International Journal

of Food Microbiology
International Journal of
ELSEVIER Food Microbiology23 (1994) 17-34

Presence, detection and growth of Listeria monocytogenes

in seafoods: a review
Peter Karim Ben Embarek
Technological Laboratory, Danish Ministry of Fisheries, Technical University, Bldg. 221,
DK-2800 Lyngby, Denmark
Received 6 April 1994; revision received 9 June 1994; accepted 25 June 1994

Abstract

Listeria monocytogenes and other Listeria spp. have been isolated from seafoods on a
regular basis since 1987. A relatively high incidence of the organism (6-36%) in ready-to-eat
cold smoked salmon and cooked fish products has raised concern about the survival and
growth potential of this organism in seafoods, as these products are not processed further
before consumption. L. monocytogenes grows well at refrigeration temperature on most
seafoods, but the sources of contamination in ready-to-eat fish products are still unknown.
This paper reviews the knowledge available in order to make recommendations on control
options and avenues for future research.

Keywords: Listeria monocytogenes; Seafoods; Incidence; Prevalence; Growth; Shellfish;

Shrimp

I. Introduction

Listeria monocytogenes has been recognised as a human p a t h o g e n since 1929

and as a food-borne p a t h o g e n since 1981, where L. monocytogenes c o n t a m i n a t e d
coleslaw was positively identified as the vehicle of infection in an o u t b r e a k of
listeriosis in Halifax, Nova Scotia, C a n a d a (Schlech et al., 1983). Several food-borne
outbreaks of listeriosis have since g e n e r a t e d world-wide concern about the pres-
ence of L. monocytogenes in foods. This increased interest has m a d e L. monocyto-
genes one of the most studied food-borne pathogens during the last ten years, as
indicated by the t r e m e n d o u s n u m b e r of scientific p a p e r s on Listeria published
during this period. Comprehensive reviews have been published by Ryser and
M a r t h (1991) and F a r b e r and Peterkin (1991).

0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

SSDI 0168-1605(94)00092-1
18 P.K. Ben Embarek / International Journal of Food Microbiology 23 (l 994) 17-34

The presence of Listeria in seafoods has only recently been examined. Surveys
initiated in 1987 in USA, showed that several seafoods contain L. monocytogenes
(Weagant et al., 1988; Ryser and Marth, 1991). L. monocytogenes has been
identified as the causative agent in three sporadic cases of seafood-borne listeriosis
(Facinelli et al., 1989; Frederiksen, 1991; Baker et al., 1993) and has been
suspected to be the causative agent in at least two other outbreaks involving
seafoods (Lennon et al., 1984; Riedo et al., 1990).
Several papers have been published on the prevalence and growth potential of
L. rnonocytogenes in seafoods and two reviews based on the initial data have dealt
with this specific topic (Fuchs and Reilly, 1992; Dillon and Patel, 1992b) but most
of the information on Listeria in seafoods has been published during the last three
years. Recalls of seafood products in North America and several European
countries because of the presence of L. monocytogenes are still common (Anony-
mous, 1993b,c,e) despite increased effort to eliminate or reduce the organism
(Anonymous, 1993f) and sporadic cases of listeriosis involving seafood are still
reported (Baker et al., 1993).
The purpose of this p a p e r is to provide an update on the knowledge available
on the source of contamination and the behavior of Listeria in different seafoods
and thereby provide recommendations for control options and further research
efforts in this area.

2. Presence of L. monocytogenes in freshwater, seawater, and live fish and shellfish

Only a few surveys have been conducted to assess the presence of L. monocyto-
genes in water (Table 1). L. monocytogenes and other Listeria spp. have been
isolated mainly from freshwater samples and from seawater from coastal areas
(Table 1) subject to pollution or contamination from industrial, human or animal
sources. In one survey (Motes, 1991), three of the four (75%) water samples
positive for Listeria (one L. innocua and three L. monocytogenes) were collected
from prohibited areas and two of them were effluent samples from processing and
sewage treatment plants. Detection of the bacteria in rivers (Colburn et al., 1990),
canals emptied in the sea (Dijkstra, 1982), and in river sediments suggest that
Listeria is supplied on a regular basis into the sea in coastal area.
In our analysis of cold seawater from salmon farm surroundings, Listeria spp.
could not be detected (Ben E m b a r e k et al., 1994a). The seawater analysed by
Rorvik et al. (1992) with a relative high detection of the bacteria (52% of the
samples positive for Listeria spp. (Table 1)) was sampled in the surroundings of a
salmon processing plant. No indications are given on possible sources of contami-
nation. In conclusion, it seems that Listeria spp. can be isolated from polluted
waters and waters with a high content of organic material, e.g, rivers and coastal
areas. Its natural presence in clean open seawater where most fish farms are to be
found, remains to be established.
Only two surveys have analysed live fish for the presence of Listeria spp. (Table
2). The bacteria could not be detected in 10 live salmon where guts, skins and gills
 P.K. Ben Embarek / International Journal of Food Microbiology 23 (1994) 17-34 19

Table 1
Prevalence of Listeria spp. in freshwater, seawater and sediments
Sampling location Number % positive for References
of Listeria L. monocytogenes
samples

Freshwater (river, 37 81 62 Colburn et al. (1990)

contact with domestic
animals) (USA)
Freshwater (UK) 7 100 Watkins and Sleath
(1981)
Canals, lakes (Netherlands) 180 37 DOkstra (1982)
Effluents (sewage treatment 8 25 Motes (1991)
and seafood processing
plants) (USA)
Seawater (coastal area) 3 33 33 Colburn et al. (1990)
(USA)
Seawater (shellfish 70 3 Motes (1991)
growing area) (USA)
Seawater (Netherlands) 43 0 0 Dijkstra (1982)
Seawater (used to transport 21 52 14 Rcrvik et al. (1992)
live salmon) (Norway)
Seawater (around salmon 8 0 0 Ben Embarek et al.
farm) (Norway) (1994a)
Sediments (freshwater) 46 30 17 Colburn et al. (1990)
(USA)
Sediments (freshwater) 15 20 0 Colburn et al. (1990)
(USA)

Table 2
Prevalence of Listeria spp. in live fish and shellfish
Fish type (country) Number of % positive for References
samples Listeria L. monocytogenes
Oysters (USA) 35 2.8 0 Colburn et al. (1990)
Oysters (USA) 75 0 0 Motes (1991)
Shrimps (USA) 74 11 11 Motes (1991)
Mussels (Italy)
Before depuration 175 b 0.9 0 Decastelli et al. (1993)
After depuration 110 b 0 0
Freshwater chanel 4 100 a Leung et al. (1992)
catfish (USA)
Freshwater hybrid ~ ? Positive ¢ Nedoluha and Westhoff
stripped bass, reared (1993)
(USA)
Salmons (Norway)
Gills 10 0 0 Ben Embarek et al.
Skin 10 0 0 (1994a)
Guts 10 0 0
a Presumptive Listeria spp. b Each sample consisting of approx. 50 mussels, c L. monocytogenes was
isolated but there was no indication of the prevalence of the organism.
20 P.K. Ben Ernbarek / lnternational Journal of Food Microbiology 23 (1994) 17 34

were analysed separately (Ben Embarek et al., 1994a) while four out of four
samples of freshwater channel catfish yielded presumptive Lister& spp. (Leung et
al., 1992). Based on this limited studies and the data from water samples (Table 1),
it is suggested that Listeria spp. are likely to occur naturally on freshwater fish but
unlikely to occur on pelagic fishes or fish reared in clean seawater. It is accepted
that the bacterial flora of fish reflects the bacterial load of the environment
(Shewan, 1977; Gram, 1989) and therefore the contamination of fish with Listeria
spp. most likely depends on the presence of the bacteria in the surrounding waters.
L. monocytogenes was not detected in two surveys with live oysters and one with
live mussels including 35, 75 and 285 samples, respectively. L. innocua was isolated
from one of 35 oyster samples and from one of 175 mussel samples before
depuration (Colburn ct al., 1990; Decastelli et al., 1993) and was the only Listeria
sp. isolated from live bivalves. Bivalve molluscs filter large quantities of water and
are therefore able to concentrate water-borne pathogens like the vibrios
(NACMCF, 1992; Huss, 1994). The surprisingly low prevalence of Listeria spp. and
the absence of L. monocytogenes in live oysters and mussels confirm that Lister&
are rare or uncommon in water and they explain why the bacteria were not
detected in two very limited surveys of processed oysters (Buchanan et al., 1989;
Weagant et al., 1988) in the USA and a larger study in Japan (Masuda et al., 1992;
Table 3).
Regarding live fish and shellfish, L. monocytogenes has only been isolated from
live shrimp (Motes, 1991; Table 2) which probably represent a source of Lister&
contamination to shrimp processing plants. Based on the few results available
(Table 2), it is not possible to give any explanation of the possible differences
between live shrimp and other live fish products.

3. Prevalence of L. monocytogenes in fresh, frozen and processed seafoods

Following the first published survey on the prevalence of Lister& spp. in frozen
fish products (Weagant et al., 1988) and the recalls of L&teria contaminated
seafood in the United States from 1987 onwards, several surveys have been
initiated in many countries (Table 3) to assess the extent of the presence of
Listeria spp. in fish products.
In flesh or frozen fish and fish-based seafood, Listeria spp. are always to be
found. The prevalence of L. monocytogenes varies from 4% to 12% for the surveys
from t e m p e r a t e areas with a representative number of samples, and is generally
lower than the prevalence in raw meat (4-60%) or flesh poultry (23-60%) and
higher than in raw milk (2.2%) (Johnson et al., 1990; Farber and Peterkin, 1991).
One exception is a survey on frozen seafood (Weagant et al., 1988) where an
incidence of 27% was observed, but no information is given by the authors on the
randomness of the sampling plan.
Interestingly, the prevalence of L. monocytogenes in tropical fresh fish and
seafood is very low ( 0 - 2 % ) while the prevalence of Listeria spp. is comparable to
others studies (Table 3) (Fuchs and Surendran, 1989; Karunasagar et al., 1992;
 P.I~ Ben Embarek / International Journal of Food Microbiology 23 (1994) 17-34 21

Table 3
Prevalence of Listeria spp. in fresh, frozen and processed seafood
Fish type (country) Number of % positive for References
samples Listeria L. monocytogenes
Fresh and frozen fish
Fish (f) (USA) 4 50 50 Buchanan et al. (1989)
Catfish (f) (USA) 1 100 0 Buchanan et al. (1989)
Fish (f) (Trinidad) 61 14.8 2 d Adesiyun (1993)
Minced fish (f) 8 12 Rorvik and Yndestad (1991)
(Norway)
Minced raw tuna (r) 37 43.2 8.1 Ryu et al. (1992)
(Japan)
Fish (f) (Japan) 382 12.6 2.4 Masuda et al. (1992)
Fish (f) (India) Manoj et al. (1991)
Landing center 12 0 0
Market 39 5.1 0
Seafood, raw (r) 28 7.1 10.7 Ryu et al. (1992)
(Japan)
Seafood (f+ fr) 57 10.5 Wong et al. (1990)
(Taiwan)
Seafood (f+ p) 26 3.9 3.9 Hartemink and Georgsson (1991)
(Iceland)
Seafood (fr) (USA) 56 (510) c 62.5 26.7 Weagant et al. (1988)
Seafood (f) (India) 10 33 0 Fuchs and Surandran (1989)
Seafood (India) 200 8 0 Karunasagar et a1.(1992)
Processed seafood
Fish salads (r) (Iceland) 37 32 16 Hartemink and Georgsson (1991)
Seafood (r) 50 48 26 Hudson et al. (1992)
(New Zealand)
Seafood a (p) (USA) 2 0 0 Buchanan et al. (1989)
Crab (r), USA (China) 7 14 Farber (1991)
Seafood b (Canada) 46 2 Farber (1991)
Seafood (c + p) (Japan) 11 0 0 Ryu et al. (1992)
Seafood (fr) (India) (d) 14 35.7 0 Fuchs and Surandran (1989)
11 0 0
Shrimp
Shrimp (f) (Japan) 70 8.6 1.4 Masuda et al. (1992)
Prawn, raw (r) (Japan) 38 15.8 2.6 Ruy et al. (1992)
Shrimp (f) (Trinidad) 41 5 2 d Adesiyun (1993)
Shrimp in brine (r) 16 18 Rorvik and Yndestad (1991)
(Norway)
Shrimp (c + fr) 11 Hartemink and Georgsson (1991)
(Iceland)
Shrimp 20 20 Farber (1991)
Shrimp, raw (fr) 17 23.5 11.8 Ravomanana et al. (1993)
(France)
Shrimp (c) (France) 35 54.3 11.4 Ravomanana et al. (1993)
Shrimp (f+ fr) (USA) 4 25 0 Buchanan et al. (1989)
Shrimp (r) (USA) 45 9 Farber (1991)
(UK, Taiwan) 4 0
Shrimp (f+ p) (India) 19 10.5 0 Manoj et al. (1991)
22 P.K. Ben Embarek /International Journal of Food Microbiology 23 (1994) 17-34

Table 3 (continued)
Fish type (country) Number of % positive for References
samples Listeria L. monocytogenes
Shellfish
Oysters and clams (p) 3 (I 0 Buchanan et al. (1989)
(USA)
Oysters (f)(Japan) 84 0 0 Masuda et al. (1992)
Oysters (fr) (USA) 1 (10) c 0 0 Weagant et al. (1988)
Non-oysters (f) (Japan) 147 11.6 1.4 Masuda et al. (1992)
Mussels (f) (Spain) 40 22.5 7.5 Sim6n et al. (1992)
(c) = cooked; (d) = dried; (f) = fresh; (fr) = frozen; (p) = processed; (r) = ready-to-eat, a Seafood salad,
shrimp salad, b Surimi, fresh and cooked seafood, c Samples (subsamples). d For seafood total (fish and
shrimps).

Adesiyun, 1993). Even if L. monocytogenes is absent in fresh tropical seafood, the

chance of c o n t a m i n a t i o n from the environment of processing plants may be the
same, as from t e m p e r a t e climates.
Using the U S D A m e t h o d with plating onto Oxford agar (Oxoid), ten raw and
h o t - s m o k e d fish samples from a fish market in C o n g o were analysed and found
negative for Listeria spp. (Ben E m b a r e k , unpublished work). Yet, other esculin-
positive organisms were able to grow on the Listeria selective Oxford plates. Fish
caught out of the Indian coast where L. monocytogenes has not been f o u n d (Table
3) have a relative high microbial load (skin, 103-107 c f u / c m 2 ; gills, 104-108 c f u / g ;
intestine, 105-109 c f u / g ) but with large seasonal variation (Surendran and
G o p a k u m a r , 1982). T h e water t e m p e r a t u r e varies from 20 ° to 32°C. Thus the high
ambient t e m p e r a t u r e and microbial competition may inhibit or mask the presence
of the psychrotrophic L. monocytogenes on tropical seafood. If the absence of L.
monocytogenes is confirmed in tropical fresh seafood, it would have wide implica-
tions in the way the hazard 'Listeria' is perceived and controlled in a H A C C P - b a s e d
safety plan for those products.
C o m p a r e d to fish, raw shrimp have a similar prevalence of L. monocytogenes
(1.5-20%), and it is of great c o n c e r n that the same contamination rate can be
f o u n d on processed cooked shrimps which u n d e r g o a listericidal process (Table 3).
Oysters are typically grown in coastal area in waters rich in nutrients and
organic materials. Therefore, the absence of Listeria in oysters is surprising and
should be further investigated. C o m p a r e d to the almost negative findings from the
analysis of live mussels in Italy, three out of 40 (7.5%) samples of fresh mussels
m a r k e t e d in Spain and two out of 147 (1.5%) samples of shellfish (other than
oysters) in J a p a n were f o u n d positive for L. monocytogenes (Table 3). T h e bacteria
have also b e e n isolated from smoked mussels in New Z e a l a n d (Anonymous, 1993d;
Baker et al., 1993). In all three cases, the origins of the bacteria were not
established since the possibility of post-harvest contamination cannot be excluded.
T h u s the situation observed for oysters may also apply for mussels.
 P.K. Ben Embarek / International Journal of Food Microbiology 23 (1994) 17-34 23

4. Prevalence of L. monocytogenes in smoked and lightly preserved fish products

L. m o n o c y t o g e n e s can survive the c o l d - s m o k i n g process ( M a c r a e a n d G i b s o n ,

1990; G u y e r a n d J e m m i , 1991) a n d p r o b a b l y o t h e r light p r e s e r v a t i o n processes like
m a r i n a t i n g a n d curing, b u t does not survive hot-smoking processes ( i n t e r n a l
t e m p e r a t u r e 65°C, 20 min; J e m m i a n d Keusch, 1992). D u r i n g the past few years, L.
m o n o c y t o g e n e s has consistently b e e n isolated from cold-smoked s a l m o n ( T a b l e 4)
a n d its p r e s e n c e in smoked fish has b e e n reviewed recently by J e m m i (1993).
Lightly p r e s e r v e d fish p r o d u c t s like cold-smoked s a l m o n do not u n d e r g o f u r t h e r
listericidal processes a n d h e n c e can be c o n s i d e r e d as a regular source of food-re-
lated ingestion of Listeria cells. T h e r e f o r e , cold smoked s a l m o n has b e e n the

Table 4
Prevalence of L. monocytogenes in lightly preserved fish products
Fish type (country) Number % positive for References
of
Listeria L. monocytogenes
samples
Cold smoked salmon 100 24 Jemmi (1990a)
(Switzerland)
Cold smoked salmon 64 6.3 Guyer and Jemmi
(Switzerland) (1990)
Cold smoked fish 324 13.6 Jemmi (1990b)
(Switzerland)
Cold smoked fish 434 11.3 Jemmi (1993)
(Switzerland)
Cold smoked salmon 33 9 Rorvik and Yndestad
(Norway) (1991)
Smoked salmon 13 23 0 Hartemink and
(Iceland) Georgsson (1991)
Cold smoked salmon 32 31.2 Farber (1991)
(Canada)
Cold smoked salmon 37 0 -80 a 0 Valenti et al. (1991)
(Italy)
Cold smoked salmon 12 75 Hudson et al. (1992)
(New Zealand)
Smoked fish (Canada) 71 11.3 Dillon et al. (1992)
Smoked fish (Canada) 20.4 4.4 Dillon and Patel
(1992a)
Hot smoked fish 496 8.9 Jemmi (1990b)
(Switzerland)
Hot smoked fish 691 8.4 Jemmi (1993)
(Switzerland)
Smoked mussels 14 35.7 Hudson et al. (1992)
(New Zealand)
Ceviche (Peru) 32 75 9 Fuchs and Sirvas (1991)
Marinated, lightly 89 25.8 Jemmi (1990b)
pickled (Switzerland)
a Depending on the origin of the salmon.
24 P.K. Ben Embarek / International Journal of Food Microbiology 23 (1994) 17-34

seafood which has attracted most of the attention from legislators, researchers and
food control agencies concerned with the presence of L. monocytogenes in seafood.
The overall prevalence on cold smoked fish is approx. 10% with a few excep-
tions such as Jemmi (1990a) and Farber (1991) who found a prevalence of 24% and
31%, respectively. In two reports, Dillon and Patel (1992a) and Dillon et al. (1992)
reported a prevalence of Listeria spp. of 20.4% and 11.3%, respectively, in smoked
fish marketed in Canada. No explanations are given for those differences.
The source of Listeria in hot-smoked fish is likely to be post-process contamina-
tion (Jemmi and Keusch, 1992). The prevalence of Listeria in hot smoked fish
(9%) is comparable to the prevalence in cold smoked fish (Table 4). This could be
an indication that contamination of cold smoked fish also occur during or after
processing as live fish do not seem to be the source (Table 1). A clear identifica-
tion of the route of contamination of these products has yet to be established
(Jemmi, 1993).

5. Detection of L. monocytogenes from seafoods

The two common isolation procedures for Listeria developed by the US Food
and Drug Administration (FDA) and the US department of Agriculture (USDA)
are both used for the detection of Listeria spp. in seafood. The FDA methods with
modifications seems to be preferred in North America (Weagant et al., 1988;
Farber 1991; Dillon et al., 1992) while modified versions of the USDA method
seems to be preferred in Europe (Guyer and Jemmi, 1990; Jemmi, 1990a;
Hartemink and Georgsson, 1991; Rorvik and Yndestad, 1991; Fuchs and Reilly,
1992).
Comparing the U S D A method and the cold enrichment method (CE), Hayes et
al. (1991) demonstrated the superiority of the USDA method for isolating L.
rnonocytogenes from naturally contaminated foods. The relative low prevalence of
L. monocytogenes found on seafood in Trinidad (Table 3) by Adesiyun (1993) could
be explained by the use of the CE-method.
Comparing the FDA and the USDA for the recovery of L. monocytogenes in
inoculated seafoods, Lovett et al. (1991) found the FDA better for the isolation of
heat-stressed cells and the U S D A better for the isolation of non heat-stressed cells
with a high background flora. Similarly, Noah et al. (1991) found the Listeria
enrichment broth (LEB) used by the FDA to be superior to the modified
University of Vermont broth (UVM) used in the U S D A method for the recovery of
L. monocytogenes from naturally contaminated seafoods.
In a large inter-laboratory study, Warburton et al. (1991a,b, 1992) found both
above methods to be comparable. A collaborative study by eleven Nordic laborato-
ries reached the same conclusions (West66 and Peterz, 1992). Hayes et al. (1992)
compared both methods and the method of the Netherlands food inspection
service (NGFIS) which use L-PALCAMY as enrichment broth (48 h) and PAL-
CAM as selective plating medium (Van Netten et al., 1989). The three methods
were found to be equivalent.
 P.I~ Ben Embarek / International Journal of Food Microbiology. 23 (1994) 17-34 25

From the above mentioned studies it can be concluded that a method including
two enrichment steps is favoured (Warburton et al., 1991a; West66 and Peterz,
1992). As enrichment broths, LEB (FDA), UVM (USDA) or L-PALCAMY
(NGFIS) seem to be comparable (Hayes et al., 1992; Warburton et al., 1992;
West66 and Peterz, 1992).
As second enrichment broth the USDA's UVM-2 can be replaced with the
modified Fraser broth (MFB) (Warburton et al., 1992). MFB is not very selective
but it gives a visual presumptive positive result. It can therefore be useful as a
screening step in situations where a large number of negative tests can be expected
in products with low interfering flora, i.e., cooked products.
Regarding selective plating media, PALCAM agar or modified Oxford (MOX)
agar should be preferred for products with non-stressed Listeria cells and a large
background flora i.e., cold-smoked salmon. They are both more selective than
Oxford agar (OX) which can be used for products with a low level of background
microorganisms.
When examining products where Listeria cells are expected to be injured, a non
selective pre-enrichment step is recommended (Mc Carthy et al., 1990). Busch and
Donnelly (1992) obtained interesting results with a recently developed repair
broth.
A high number of false-positive results have been encountered by several
workers when analysing seafoods for the presence of Listeria spp.. Ben Embarek
et al. (1994a) found Corynebacterium aquaticum to give Listeria-like results from
salmon samples with the USDA method. Rcrvik and Heidenreich (1993) reported
similar interference with Corynebacterium brevis (sic) and Actinomyces pyogenes in
an ELISA-test kit used on fresh and frozen fish and samples from a fish processing
plant.
Paranjpye et al. (1992) experienced interference with lactic acid bacteria on
Oxford agar while Dillon and Patel (1992b) and Warburton et al. (1991a) reported
around a 70% false-positive rate when using Fraser broth in the isolation protocols
for smoked salmon and different food products, respectively.
Jorgensen and Spanggaard (personal communication) also reported seeing Lis-
teria-like colonies on Oxford agar when examining samples from Danish trout
farms.
According to several reports, L. innocua is the Listeria spp. most often isolated
from seafoods (Weagant et al., 1988; Masuda et al., 1992; de Sim6n et al., 1992;
Ryu et al., 1992). This is even more pronounced for reports from tropical areas
(Fuchs and Sirvas, 1991; Adesiyun, 1993) where L. innocua was almost the only
species isolated (Fuchs and Surendran, 1989; Karunasagar et al., 1992). L. innocua
was also the only Listeria spp. isolated from live mussels and oysters (Colburn et
al., 1990; Decastelli et al., 1993). A recent study shows that in the enrichment
broths commonly used, the growth of L. innocua is favoured when grown together
with L. monocytogenes (Petran and Swanson, 1993). Thus, species identification
from a definite number of colonies per plate can be biased. An improvement could
be the replacement of the selective media commonly used (OX, MOX or PAL-
26 P.K. Ben Embarek / International Journal of Food Microbiology 23 (1994) 17-34

CAM) by a recently developed media which visually differentiates the two species
(Poysky et al., 1993).
In most seafoods, Listeria can be expected to be injured or stressed in one way
or another. The use of selective agar for a direct plate count of injured cells is
inappropriate (Ben E m b a r e k and Huss, 1993). Therefore, quantitative isolation of
Listeria on selective plates is at best doubtful. For quantitative estimates, an MPN
count from a repair broth is presently preferred. Farber (1993) found the sensitive
and quantitative kit Listertest T M (VICAM, MA, USA) effective, but the method
can be tedious and laborious if a large number of samples have to be analysed at
the same time (Ben E m b a r e k et al., 1994a).
Following species confirmation, L. monocytogenes isolates can be further identi-
fied for epidemiological purposes. Unfortunately, from the data available in the
literature, the predominance of certain serotypes, phage types (Estela et al., 1992)
or electrophoretic types (Norrung and Skovgaard, 1993) in L. monocytogenes
isolated from seafoods do not differ from those of other foods.

6. Growth and inhibition of L. monocytogenes in seafoods

U n d e r laboratory conditions at otherwise optimal conditions, L. monocytogenes

is capable of growth at temperatures as low as 1°C (Ryser and Marth, 1991).
Inoculated L. monocytogenes Scott A could survive but was unable to grow on two
raw Atlantic whitefish species stored on ice for 3 weeks (Harrison et al., 1991).
L. monocytogenes is able to grow in a wide range of raw and processed products
at refrigeration temperature. However, initial data indicated that inoculated L.
monocytogenes Scott A could survive but was unable to grow on raw salmon
(Macrae and Gibson, 1990) stored at 4°C for 3 weeks. Marginal growth was
obtained with a pool of L. monocytogenes strains on channel catfish fillets stored
at 4°C for 16 d (Leung et al., 1992) and on raw salmon stored at 5°C for 6 d (Ben
E m b a r e k and Huss, 1992). One study (Wang and Shelef, 1992) indicated that L.
monocytogenes Scott A was able to grow at 5°C on inoculated raw cod fillets. The
Listeria count remained unchanged for 10 d, then the numbers increased by more
than 1 log by the end of storage (day 17). At 10°C on raw salmon (Ben E m b a r e k
and Huss, 1992), and at 20°C on raw cod fish (Wang and Shelef, 1992), growth of
L. monocytogenes occurred rapidly, increasing in numbers by approx. 2.2 logs in 4 d
and 5 logs in 48 h, respectively. However, at high temperatures, food spoilage
would occur quickly negating the importance of the growth potential of L.
monocytogenes. In all the studies cited above, L. monocytogenes was grown
overnight at 30°C-370C before inoculation.
The apparent failure of raw fish to support the growth of Listeria spp. at low
t e m p e r a t u r e could be due to the competition from the natural flora of the fish. L.
monocytogenes cells inoculated on pre-sterilized fish stored at 7°C increased 5 logs
in n u m b e r within 2 weeks (Lovett et al., 1990). No information was given on the
sterilization process. Raw salmon with 3 - 4 % ( w / w ) added NaC1 in the water
phase supported the growth of L. monocytogenes at 5°C; i.e., a 3.6 log increase in 2
 P.K. Ben Embarek / International Journal of Food Microbiology 23 (1994) 17-34 27

weeks was observed in one study (Ben Embarek and Huss, 1992) and a 3 log
increase in 20 d in another (Peterson et al., 1993). In our study (Ben Embarek and
Huss, 1992), both raw and brined salmon were vacuum-packed. In the study by
Peterson et al. (1993) little difference was observed between fish packed in an
oxygen-permeable film and fish vacuum packed in an impermeable film. On both
fish, a high aerobic plate count was observed. These data suggest that if part of the
natural flora of fish is inhibitory to L. monocytogenes, it could be sensitive to low
levels of NaC1. Another possibility is that the growth of L. monocytogenes could be
enhanced or favoured by the addition of low levels of NaCI.
Growth of L. monocytogenes on cold-smoked salmon (Farber 1991; Guyer and
Jemmi, 1991; Rorvik et al., 1991; Ben Embarek and Huss, 1992; Hudson and Mott,
1993) as well as on cold-smoked cod (Dillon and Patel, 1993) has been observed at
refrigeration temperatures of 4-10°C.
L. monocytogenes can also frequently be isolated from cooked crab meat
(Weagant et al., 1988; Farber 1991) and is able to grow on inoculated refrigerated
crab meat as well as on cooked shrimp, cooked crawfish tail meat and canned
lobster meat stored at 4°C-5°C; i.e., an increase in these products of about 2 to 3
logs in 1-2 weeks was observed (Brackett and Beuchat, 1990; Farber, 1991; Dorsa
et al., 1993).
Experiments done in broth have shown that the organism can grow in combina-
tions of factors (temperature, pH, NaCl, K-sorbate and Na-sorbate) found in
lightly preserved fish products (Ben Embarek and Huss, 1992). The pH of these
products is typically 5.5-6.5, the NaC1 content between 3% and 5% ( W / W in the
water phase). Due to their extended shelf life at refrigeration temperatures of 5°C
(4 to 12 weeks), cold-smoked salmon and other lightly preserved fish products have
to be considered as potential high risk foods.

7. Heat resistance of L. monocytogenes in seafoods

Seafoods are usually cooked at lower temperatures than meat products. The
regular isolation of L. monocytogenes from cooked and pasteurised seafoods and
the conflicting results obtained on the heat resistance of the bacterium has raised
concern about the effectiveness of pasteurisation processes employed by the
seafood industry. Recent studies have shown that L. monocytogenes can be easily
destroyed by pasteurisation treatments employed in the industry (Table 5). How-
ever, in our study (Ben Embarek and Huss, 1993) we showed that product
characteristics like fat content, can play an important role in the heat resistance of
L. monocytogenes in seafoods. Thus, applying heat resistance results from one
product to another may be hazardous.
Jemmi and Keusch (1992) showed that L. monocytogenes (inoculated at level
10 6 M P N / g ) will not survive a hot-smoking process in trout (65°C for 20 min).
Preliminary studies in our laboratory have shown similar results with trout inocu-
lated with L. monocytogenes at level 102 c f u / g and receiving a hot-smoke treat-
28 P.K. Ben Embarek / In ternational Journal o["Food Microbiology 23 (1994) 17 34

Table 5
Heat resistance of L. monocytogenes in seafoods at 60°C
Strain Product D-values Reference
at 60°C
Scott A Crabmeat 2.61 Harrison and Huang (1990)
Mix of 3 strains Crawfish 1.98 Dorsa et al. (1993)
Mix of 5 strains Lobster meat 2.39 Budu-Amoako et al. (1992)
062 (human isolate) Cod 1.98 Ben Embarek and Huss (1993)
057 (fish isolate) Cod 1.95 Ben Embarek and Huss (1993)
062 (human isolate) Salmon 4.48 Ben Embarek and Huss (1993)
057 (fish isolate) Salmon 4.23 Ben Embarek and Huss (1993)

ment of 65°C for 15 min. However, the organism was able to survive a lower heat
treatment, i.e. 15 min at 60°C (Ben Embarek, unpublished results).

8. Elimination and control of L. monocytogenes in seafoods: research needs

Ready-to-eat seafoods show a high prevalence of L. rnonocytogenes (Tables

2-4) and some support the growth of the organism to high levels during their
normal shelf life, making them potentially high-risk foods. The processing of such
products (i.e., cold smoked salmon) involves several 'non-aseptic' steps which
makes it unlikely that complete elimination of the organism can be achieved,
however a reduction in levels is a possible goal.
It is now suggested that the hazard analysis critical control point (HACCP)
approach should be used to control Listeria in the food supply (Farber, 1993). The
application of the HACCP-system to the production of seafoods has been de-
scribed (Huss, 1992; Huss, 1994). Since 1993, HACCP-based quality management
plans are mandatory in the seafood industry in Canada. In Europe, following a
council directive on seafoods (EEC, 1991) the seafood industry is moving toward a
HACCP-based system. The same approach is required from foreign producers
exporting to the European Union (Ababouch, 1993; Lima dos Santos et al., 1993).
A similar approach is expected to be introduced in the United States.
Whether L. m o n o c y t o g e n e s is a natural part of the bacterial load of seafood or
arise as a contaminant during processing will dictate how the organism can be
controlled in a HACCP-based system. Before HACCP-plans can be effectively
applied to eliminate or control Listeria in seafoods, some issues remain to be
resolved: (a) The suggestion from the available literature that the organism is not
present on live fish has to be confirmed (Table 1). (b) Apparent differences in the
isolation of the organism from freshwater and from open, clean seawater remain to
be resolved. (c) Failure to isolate L. m o n o c y t o g e n e s from tropical seafoods in India
should be confirmed for other tropical areas and the reasons elucidated. (d) The
same questions have to be answered for bivalves and other shellfish.
 P.K. Ben Embarek / International Journal of Food Microbiology 23 (1994) 17-34 29

Table 6
Seafoods involved in recalls and import alerts issued in the United States in 1993 due to contamination
with L. monocytogenes
Fish type Origin Quantity Reference
Cold smoked salmon Canada, Scotland, Anonymous (1993c)
Chile, Denmark,
United Kingdom,
Faroe Island, Norway
Cold smoked salmon, USA 600 pounds Anonymous (1993e)
sliced
Cold smoked tuna USA 41.93 pounds Anonymous (1993a)
Frozen salmon caviar Canada Anonymous (1993c)
Frozen cooked lobster Canada Anonymous (1993c)
meat
Stone crab meat Chile Anonymous (1993c)
IQF shrimp Chile Anonymous (1993c)
IQF shrimp USA 50272 cases Anonymous (1993e)
Toasted Sea Eel Japan Anonymous (1993c)
Frozen broiled eel Taiwan Anonymous (1993c)
Frozen seasoned Korea Anonymous (1993c)
Pollack roe
Surimi products Korea Anonymous (1993c)
Smoked mussels New Zealand Anonymous (1993c, 1993e)
Cooked crab meat Russia Anonymous (1993c)
Crab meat USA 394 pounds Anonymous (1993e)

Several workers have noticed a significant proportion of false-positive results

from Listeria analysis of seafoods (see above) prompting the need for methods and
media developed for the isolation of L. monocytogenes from these products.
Another way of controlling the presence and growth of the organism could be
by the introduction of appropriate 'hurdles' in the foods (Wang and Shelef, 1992;
Farber, 1993). The use of naturally occurring bacteria in seafoods with antagonistic
activity against Listeria spp. at low temperature has shown interesting results (Ben
Embarek et al., 1994b; Jeppesen and Huss, 1993a,b) and could eventually be more
acceptable to regulatory agencies than the direct use of bacteriocins.
While some European countries (Germany, United Kingdom, Denmark), as well
as Australia and Canada have shifted to a food group risk-based approach toward
the control of Listeria, the United States still has a 'zero-tolerance' policy. On a
regular basis (Table 6), seafoods from national and foreign producers are recalled
due to the presence of L. monocytogenes with a resulting severe economic loss for
the producers. This situation emphasises the need for the seafood industry to
assure a supply of Listeria-free seafood especially for products which receive a
listericidal process such as hot-smoking, cooking or pasteurisation. It also stresses
the need to determine whether and how cold smoked products can be produced
free of L. monocytogenes.
30 P.K. Ben Embarek / hzternational Journal of" Food Microbiology, 23 (1994) 17-34

Acknowledgements

T h e a u t h o r wish to t h a n k p r o f e s s o r H a n s H e n r i k Huss, D r L o n e G r a m and D r

V i b e k e F r o m J e p p e s e n for their valuable c o m m e n t s to t h e m a n u s c r i p t .

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