence potential. These tests determine presence of genes coding for listeriolysin and actin polymerizing protein, adherence to CaCo2 cells, plaque formation on mouse L-cells, and infection of chick embryos. Wiedmann and others (1997) suggested that L. monocytogenes should be subdivided into three different lineages, of which mainly lineage I contains virulent strains. In a recent study Norton, Timothe and others (2000) found that the majority (63.2%) of L. monocytogenes isolated from cold-smoked salmon belonged to lineage II, whereas clinical isolates and 30% of fish isolates belonged to lineage I. These findings suggest that the pathogenicity potential of isolates from cold-smoked fish may be lower than predicted from its prevalence.

2. Prevalence in water, raw fish, and smoked fish 

Listeria monocytogenes is a ubiquitous organism (Farber and Peterkin 2000). Although its natural niche is probably soil and vegetation, it can readily be isolated from fresh and marine waters. Ben Embarek (1994) reviewed a number of studies and found that the prevalence in a variety of water bodies (river waters, seawater, surface water, spring water) varied from 0 to 62%. Notably, the highest number of positive samples was found in waters exposed to runoff from agricultural or urban areas, whereas waters such as spring water or free ocean waters were negative for the organism (Table 1). Fenlon and others (1996) investigated river waters in the United Kingdom and found 17 of 36 samples to be positive for L. monocytogenes, with levels in positive samples ranging from 10 to 350 cfu / liter.

 

Table II-1. Prevalence of Listeria spp. in freshwater, seawater and sediments (modified from Ben Embarek 1994; Jemmi and Keusch 1994; Huss and others 1995). nd = not determined

Sampling location

No. of samples

% positive for

Listeria spp.

L. monocytogenes

Freshwater   
 river, domestic animals (USA)

37

81

62

 not specified (UK)

7

100

nd

 river, populated (UK)

36

nd

47

 canals, lakes (Holland)

180

nd

37

 ground water (Switzerland)

12

0

0

 spring water (Switzerland)

12

0

0

Seawater   
 coastal area (USA)

3

33

33

 shellfish growing area (USA)

70

3

nd

 not specified (Holland)

43

0

0

 used for salmon transport (Norway)

21

52

14

 around salmon farm (Norway)

8

0

0

Sediments   
 freshwater (USA)

46

30

17

 freshwater (USA)

15

20

0

 

Listeria monocytogenes is commonly present in certain waters, as demonstrated by the following research findings. Farber (1991) reported the presence of L. monocytogenes in salmon from the United States, Chile, Norway, and Canada. The prevalence of L. monocytogenes in raw fish, as reported in published studies, is low, ranging from 0-1% to 10% (Johansson and others 1999; Autio and others 1999; Jemmi and Keusch 1994; Jinneman and others 1999; Weagant and others 1988). Only 1.3% of 781 samples of Japanese fish contained L. monocytogenes (Iida and others 1998), and none of 60 raw salmon sampled in Japan was positive for the bacterium (Jin and others 1994). The literature reviewed by Ben Embarek (1994) indicated large variations, with 0 to 50% of fresh fish samples positive. Unpublished information indicates that the prevalence in fish in mud freshwater ponds or fish in seawater nets close to land runoff can be as high as 100%. Based on published data, the frequency appears to be low; however, surveys are needed to evaluate the prevalence and levels on raw, live fish from different sources. To the panel's knowledge, quantitative data are not available on live, fresh fish. In areas where the prevalence is low, it must be assumed that levels are very low (<10 L. monocytogenes / g). It could be speculated that fish reared in waters close to agricultural runoff would carry a higher load of L. monocytogenes than fish cultured in waters free from such soil and vegetation sources. As shown in Table II-2, this can be neither confirmed nor excluded based on current data.

 

Table II-2. Prevalence of Listeria spp. and Listeria monocytogenes in live or newly slaughtered fish (modified from Farber 1991; Ben Embarek 1994; Jemmi and Keusch 1994; Eklund and others 1995; Fonnesbech Vogel, Ojeniyi, Ahrens and others 2001). nd = not determined

Sampling location

No. of samples

% positive for

Listeria spp.

L. monocytogenes

Freshwater   
 skin of live trout (Switzerland)453311
 channel catfish (USA)4100nd
 slaughtered trout (Switzerland)272215
Seawater   
 salmon, at harvest (Norway)1000
 salmon, at processing plant (Norway)1800
 salmon (Faroe islands)18nd1
 frozen salmon (received at plant) (USA)65nd34
 salmon (USA, Chile, Norway, Canada, Scotland)32nd10

After cold smoking, there is an increase in the percentage of samples containing L. monocytogenes. The variation is high, ranging from 0 to 100% (Ben Embarek 1994; Eklund and others 1995; Dillon and others 1994; Heinitz and Johnson 1998; Jørgensen and Huss 1998; Fonnesbech Vogel, Ojeniyi, Ahrens and others 2001). Given this vast range, average values are meaningless, but typical prevalence is between 15 and 40%. While Eklund and others (1995) found positive samples from all of 6 cold-smoked salmon plants sampled, other authors have seen a large plant-to-plant variation. The prevalence in products such as cold-smoked salmon seems to be similar in several countries. Thus, in Danish samples from 10 smoke houses, the number of positive samples of finished product varied from 0 to 100%, whereas a survey of 6 United States smoke houses showed L. monocytogenes in the product in 50 to 100% of the samples (Eklund and others 1995). Heinitz and Johnson (1998) reported that an average of 17.5% of cold-smoked fish (291 samples) and 8.1% of hot-smoked fish (234 samples) from the United States contained L. monocytogenes, and 7.3% of 96 cold-smoked fish samples from 5 United States smoke houses were positive (Norton, McCamey and others 2000). Similar levels have been reported from Switzerland (Jemmi 1990), where 12.2% were positive; from Norway, where 9% were positive (Rørvik and Yndestad 1991); and from a range of other studies (Ben Embarek 1994). Jin and others (1994) did not detect the bacterium in raw salmon; however, they found 16% of smoked salmon positive for L. monocytogenes (see Table II-3 for a selection of data).

 

Table II-3. Prevalence of Listeria spp. and Listeria monocytogenes in cold-smoked fish (modified from Ben Embarek 1994; Nilsson 1999; Norton, McCamey and others 2000). nd = not determined

Sampling location

No. of samples

% positive for

Listeria spp.

L. monocytogenes

Salmon   
 Norway33Nd9
 Norway408033
 Norway651111
 Italy630-1000-29
 Italy165Nd19
 Switzerland100Nd24
 Switzerland64Nd6
 New Zealand12Nd75
 UK2214nd
 USA61Nd79
 Iceland13230
 Canada, USA, Chile, Scotland, Norway32Nd31
 Japan (origin ?)763016
 Denmark188Nd34
Trout   
 Switzerland4942
Species not specified   
 USA291Nd18
 USA96Nd7
 Switzerland324Nd14

Jørgensen and Huss (1998) reported that products from some smoke houses repeatedly were free from the organism while samples from other smoke houses always were positive. More recently (Fonnesbech Vogel, Jorgensen and others 2001; Fonnesbech Vogel, Ojeniyi, Ahrens and others0 2001), the bacterium could be detected from plants and products that were negative in the former study by Jørgensen and Huss (1998), indicating that although eliminated during some periods, the bacterium can be re-introduced in processing plants and, therefore, in the final product.

Although the prevalence of L. monocytogenes in cold-smoked fish is often high, numbers of microorganisms are typically low. For instance, Eklund and others (1995) found that although as many as 48 out of 61 samples of U.S. cold-smoked salmon contained L. monocytogenes, numbers of the microorganism ranged from 0.3 to 34.3 cells / g with a mean of 6.2 cells / g. Similarly low levels were found in Danish cold-smoked salmon, where 34 of 64 samples were positive, with 28 of them containing fewer than 10 cells / g, 5 samples containing between 10 and 100 cells / g, and one sample containing between 100 and 1,000 cells / g (Jørgensen and Huss 1998). In a survey of smoked fish (not specified hot- or cold-smoked) on the German market, 27 samples (of 380) were positive for L. mMonocytogenes, with 5 samples containing fewer than 1 cell / g, 14 samples containing between 1 and 100 cells / g, and 4 samples containing between 100 and 104 cells / g. In four samples, levels exceeding 104 cells / g were found (Teufel and Benzulla 1993; Notermans and others 1998). The levels of L. monocytogenes found in cold-smoked fish are not different from those in other RTE food products (Table II-4).

 

Table II-4. Concentration of Listeria monocytogenes in ready-to-eat food products on the German market (based on testing of 7,063 samples and modified from Teufel and Benzulla [1993]; Notermans and others [1998])

Type of ready-to-eat product

 

% positive

% samples (of positives) containing different levels of L. monocytogenes in cfu/g

0.04-11 - 102102 - 104> 104
Meat products23.1593461
Fish products9.4642366
Cheese3.15823163
Salads5.2603740

3. Effect of various processing steps 

This section describes the parameters and steps of the fish-smoking process that may influence the survival and growth of L. monocytogenes. It has been suggested that rinsing of the thawed and brined fish is important to reduce numbers of L. monocytogenes; however, no data on the effect of this procedure were found in the scientific literature, and consequently the effect of such steps cannot be evaluated. One U.S. processor reported that the raw material (frozen, thawed fish) are "treated with a pH control at 12.5," which eliminates the bacterium. This processor further treats the finished product with chlorine dioxide prior to packing. These two steps, combined with chlorine-dioxide disinfection of the plant, enables the processor to produce cold-smoked salmon free of L. monocytogenes. Bremer and Osborne (1998) reported that rinsing raw fish (salmon) in chlorine solution with 200 ppm free chlorine caused a significant reduction of surface-inoculated L. monocytogenes; however, this treatment could not ensure listeria-free raw materials. The procedure was not evaluated on naturally contaminated salmon, so it is not known if this treatment would be more or less efficient on a naturally present population.

3.1. Freezing 

A freezing step often is included during the smoking process. Either the raw fish or the smoked product is frozen. El-Kest and others (1991) found that freezing (at -18 °C, -0.4 °F) L. monocytogenes in buffer caused a one log reduction, whereas freezing in nutrient broth resulted in a 50% reduction. It is well known that lipids and dry matter protect bacteria against freezing damage. It must be assumed that fish, particularly fatty fish, offers a good protection and that reduction of numbers of L. monocytogenes due to freezing is marginal. Preliminary work at the Danish Institute for Fisheries Research found that growth of Listeria monocytogenes in cold-smoked salmon was not affected by an initial freezing period. It is, however, not known if prolonged frozen storage, either of the raw fish or packed product, can affect subsequent growth at chill temperature of L. monocytogenes in the smoked-fish product.

3.2. Salting/drying 

Salting of fish before smoking is done either by brine-injection, bath brining, or dry salting. As L. monocytogenes is a halotolerant bacterium, salting is not likely to reduce the number of microorganisms. On the contrary, several studies have isolated the organism from brine (Eklund and others 1995), needles used for brine injection (Fonnesbech Vogel 2000; personal communication; unreferenced), and in fish flesh that had been injected with contaminated brine (Eklund and others 1995). Autio and others (1999) found that the brining step caused a major increase in contamination of L. monocytogenes during cold-smoked trout processing. Salt levels (salt in water phase) in the final product range from 3% to in a few cases as high as 12%, although salt levels typically range from 3.5% - 5% (Truelstrup Hansen and others 1998; Jørgensen and others 2000;). This level of salt (3.5% - 5%) has no inhibitory effect on the bacterium (Peterson and others 1993). At levels above 6% NaCl and with a low initial inoculum, growth is prevented at 5 °C (Peterson and others 1993); however, this level of salt is generally too high for consumer preferences. The level of salt may also affect the growth of an accompanying lactic acid bacterial flora, and high levels (>5.5%) can significantly delay growth of the lactics, thereby reducing their potential inhibitory effect against L. monocytogenes (Nilsson 2001; personal communication; unreferenced).

3.3. Smoking process 

After salting, the fish is often dried and smoked. The hot-smoking process (usually 60 °C for 30 min) is sufficient to kill L. monocytogenes (Jemmi and Keusch 1992; Ben Embarek and Huss 1993). Eklund and others (1995) found that cells of L. monocytogenes inoculated into the fish flesh could grow during a cold-smoking process (20 - 30 °C [68 - 90 °F]). If cells of L. monocytogenes were inoculated on the surface of the fish, cold-smoking at 22 - 30 °C (72 - 90 °F) for 18 h caused a 2 log reduction in numbers (Eklund and others 1995). Smoke must be applied to the product before the surface dries, otherwise L. monocytogenes will be embedded under the pellicle where the effect of smoke is markedly reduced (Eklund 2001, personal communication; unreferenced). Consistent with the results by Eklund and others (1995), Autio and others (1999) did not find a significant increase in the number of positive samples during the cold-smoking step of trout. In cold smoking of salmon, no positive samples were detected immediately following the smoking procedure of 16 h at 22 °C (Fonnesbech Vogel, Ojeniyi, Ahrens and others 20010). In a Norwegian study, L. monocytogenes was detected in salmon samples before filleting and after salting but not immediately after cold smoking (Rørvik and others 1995). In a subsequent study, Rørvik (2000) reported that 54% of 200 samples of salmon were positive for L. monocytogenes just before smoking, whereas only 9.5% were positive after smoking. Of 11 samples that contained between 10 and 300 L. monocytogene / g before smoking, none was above 10 cfu / g after smoking. In conclusion, the studies indicate that short term cold-smoking (<24 h, as recommended by the Association of Food and Drug Officials [AFDO] guidelines, AFDO 1991) reduces rather than increases numbers of L. monocytogenes.

Some processors use a drying procedure combined with addition of liquid smoke instead of the more traditional smoke-generated process. The potential inhibitory properties of liquid smoke will vary depending on type of wood, method of preparation, and target organism. In a study of 7 commercial smoke preparations (Suñen 1998), large variations in minimum inhibitory concentration (MIC) values against a range of microorganisms were seen. Listeria monocytogenes was inhibited by two preparations (one liquid and one solid), both high in aldehydes, phenols, furan, and pyran derivatives and acids, whereas other preparations had no effect even at high concentrations (>8%). In conclusion, any antilisterial effect of liquid smoke will depend on the particular product in use.

4. Growth in refrigerated smoked fish 

Innumerable studies have documented that cold-smoked fish is an excellent substrate for L. monocytogenes. When inoculated, the organism will grow to high numbers even when stored at 5 °C (41 °F) under vacuum (Farber 1991; Rørvik and others 1991; Jemmi and Keusch 1992; Hudson and Mott 1993; Jin and others 1994; Nilsson and others 1997). Some variation in the growth rates was reported. For example, some studies have found that levels may increase from 103 to 105 logs in a few weeks (Farber 1991; Jin and others 1994; Nilsson and others 1997, 1999), while in other studies a similar increase takes several weeks (Rørvik and others 1991; Hudson and Mott 1993). Variations in strains and preculture conditions (that is, with or without adaptation to the saline, cold environment) may explain some of these differences.

One should be cautious, though, about drawing definite conclusions from inoculated studies which significantly overestimate both real growth rates and maximum cell numbers. Growth in naturally contaminated samples of cold-smoked fish does not follow the predictions from the inoculation studies (Dalgaard and Jørgensen 1998). Jørgensen and Huss (1998) reported that initial numbers of L. monocytogenes in cold-smoked salmon were <10 cells / g (53 out of 64 positive) and only 2 samples (of 32 positive) contained between 103 and 104 cfu / g after 3-7 wk of storage. Cortesi and others (1997) did not find growth of L. monocytogenes in naturally contaminated cold-smoked salmon stored up to 5 wk at 2 or 10 °C (36 or 50 °F). In 380 samples of smoked fish, 27 were positive for L. monocytogenes (Teufel and Bendzulla 1993). Of these, 4 samples contained numbers between 100 and 10,000, and 4 samples contained levels above 10,000 cells of L. monocytogenes / g.

Current predicting models for growth of L. monocytogenes are based on combinations of salt, temperature, and atmosphere. The appropriateness of these models for cold-smoked fish is questionable, because other parameters may influence the behavior of the organism, that is, structure, smoking, drying, and associated microflora. Also, the physiological state of cells naturally contaminating the product (having been exposed to drying, freezing, cleaning, and sanitizing agents) is likely to be different than that of cells used for inoculation experiments. A combination of these factors may explain the discrepancy seen between levels predicted by models and actual levels reached in fish products.

Following the growth of L. monocytogenes in 10 different batches of cold-smoked salmon, both maximum numbers and average growth rate were much lower than predicted by Food MicroModel (UK) based on the water phase salt (WPS), pH, and atmosphere (Table II-5). As outlined below, several factors may explain this lack of growth, one being the competitive action of the natural lactic acid bacterial flora. Rørvik and others (1991) also noted that even inoculated strains grew slowly if salmon with a high background flora was used for the study.

Keeping initial numbers low is important in order to limit numbers at, for example, time of spoilage. Thus, Rørvik (2000) reports that concentrations of L. monocytogenes in stored product (3 wk at 5 °C, 41 °F) were below 100 cfu / g if levels on the freshly produced product were less than 100 / g. In contrast, if levels in the freshly produced product were 300 - 400 cfu / g, L. monocytogenes grew to 3 x 104 cfu / g during the same storage period.

Table II-5. Growth of Listeria monocytogenes in naturally contaminated cold-smoked salmon stored at 5 °C (modified from Dalgaard and Jørgensen 1998). Predictions based on Food MicroModel using lot characteristics. WPS = water phase salt; WPL = water phase lactate
Product characteristics

L.m. growth during storage log(cfu/g)

Average growth rate log(cfu/g)/week

Lot

storage time

(days)

WPS (%)WPL (g/l)pHInitial TVC log(cfu/g)Initial L.m. (log(MPN/g)observedhighest number in single packs1predictedobservedpredicted
A85.06.76.23.60.60.82-32.20.71.9
B143.59.46.2<2<00.82.34.50.42.3
C144.07.86.22.8<00.82-34.30.42.2
D165.08.86.15.20.91.12-31.70.50.7
E164.28.26.16.10.80.51-23.70.21.6
F204.17.16.24.8<02.12.44.70.71.6
G213.910.46.15.50.60.41-25.70.11.9
H215.48.56.25.9<00.31-23.80.11.3
I215.29.36.36.00.90.93.25.00.31.7
J218.98.86.33.9<00<1000
K234.49.26.23.9<00<16.201.9
L433.711.16.22.5<00<17.701.3
M495.87.56.23.6<01.12.77.70.21.1
1. Numbers indicated as a range, that is, 2-3, were determined by a semiquantitative method. Numbers indicated with one decimal point were determined by a quantitative method.

 

 

 

 5. Source of contamination

Listeriosis seems to be caused almost exclusively by industrialized, highly processed foods. Therefore, in recent years, much attention has been focused on the prevalence of the bacterium in raw materials and food products and to tracing its contamination routes in modern food processing plants. An important prerequisite for control of L. monocytogenes is the knowledge and understanding of its niches during food production. Several studies have shown that L. monocytogenes is able to reside in food processing plants ranging from poultry production (Wenger and others 1990; Lawrence and Gilmour 1995; Ojeniyi and others 1996), meat processing industries (Nesbakken and others 1996; Giovannacci and others 1999), ice cream plants (Miettinen, Bjorkroth, Korkeala and others 1999), shrimp peeling operations (Destro and others 1996), "gravad" (Autio and others 1999), and smoked-fish (Rørvik and others 1995; Norton, McCamey and others 2000; Fonnesbech Vogel, Ojeniyi, Ahrens and others 20010) processing plants.

Early studies of contamination routes depended solely on isolating and counting the organism at different places along the processing line (Eklund and others 1995; Jemmi and Keusch 1992). Recent studies have been greatly facilitated by the use of molecular typing methods of high discriminatory power. These have included Pulsed Field Gel Electrophoresis (PFGE) (Autio and others 1999; Destro and others 1996; Miettinen, Bjorkroth, Korkeala 1999; Ojeniyi and others 1996), ribotyping (Norton, McCamey and others 2000; Norton, Timothe, and others 2000) and Randomly Amplified Polymorphic DNA (RAPD) profiles (Lawrence and Gilmour 1995; Destro and others 1996; Wagner and others 1999; Fonnesbech Vogel, Jorgensen and others 2001; Fonnesbech Vogel, Ojeniyi, Ahrens and others 2001). Comparing DNA-types of L. monocytogenes isolates from raw fish to finished products allows identification of spots along the whole processing line where contamination of finished product occurs.

Conclusions from published studies attempting to identify the source of L. monocytogenes contamination vary. In studies of meat products, some authors (Giovannacci and others 1999) found that the raw materials were the source of product contamination with L. monocytogenens. Eklund and others (1995) reached a similar conclusion in their study of cold-smoked salmon where the raw fish entering the plant was identified as the primary source of L. monocytogenes. Several other studies (Autio and others 1999; Johansson and others 1999; Wenger and others 1990; Rørvik and others 1995; Fonnesbech Vogel and others 2001a,b; Dauphin and others 2001) have found that the major source of direct product contamination is the process environment and equipment (Autio and others 1999; Johansson and others 1999; Wenger and others 1990; Rørvik and others 1995; Fonnesbech Vogel, Jorgensen and others 20010; Fonnesbech Vogel, Ojeniyi, Ahrens and others 2001). Rørvik and others (1995) investigated 475 samples from raw fish, water, products, and environment of a cold-smoked salmon processing plant. Listeria monocytogenes could not be detected in any of 50 raw fish samples, whereas almost one third of samples from the smoke-house products and environment were positive. Using multilocus enzyme electrophoresis (MEE) to compare the strains, a particular electrophoretic type (ET6) that was found in the finished product was repeatedly isolated from the smoke-house environment. Similar conclusions were reached by Autio and others (1999), who used PFGE to compare strains of L. monocytogenes isolated from a cold-smoked trout processing plant. In this study prevalence on raw fish was also low, with only 1 in 60 samples being positive. The PFGE type of these raw fish strains was different from the dominant type found in the product (type I). On the other hand, type I was isolated from several areas of the plant, pointing to the environment as a source of contamination. Dauphin and others (2001) used PFGE to trace contamination of L. monocytogenes in three cold-smoked salmon processing plants. In plant I, one pulsotype dominated in the plant and was detected in the product even though no L. monocytogenes were found in the raw fish. In plant II, 87% of the raw fish were contaminated; however, no L. monocytogenes were detected in the final product.

In a recent study, L. monocytogenes strains were isolated from products from 6 Danish smoke houses, some of which were sampled on two occasions with a time interval of 6-8 mo (Jørgensen and Huss 1998). RAPD-typing of these strains revealed that different DNA-types were found in products from different smoke houses, but that the same DNA-type was found in products from the same smoke house over this period of time (Fonnesbech Vogel, Jorgensen, and others 20010). These data indicated that the same type or clone persisted in a particular smoke house over time. A similar conclusion was reached by Norton, McCamey and others (2000) who visited 5 United States smoke houses over a 6-mo period. Using ribotyping, the authors found that each smoke house harbored its own specific ribotype(s) of L. monocytogenes (Table II-6).

Two out of the 6 Danish smoke houses were selected for further study over a 4-year period (Fonnesbech Vogel, Ojeniyi, Ahrens and others 20010). Raw fish and environmental samples were tested at two repeated visits. As shown in Table II-7, in plant II, one DNA-type dominated in the finished product over the 4-year period. That particular DNA-type was also isolated from the slicing lines, whereas other DNA-types were found in the raw fish handling room. In plant I, the prevalence of L. monocytogenes was lower than in plant II (Table II-8). The pattern was somewhat similar to the pattern of plant I, in that one particular DNA-type dominated the slicing areas and was re-isolated on the finished product. That type, however, was not found on the raw fish. In both plants, experiments were conducted in which samples were taken of 18 marked fish from beginning of processing until finished product. Listeria monocytogenes was not detected in the raw fish but could be found immediately after slicing (Fonnesbech Vogel, Ojeniyi, Ahrens and others 20010). In a similar study, Norton, Timothe and others (2000) sampled from different U.S. smoke houses on repeated visits. Ribotyping showed that in one plant, a particular ribotype isolated from finished product could be traced to both the raw fish and the processing line, whereas in another plant the ribotype found in the product was only isolated from the processing environment, not from the raw fish.

Table II-6. Prevalence of Listeria monocytogenes ribotypes in each processing facility over five sampling visits (Norton, McCamey and others 2000)

 

Ribotype

% prevalence

Processor B

(129 samples)

Processor C

(173 samples)

Processor D

(229 samples)

1039C0.00.010.0
1042B0.81.20.4
1042C6.20.60.4
1044A0.02.33.1
10455.40.00.9
1046B0.02.30.0
10530.00.61.7
10620.80.62.6

 

Table II-7. Number of samples, number of Listeria monocytogenes positive and Randomly Amplified Polymorphic DNA (RAPD) type of L. monocytogenes of processing plant II. P = product, R = raw fish, R-E = Raw fish environment, S-E = smoking environment, Sl-E = Slicing environment. X = Unique types; isolated only once each (Fonnesbech Vogel, Ojeniyi, Ahrens and others 2001)

 

 

RAPD type

by HLWL 85

Number of isolates with RAPD type

 19951996

Nov. 1998

March 1999

 PPRR-ES-ESl-E1Sl-E2PRR-ES-ESl-E1Sl-E2P
 2   4 3 7      
3   1     3    
52             
6   36 1        
74  4     5   6
122512 1 63377   622
13   1 1        
15   6 1011 6 317
110      1       
x   2 2   3    

No. samples

positive for L.monocytogenes3112055080391501709315
total??20362398150147401210527510048

 

Table II-8. Number of samples, number of Listeria monocytogenes positive and Randomly Amplified Polymorphic DNA (RAPD) type of L. monocytogenes of processing plant I. P = product, R = raw fish, R-E = Raw fish environment, S-E = smoking environment, Sl-E = Slicing environment. X = Unique types; isolated only once each (Fonnesbech Vogel, Ojeniyi, Ahrens and others 2001)

 

 

RAPD type

by HLWL 85

Number of isolates with RAPD type

 19951996

Oct. 1998

March 1999
 PPRR-ES-ESl.-E.PR.R-ES-ESl-EP
 2  125578 2   
X   4 2  3  1
No. samplespositive for L. monocytogenes0012959805001
total202015197602114514151613535

The majority of published studies suggest that the processing line (with a "colonized" L. monocytogenes flora) is the immediate, direct source of L. monocytogenes in cold-smoked fish, as opposed to raw fish being the direct source of contamination in the final product. None of the studies, however, has determined the initial source of L. monocytogenes. Listeria monocytogenes may originate from the raw fish, which seeds the processing line, but may also be introduced by staff (2 - 6% of healthy individuals are carriers of the bacterium [cited from Rocourt and others, 2000]), or transporting devices entering the processing area.. As mentioned above, however, some studies also implicate raw fish as the direct source of final product contamination. Based on current data, it is not possible to determine which of these are most important. Given the ubiquitous nature of L. monocytogenes, it is likely that many sources contribute to the entrance of the organism into the processing environment and that the most important source of immediate product contamination is the processing environment.

In support of this last scenario, L. monocytogenes is capable of adhering to food processing surfaces like stainless steel (Hood and Zottola 1997; Jeong and Frank 1994). In addition, L. monocytogenes cells in the adhering state may be more resistant to cleaning and disinfecting procedures than cells in the planktonic state (Wirtanen and Mattila-Sandholm 1992; Norwood and Gilmour 2000). For example, Fonnesbech Vogel, Ojeniyi, Ahrens and others (2001) repeatedly isolated the same organism from cleaned and disinfected smoke houses. Such resistance may explain the finding that the same DNA-types of L. monocytogenes can persist in a food processing plant over years (Miettinen, Bjorkroth, Korkeala 1999).

6. Control of Listeria monocytogenes 

6.1. Control in the processing environment 

The complete and indefinite erradication of L. monocytogenes from processing environments in which RTE foods are produced is currently considered impossible. As outlined above, this is the case not only for cold-smoked fish but for also for meat products. Therefore, even cooked RTE meat products, where a heat-step which kills L. monocytogenes is included, have a high prevalence of the organism. This high prevalence in cooked RTE products is attributed to post-process contamination (Tompkin and others 1999). Re-contamination following a heating step or increase in contamination on, for example, cold-smoked fish during processing is often the consequence of the establishment of L. monocytogenes in a particular niche, or "hot spot," in which the bacteria multiply and spread downstream (Tompkin and others 1999; Autio and others 1999; Fonnesbech Vogel, Ojeniyi, Ahrens 20010).

Under the HACCP concept, a critical control point is a point or process at which the organism of concern is eliminated, ()or combinations of preserving factors can guarantee that growth of the organism does not occur. Cold smoking does not include such a control point for L. monocytogenes. Therefore, reduction of L. monocytogenes to the lowest possible levels must rely on prerequisite programs adhering strictly to Good Hygienic Practices (GHPs) and Good Manufacturing Practices (GMPs). For more details, see guidelines by Tompkin and others (1999) and the Food and Agriculture Organization (FAO 1999) specifically for control of re-contamination with L. monocytogenes in RTE foods. In general, focus must be on education of staff, cleaning and sanitation, redesign of equipment, and proper flow and separation in the processing plant.

In support of the implementation of these guidelines, Fonnesbech Vogel, Ojeniyi, Ahrens and others (2001) found that the prevalence of L. monocytogenes could be dramatically reduced in a smoke house by strictly adhering to GMPs and by targeting spots where the organism had been found to reside with appropiate cleaning and disinfection procedures. Despite the isolation of sporadic positive samples, long-time persistence was eliminated, as indicated by the variations of RAPD types.

The following are areas of upmost importance in the environmental control of L. monocytogenes:

 

6.1.1. Training of staff to understand the necessity of hygienic handling and avoidance of re-contamination is of major importance. Industry experiences indicate that the more aware the staff is of the problem and hygienic practices, the lower the prevalence of L. monocytogenes.

 

6.1.2. Reduction or elimination of L. monocytogenes in the niches in which it becomes established is a continuous effort. Only a limited range of thorough investigations on L. monocytogenes contamination has been carried out in the smoked-fish industry; however, these indicate that particular attention should be paid to the following points:

  • brine
  • injection needles
  • slicers

Several approaches may be used to reduce or eliminate the organism when found. Quaternary ammonium compounds have been found efficient against L. monocytogenes. Peracetic acid and peroctanoic acid have been effective against L. monocytogenes in biofilms (Tompkin and others 1999). If possible, disinfection using steam (that is, 80 °C, 176 °F for 60 min) is effective in eliminating the organism in processing plants (Autio and others 1999; Tompkin and others 1999).

Other areas along the processing line where the product comes in direct contact with surfaces, for example conveyor belts, should also be closely monitored. Experience has shown that floors and particularly floor drains often harbor L. monocytogenes. Attention must be paid to cleaning these areas. Spreading citric acid powder on the floor (to pH of 5) may reduce numbers (Tompkin and others 1999). This procedure, however, should only be used if corrosion or other damage to equipment can be avoided. Foot baths with disinfectants can help reduce the spread of the bacterium in this particular environment.

 

6.1.3. Monitoring of contamination with L. monocytogenes must be part of the quality check of the processing plant. Some plants may not want to test for the pathogen directly. In those cases, Listeria spp. has been found to be a useful indicator organism.

It is the opinion of this panel that cold-smoked fish products consistently free from L. monocytogenes cannot be produced; however, by adhering to GMPs (including training staff), it is possible to reduce prevalence. Smoke houses with strict adherence to GMPs are capable of producing cold-smoked salmon with very low levels of L. monocytogenes, often less than 1 cell / g. Although not eliminated, such low levels would ensure that the number of L. monocytogenes does not increase to above 100 cfu / g at time of consumption given that appropriate temperature (5 °C) and time (3 - 4 wk) limits are met.

6.2 Prevention of growth in the product

Based on levels of L. monocytogenes in smoked fish and the number of cases of listeriosis in Germany, Buchanan and others (1997) developed a dose-response relationship for the organism and listeriosis. Although a threshold value could not be indicated, a linear relationship was seen between the logarithm of (assumed) ingested numbers and of the logarithm of probability of disease. It was concluded that the risk of contracting listeriosis was extremely low from foods containing low numbers (< 100 cfu / g). In addition, the model predicted that 198 of 200 yearly cases of listeriosis resulted from ingestion of samples containing 104 (or more) L. monocytogenes / g. Based on their analysis, the authors concluded that "the initial focus for risk-management decisions should be the prevention of the growth of this pathogen in foods to high levels" (Buchanan and others 1997).

Because the combination of salt and temperature used in smoked-fish products is not sufficient to guarantee "no growth," several studies have evaluated the growth-inhibitory effect of principles other than salt and low temperature. Lately, several physical methods (irradiation, high pressure, pulsed light) have been suggested as general decontamination procedures (Lucore and others 2000; Thayer and Boyd 1999; Rowan and others 1999); however, these procedures remain to be evaluated in real products and therefore will not be addressed in this section. The following text focuses on procedures and principles that have been evaluated against L. monocytogenes in substrates mimicking fish or in real products.

6.2.1. Frozen storage

Frozen storage is an efficient way to completely prevent growth of L. monocytogenes. If the product is vacuum-packaged, frozen storage will have few adverse effects on sensory quality (that is, lipid oxidation). One study has reported growth of L. monocytogenes at -1.5 °C (29 °F) in roast beef, but the generation time at this temperature was 129 h and other studies have found minimum temperature for growth at 0.1°C - 1.1 °C (32 - 34 °F) (Farber and Peterkin 2000).

 

6.2.2. Carbon dioxide

Carbon dioxide has an inhibitory effect on L. monocytogenes. Levels of 70 - 100% CO2 cause a significant increase in lag phase as well as a reduction in growth rate at chill temperatures (Hendricks and Hotchkiss 1997; Nilsson and others 1997; Szabo and Cahill 1998). Given the limited growth in naturally contaminated samples, it is likely that CO2 packing would offer complete elimination of growth of the organism at low temperatures. The use of 70% CO2 for packaging cold-smoked salmon was found not to impart any changes in sensory quality (Nilsson and others 1997). A predictive model describing the influence of CO2, pH, NaCl and incubation temperature on growth of L. monocytogenes has been developed and validated in selected foods (Fernandez and others 1997).

Regardless of its potential to control L. monocytogenes, the vast majority of smoked fish plants today use vacuum packing. Changing to CO2-packing would require investing in new equipment. Also, the volume/pack would increase, which may increase transportation costs. For these reasons, CO2-packing currently may not be a feasible control method. Nonetheless, the panel recommends investigating the technological requirements to introduce this packaging method on a broader scale.

6.2.3. Nitrite 

Nitrite (at 200 ppm) has, in combination with salt, an inhibitory effect against L. monocytogenes at 5 °C (41 °F), where no increase in cell numbers was seen when nitrite was added. In contrast, at 10 °C (50 °F) growth occurred almost at the same rate with and without nitrite (Pelroy, Peterson, Paranjpye and others 1994). Sodium nitrite used in combination with salt and sodium lactate increased the inhibitory effect (Pelroy, Peterson, Holland and others 1994).

 

6.2.4. Lactate

Experiments have been conducted on lactate added to raw, salted salmon. Growth of L. monocytogenes was completely inhibited by 2% lactate at 5 °C (41 °F), whereas 3% lactate was required to inhibit growth at 10 °C (50 °F) (Pelroy, Peterson, Holland and others 1994). While lactate is used as a flavor enhancer in some products, it is not known what the sensory effect of lactate would be on smoked fish. Also, it may be difficult to adsorb 2% lactate into the water phase of the fish.

 

6.2.5. Sorbate

Sorbate is Generally Recognized as Safe (GRAS) and therefore may be used as an additive. No studies have been published on the effect of sorbate on growth of L. monocytogenes in cold-smoked fish. El-Shenawy and Marth (1988) reported that 0.05% sorbate at pH 5.6 caused a marked reduction in growth rate of L. monocytogenes at 4 °C (39 °F). In control samples growth increased from 103 to 108 cells in 24 d, whereas a count of 106 was reached after 45 d when sorbate was added. In vacuum-packed crayfish stored at 4 °C, 0.3% potassium sorbate caused a lag phase of 2 d in L. monocytogenes, but growth rate was not affected (Dorsa and others 1993). In different types of meat sausage products (which typically include salt and nitrite), addition of potassium sorbate delays or inhibits growth of the organism (Hu and Shelef 1996). As with lactate, the application of sorbate may be difficult from a technological point of view.

 

6.2.6. Bacteriocins

Innumerable studies have determined the antilisterial effect of lactic acid bacteria and their bacteriocins. For a recent overview of this, see Farber (2000). Listeria monocytogenes has been inhibited by nisin and divercin in fish products, by nisin and enterocin in dairy products, by nisin and pediocin in meat products, and by pediocin in vegetable products (Nilsson 1999). Nilsson and others (1997) found that the addition of 1000 ppm nisin to cold-smoked salmon caused an initial reduction in numbers of L. monocytogenes, but after 2 wk, growth in vacuum packs at 5 °C (41 °F) resumed as in the control. Duffes and others (1999) similarly found that nisin caused a significant delay in growth of L. monocytogenes on vacuum-packaged, cold-smoked salmon. The inhibition was less pronounced at 8 °C (46 °F) than at 4 °C (39 °F). Combining nisin with 70% CO2 packaging eliminated growth of the bacterium in cold-smoked salmon (Nilsson and others 1997). This was later explained by the synergistic effect of nisin and CO2 both acting on the cell membrane of the bacterium (Nilsson and others 2000). French studies have demonstrated that bacteriocins from Carnobacterium spp. are efficient in preventing growth of L. monocytogenes on cold-smoked salmon (Duffes and others 1999) (Figure II-1). While the inhibitory effect of several bacteriocins against L. monocytogenes is well documented, practical application of bacteriocins will be hampered by several aspects. First, only some countries allow the use of bacteriocins for certain products. Second, bacteriocins' stability and activity in food products are unpredictable (bacteriocins may be degraded by proteases or absorb to food matrix components). Finally, resistance to bacteriocins in L. monocytogenes has been shown to occur quite readily.

 

line chart plotting days of vacuum-packed storage vs. log (L. monocytogenes / gram) Figure II-1

Growth of Listeria monocytogenes in vacuum-packed, cold-smoked salmon at 4 or 8 °C with or without the addition of crude bacteriocin from a Carnobacterium divergens (data from Duffes and others 1999)

 

6.2.7. Background microflora

Several studies have shown that growth of L. monocytogenes in smoked fish is hampered by a high background microflora (Rørvik and others 1991). The deliberate use of non-pathogenic, non-spoiling lactic acid bacteria to control L. monocytogenes is a promising area of targeted preservation. Growth of L. monocytogenes could be significantly delayed in brined shrimp by the addition of a Lactobacillus saké/curvatus strain (From Jeppesen and Huss 1993). In a pork product, an L. saké strain caused a reduction in maximum cell density of L. monocytogenes of 6 log units compared to the control (lactic acid bacteria was not added to the control) (De Martinis and Franco 1998). Despite strong antilisterial activity, the strain used in brined shrimp was not suited for use on cold-smoked salmon, as it produced pronounced sulfur off odors. In contrast, Carnobacterium piscicola and Carnobacterium divergens have no adverse effects on the sensory quality (Duffes and others 1999; Nilsson and others 1999; Paludan-Müller and others 1998). Several laboratories have shown that carnobacteria inhibit growth of L. monocytogenes in food products (Buchanan and Klawitter 1992) and that growth of L. monocytogenes in cold-smoked salmon can be completely inhibited by addition of C. piscicola or C. divergens (Duffes and others 1999; Nilsson and others 1999).

7. Conclusions 

Based on the scientific data available, the panel concludes that:

  • Given the ubiquitous nature of L. monocytogenes, the lack of listericidal steps in the cold-smoking procedure, and the ability of the organism to become established in the processing environment and re-contaminate products, it is not possible to produce cold-smoked fish consistently free of L. monocytogenes. This is not unique to cold-smoked fish because this microorganism can be isolated from a wide range of ready-to-eat (RTE) foods.
  • By adhering strictly to GMPs and GHPs, it is possible to produce cold-smoked fish with low levels of L. monocytogenes, preferably at <1 cell / g at the time of production.
  • Growth of L. monocytogenes in naturally contaminated fish products is significantly slower than predicted by models (using combinations of pH, NaCl, temperature, and lactate) and inoculation studies.
  • Prevention of growth in cold-smoked fish cannot be guaranteed using current combinations of NaCl and low temperatures; however growth can be prevented by freezing. Other alternatives (for example, lactate, nitrite or bioprotective cultures) are currently being researched.
  • If the organism cannot be eliminated and growth inhibiting steps are not introduced, the hazard must be controlled by limiting shelf life (at 4.4 °C, 40 °F) to ensure that no more than 100 cells / g are present at time of consumption. The limitation of shelf life has been suggested by several investigators (Rørvik 2000, Nørrung 2000, Farber 2000). Time limits may need to be established by each processor, reflecting the initial level of the organism in freshly produced product.
  • Some countries, such as Australia, warn pregnant women about listeriosis and offer a list of food items to be avoided during the pregnancy. Labels on cold-smoked fish as well as other RTE foods indicating that these products may constitute a health hazard for immuno-compromised individuals and pregnant women could be considered.

8. Research needs 

The panel concluded that research is needed to:

  • Conduct epidemiological investigations to determine if and to what extent cold-smoked fish is involved in cases of listeriosis. Despite prediction of a risk, only a limited number of cases have been associated with cold-smoked fish.
  • Assess the virulence potential of L. monocytogenes isolated from cold-smoked fish.
  • Measure behavior of L. monocytogenes in naturally contaminated products. Listeria monocytogenes appears to grow more slowly and to lower numbers than anticipated based on model predictions and inoculation trials. An understanding of which factors cause these differences may be used to design appropriate control measures in the product.
  • Determine the robustness and applicability of alternative growth inhibitory measures such as bioprotective cultures, bacteriocins, lactate and others.
  • Determine how L. monocytogenes becomes established in the smoke houses and processing facilities. Several studies show that particular DNA-types become established in niches in the processing environments. Research is needed to evaluate what parameters determine which types reside; whether it be particular adhesion properties, particular resistance properties, or other factors.
  • Investigate the source of contamination in smoke houses and processing environments in order to introduce procedures specifically targeted at eliminating or limiting introduction of the organism.
  • Identify GMP practices that would minimize the contamination and growth of L. monocytogenes.
  • Determine the effectiveness of intervention strategies to reduce or eliminate L. monocytogenes, such as using chlorinated water to thaw and rinse incoming fish, and for rinsing fish following the brining operation.
  • Develop cleaning and disinfection procedures targeted at adhered or established cells for removal of L. monocytogenes from surfaces.
  • Determine if particular types of surfaces reduce numbers of adhering L. monocytogenes or if particular treatments (that is, spraying with lactic acid bacteria or lactate) can reduce surface contamination by minimizing adhesion and biofilm formation.
  • Evaluate the robustness and the sensory acceptability of various procedures under investigation (that is, bioprotection, lactate, and so on) for the elimination of growth in the cold-smoked fish product.
  • Determine the effectiveness of post-processing methods such as irradiation and high pressure for the elimination of L. monocytogenes in cold smoked fish.

 

References

[AFDO] Association of Food and Drug Officials. 1991 June. Cured, salted, and smoked fish establishments good manufacturing practices [model code]. [York (PA)]: Association of Food and Drug Officials. 7 p.

Autio T, Hielm S, Miettinen M, Sjoberg A-M, Aarnisalo K, Bjorkroth J, Mattila-Sandholm T, Korkeala H. 1999. Sources of Listeria monocytogenes contamination in a cold-smoked rainbow trout processing plant detected by pulsed-field gel electrophoresis typing. Appl Environ Microbiol 65(1):150-5.

Barakat RK, Harris LJ. 1999. Growth of Listeria monocytogenes and Yersinia enterocolitica on cooked modified-atmosphere-packaged poultry in the presence and absence of a naturally occurring microbiota. Appl Environ Microbiol 65(1):342-5.

Ben Embarek PK. 1994. Presence, detection and growth of Listeria monocytogenes in seafoods: a review. Int J Food Microbiol 23:17-34.

Ben Embarek PK, Huss HH. 1993. Heat resistance of Listeria monocytogenes in vacuum packaged pasteurized fish fillets. Int J Food Microbiol 20:85-95.

Bremer PJ, Osborne CM. 1998. Reducing total aerobic counts and Listeria monocytogenes on the surface of king salmon (Oncorhynchus tshawytscha). J Food Prot 61:849-54.

Brett MSY, Short P, McLauchlin J. 1998. A small outbreak of listeriosis associated with smoked mussels. Int J Food Microbiol 43:223-9.

Buchanan RL, Damert WG, Whiting RC, van Schothorst M. 1997. Use of epidemiologic and food survey data to estimate a purposefully conservative dose-response relationship for Listeria monocytogenes levels and incidence of listeriosis. J Food Prot 60(8):918-22.

Buchanan RL, Klawitter LA. 1992. Effectiveness of Carnobacterium piscicola LK5 for controlling the growth of Listeria monocytogenes Scott A in refrigerated foods. J Food Saf 12:219-36.

Cortesi ML, Sarli T, Santoro A, Murru N, Pepe T. 1997. Distribution and behavior of Listeria monocytogenes in three lots of naturally-contaminated vacuum-packed smoked salmon stored at 2 and 10° C. Int J Food Microbiol 37:209-14.

Dalgaard P, Jorgensen LV. 1998. Predicted and observed growth of Listeria monocytogenes in seafood challenge tests and in naturally contaminated cold-smoked salmon. Int J Food Microbiol 40:105-15.

Dauphin G, Ragimbeau C, Malle P. 2001. Use of PFGE typing for tracing contamination with Listeria monocytogenes in three cold-smoked salmon processing plants. Int J Food Microbiol 64:51-61.

De Martinis ECP, Franco BDGM. 1998. Inhibition of Listeria monocytogenes in a pork product by a Lactobacillus sake strain [research communication]. Int J Food Microbiol 42:119-26.

Destro MT, Leitao MFF, Farber JM. 1996. Use of molecular typing methods to trace the dissemination of Listeria monocytogenes in a shrimp processing plant. Appl Environ Microbiol 62(2):705-11.

Dillon R, Patel T, Ratnam S. 1994. Occurrence of Listeria in hot and cold smoked seafood products. Int J Food Microbiol 22:73-7.

Dorsa WJ, Marshall DL, Semien M. 1993. Effect of potassium sorbate and citric acid sprays on growth of Listeria monocytogenes on cooked crawfish (Procambarus clarkii) tail meat at 4 C. Lebensm Wiss u Technol 26:480-2.

Duffes F, Corre C, Leroi F, Dousset X, Boyaval P. 1999. Inhibition of Listeria monocytogenes by in situ produced and semipurified bacteriocins on Carnobacterium spp. on vacuum-packed, refrigerated cold-smoked salmon. J Food Prot 62(12):1394-1403.

Eklund MW, Poysky FT, Paranjpye RN, Lashbrook LC, Peterson ME, Pelroy GA. 1995. Incidence and sources of Listeria monocytogenes in cold-smoked fishery products and processing plants. J Food Prot 58(5):502-8.

El-Kest SE, Yousef AE, Marth EH. 1991. Fate of Listeria monocytogenes during freezing and frozen storage. J Food Sci 56(4):1068-71.

Elliot EL, Kvenberg JE. 2000. Risk assessment used to evaluate the US position on Listeria monocytogenes in seafood. Int J Food Microbiol 62:253-60.

El-Shenawy MA, Marth EH. 1988. Inhibition and inactivation of Listeria monocytogenes by sorbic acid. J Food Prot 51:842-7.

Ericsson H, Eklow A, Danielsson-Tham ML, Loncarevic S, Mentzing LO, Persson I, Unnerstad H, Tham W. 1997. An outbreak of listeriosis suspected to have been caused by rainbow trout. J Clin Microbiology 35(11):2904-7.

[FAO] Food and Agriculture Organization. 1999 May. Report of the FAO expert consultation on the trade impact of Listeria in fish products. Rome: FAO. FAO Fisheries Report nr 604. 34 p.

Farber JM. 1991. Listeria monocytogenes in fish products. J Food Prot 54(12):922-4, 934.

Farber JM. 2000. Present situation in Canada regarding Listeria monocytogenes and ready-to-eat seafood products. Int J Food Microbiol 62:247-51.

Farber JM, Peterkin PI. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol Rev 55:476-511.

Farber JM, Peterkin PI. 2000. Listeria monocytogenes. In: Lund BM, Baird-Parker TC, Gould GW, editors. The microbiological safety and quality of foods. Gaithersburg (MD): Aspen. p 1178-1232.

Farber JM, Ross WH, Harwig J. 1996. Health risk assessment of Listeria monocytogenes in Canada. Int J Food Microbiol 30:145-56.

Fenlon DR, Wilson J, Donachie W. 1996. The incidence and level of Listeria monocytogenes contamination of food sources at primary production and initial processing. J Appl Bacteriol 81:641-50.

Fernandez PS, George SM, Sills CC, Peck MW. 1997. Predictive model of the effect of CO2, pH, temperature and NaCl on growth of Listeria monocytogenes. Int J Food Microbiol 37:37-45.

Fonnesbech Vogel B, Jorgensen LV, Ojeniyi B, Huss HH, Gram L. 2001. Diversity of Listeria monocytogenes isolates from cold-smoked salmon produced in different smoke houses as assessed by randomly amplified polymorphic DNA analyses. Int J Food Microbiol 65:83-92.

Fonnesbech Vogel B, Ojeniyi B, Ahrens P, Due Skov L, Huss HH, Gram L. 2001. Elucidation of Listeria monocytogenes contamination routes in cold-smoked salmon processing plants detected by DNA-based typing methods. Appl Environ Microbiol. Forthcoming.

From Jeppesen V, Huss HH. 1993. Antagonistic activity of two strains of lactic acid bacteria against Listeria monocytogenes and Yersinia enterocolitica in a model fish product at 5° C. Int J Food Microbiol 19:179-86.

Giovannacci I, Ragimbeau C, Queguiner S, Salvat G, Vendeuvre J-L, Carlier V, Ermel G. 1999. Listeria monocytogenes in pork slaughtering and cutting plants use of RAPD, PFGE, and PCR-REA for tracing and molecular epidemiology. Int J Food Microbiol 53(1999):127-40.

Heinitz ML, Johnson JM. 1998. The incidence of Listeria spp., Salmonella spp., and Clostridium botulinum in smoked fish and shellfish. J Food Prot 61(3):318-23.

Hendricks MT, Hotchkiss JH. 1997. Effect of carbon dioxide on the growth of Pseudomonas fluorescens and Listeria monocytogenes in areobic atmospheres. J Food Prot 60(12):1548-52.

Hood SK, Zottola EA. 1997. Adherence to stainless steel by foodborne microorganisms during growth in model food systems. Int J Food Microbiol 37:145-53.

Hu AC, Shelef LA. 1996. Influence of fat content and preservatives on the behavior of Listeria monocytogenes in beaker sausage. J Food Saf 16:175-81.

Hudson JA, Mott SJ. 1993. Growth of Listeria monocytogenes, Aeromonas hydrophila and Yersinia enterocolitica on cold-smoked salmon under refrigeration and mild temperature abuse. Food Microbiol 10:61-8.

Huss HH, Ben Embarek PK, From Jeppesen V. 1995. Control of biological hazards in cold smoked salmon production. Food Control 6(6):335-40.

Iida T, Kanzaki M, Nakama A, Kokubo Y, Maruyama T, Kaneuchi C. 1998. Detection of Listeria monocytogenes in humans, animals, and foods. J Vet Med Sci 60(12):1341-3.

Jemmi T. 1990. Zum vorkommen von Listeria monocytogenes in importierten geraucherten und fermentierten fischen. Arch Lebensmittelhyg 41:107-9.

Jemmi T, Keusch A. 1992. Behavior of Listeria monocytogenes during processing and storage of experimentally contaminated hot-smoked trout. Int J Food Microbiol 15:339-46.

Jemmi T, Keusch A. 1994. Occurrence of Listeria monocytogenes in freshwater fish farms and fish-smoking plants. Food Microbiol 11:309-16.

Jeong DK, Frank JF. 1994. Growth of Listeria monocytogenes at 10° C in biofilms with microorganisms isolated from meat and dairy processing environments. J Food Prot 57(7):576-86.

Jin M, Kusunoki K, Ikejima N, Arai T, Irikura Y, Suzuki K, Hirata I, Kokubo Y, Maruyama T. 1994. Incidence of Listeria moncytogenes in smoked salmon. Jpn J Food Microbiol 11(2):107-11.

Jinneman KC, Wekell MM, Eklund MW. 1999. Incidence and behavior of Listeria monocytogenes in fish and seafood. In: Ryser ET, Marth ELH, editors. Listeria, listeriosis and food safety. New York: M Dekker. p 601-30.

Johansson T, Rantala L, Palmu L, Honkanen-Buzalski T. 1999. Occurrence and typing of Listeria monocytogenes strains in retail vacuum-packed fish products and in a production plant. Int J Food Microbiol 47:111-9.

Jorgensen LV, Dalgaard P, Huss HH. 2000. Multiple compound quality index for cold-smoked salmon ( Salmo salar ) developed by multivariate regression of biogenic amines and pH. J Agric Food Chem 48:2448-53.

Jorgensen LV, Huss HH. 1998. Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood. Int J Food Microbiol 42:127-31.

Lawrence LM, Gilmour A. 1995. Characterization of Listeria monocytogenes isolated from poultry products and from the poultry-processing environment by random amplification of polymorphic DNA and multilocus enzyme electrophoresis. Appl Environ Microbiol 61(6):2139-44.

Lucore LA, Shellhammer TH, Yousef AE. 2000. Inactivation of Listeria monocytogenes Scott A on artificially contaminated frankfurters by high-pressure processing. J Food Prot 63(5):662-4.

McLauchlin J. 1997. The pathogenicity of Listeria monocytogenes: a public health perspective. Rev Med Microbiol 8(1):1-14.

Miettinen MK, Bjorkroth KJ, Korkeala HJ. 1999. Characterization of Listeria monocytogenes from an ice cream plant by serotyping and pulsed-field gel electrophoresis. Int J Food Microbiol 46:187-92.

Miettinen MK, Siitonen A, Heiskanen P, Haajanen H, Bjorkroth KJ, Korkeala HJ. 1999. Molecular epidemiology of an outbreak of febrile gastroenteritis caused by Listeria monocytogenes in cold-smoked rainbow trout. J Clin Microbiol 37(7):2358-60.

Nesbakken T, Kapperud G, Caugant DA. 1996. Pathways of Listeria monocytogenes contamination in the meat processing industry. Int J Food Microbiol 31:161-71.

Nilsson L. 1999. Control of Listeria monocytogenes in cold-smoked salmon by biopreservation [DPhil thesis]. Lyngby: Technical University of Denmark, Danish Institute for Fisheries Research. 136 p.

Nilsson L, Chen Y, Chikindas ML, Huss HH, Gram L, Montville TJ. 2000. Carbon dioxide and nisin act synergistically on Listeria monocytogenes. Appl Environ Microbiol 66(2):769-74.

Nilsson L, Gram L, Huss HH. 1999. Growth control of Listeria monocytogenes on cold-smoked salmon using a competitive lactic acid bacteria flora. J Food Prot 62(4):336-42.

Nilsson L, Huss HH, Gram L. 1997. Inhibition of Listeria monocytogenes on cold-smoked salmon by nisin and carbon dioxide atmosphere. Int J Food Microbiol 38:217-27.

Norrung B. 2000. Microbiological criteria for Listeria monocytogenes in foods under special consideration of risk assessment approaches. Int J Food Microbiol 62:217-21.

Norton DM, McCamey MA, Gall KL, Scarlett JM, Boor KJ, Wiedmann M. 2000. Molecular studies on the ecology of Listeria monocytogenes in the smoked fish processing industry and implications for control strategies. Forthcoming.

Norton DM, Sue D, Timothe J, Scarlett JM, Boor KJ, Wiedmann M (Cornell Univ, Ithaca, NY). 2000. Characterization and pathogenic potential of L. monocytogenes isolates from the smoked fish industry [abstract]. Abstract submitted for presentation at the 2000 Annual Meeting of the American Society of Microbiology (ASM).

Norwood DE, Gilmour A. 2000. The growth and resistance to sodium hypochlorite of Listeria monocytogenes in a steady-state multispecies biofilm. J Appl Microbiol 88:512-20.

Notermans S, Dufrenne J, Teunis P, Chackraborty T. 1998. Studies on the risk assessment of Listeria monocytogenes. J Food Prot 61(2):244-8.

Ojeniyi B, Wegener HC, Jensen NE, Bisgaard M. 1996. Listeria monocytogenes in poultry and poultry products: epidemiological investigations in seven Danihs abattoirs. J Appl Bacteriol 80:395-401.

Paludan-Muller C, Dalgaard P, Huss HH, Gram L. 1998. Evaluation of the role of Carnobacterium piscicola in spoilage of vacuum- and modified packed cold-smoked salmon stored at 5° C. Int J Food Microbiol 39:155-66.

Pelroy GA, Peterson ME, Holland PJ, Eklund MW. 1994. Inhibition of Listeria monocytogenes in cold-process (smoked) salmon by sodium lactate. J Food Prot 57(2):108-13.

Pelroy GA, Peterson ME, Paranjpye R, Almond J, Eklund M. 1994. Inhibition of Listeria monocytogenes in cold-process (smoked) salmon by sodium nitrite and packaging method. J Food Prot 57(2):114-9.

Peterson ME, Pelroy GA, Paranjpye RN, Poysky FT, Almond JS, Eklund MW. 1993. Parameters for control of Listeria monocytogenes in smoked fishery products: sodium chloride and packaging method. J Food Prot 56(11):938-43.

Rocourt J, Jacquet C, Reilly A. 2000. Epidemiology of human listeriosis and seafood. Int J Food Microbiol 62:197-209.

Rorvik LM. 2000. Listeria monocytogenes in the smoked salmon industry. Int J Food Microbiol 62:183-90.

Rorvik LM, Caugant DA, Yndestad M. 1995. Contamination pattern of Listeria monocytogenes and other Listeria spp. in a salmon slaughterhouse and smoked salmon processing plant. Int J Food Microbiol 25:19-27.

Rorvik LM, Yndestad M. 1991. Listeria monocytogenes in foods in Norway. Int J Food Microbiol 13:97-104.

Rorvik LM, Yndestad M, Skjerve E. 1991. Growth of Listeria monocytogenes in vacuum-packed, smoked salmon during storage at 4° C. Int J Food Microbiol 14:111-8.

Rowan NJ, MacGregor SJ, Anderson JG, Fouractre RA, McIlvaney L, Farish O. 1999. Pulsed-light inactivation of food-related microorganisms. Appl Environ Microbiol 65:1312-5.

Seeliger HPR, Jones D. 1986. Genus Listeria Pirie 1940, 383al. In: Sneath PHA, Mair SN, Sharpe ME, Holt JG, editors. Bergey's Manual of Systematic Bacteriology. 9th ed. Baltimore (MD): Williams and Wilkins. p 1235-45.

Sunen E. 1998. Minimum inhibitory concentration of smoke wood extracts against spoilage and pathogenic micro-organisms associated with foods. Letts Appl Microbiol 27:45-8.

Szabo EA, Cahill ME. 1998. The combined affects of modified atmosphere, temperature, nisin and ALTA[tm] 2341 on the growth of Listeria monocytogenes. Int J Food Mircobiol 43:21-31.

Teufel P, Bendzulla C. 1993. Bundesweite Erhebung zum vorkommen von L. monocytogenes in Lebenmitteln. Berlin: Bundesinstitut fur gesundheitlichen Verbraucherschutz und Veterinarmedizin. NB.

Thayer DW, Boyd G. 1999. Irradiation and modified atmosphere packaging for the control of Listeria monocytogenes on turkey meat. J Food Prot 62:1136-42.

Tompkin RB, Scott VN, Bernard DT, Sveum WH, Gombas KS. 1999. Guidelines to prevent post-processing contamination from Listeria monocytogenes. Dairy, Food Environ San 19(8):551-62.

Truelstrup Hansen L, Drewes Røntved S, Huss HH. 1998. Microbiological quality and shelf life of cold-smoked salmon from three different processing plants. Food Microbiology 15:137-50.

Wagner M, Maderner A, Brandl E. 1999. Development of a multiple primer RAPD assay as a tool for phylogenetic analysis in Listeria spp. strains isolated from milkproduct associated epidemics, sporadic cases of listeriosis and dairy environments. Int J Food Microbiol 52:29-37.

Weagant SD, Sado PA, Colburn KG, Torkelson JD, Stanley FA, Krane MH, Shields SC, Thayer CF. 1988. The incidence of Listeria species in frozen seafood products. J Food Prot 51:655-7.

Wenger JD, Swaminathan B, Hayes PS, Green SS, Pratt M, Pinner RW, Schuchat A, Broome CV. 1990. Listeria monocytogenes contamination of turkey franks: evaluation of a production facility. J Food Prot 53(12):1015-9.

Wiedmann M, Bruce JL, Keating C, Johnson AE, McDonough PL, Batt CA. 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infec Immun 65(7):2707 - 16.

Wirtanen G, Mattila-Sandholm T. 1992. Removal of foodborne biofilms-comparison of surface and suspension tests. Part I. Lebensm Weiss Technol 25(1):43-9.