Aalberts, C.H.J.

Luten, J.B.

Pelt, H.M.L., van

English

Wageningen Yield 

60 
CONTAMINATION OF COLD-SMOKED FISH WITH 
LISTERIA MONOCYTOGENES 
Camiel Aalberts, Hilde van Pelt, Joop Luten 
Netherlands Institute for Fisheries Research (RIVO), P.O. Box 68, 1970 AB IJmuiden, 
The Netherlands 
Keywords: cold-smoked fish, Listeria monocytogenes 
Summary 
In the Netherlands, 41 % of ready-to-eat cold-smoked fish is contaminated with the 
human pathogenic bacterium Listeria monocytogenes. Therefore sources of 
contamination in fish smoking plants were investigated. A survey in three plants shows 
that both L. monocytogenes as L. spp are easy to isolate from floors, machines, tables, 
and hands and aprons of employees. Total elimination of contamination will not be 
possible, so the focus will be on measures to control contamination like the use of risk 
assessment in the evaluation of product safety, improvement of cleaning and disinfection 
procedures, and monitoring of processing steps. To enable tracing of contamination 
routes, two methods of typing of L. monocytogenes are set up. A combination of limited 
serotyping and resistance to tetracycline, cadmium, and arsenic provides a set of 9 
biotypes. Analysis of samples from different European countries shows large differences 
in the presence of the various biotypes. A more discriminatory typing method, AFLP, 
will be used further in attempt to identify contamination routes. 
Introduction 
L. monocytogenes, a human pathogen, is mainly found in contaminated food (1). 
Reported cases are associated with industrially processed food (2). Other suspected 
sources are cross-contamination in the distribution of foods (3) and the kitchen (4). A 
survey on Dutch fish and fish products, carried out in the past year by our institute, 
shows a high incidence of contamination with Lmonocytogenes (Table 1). Fresh fish 
fillets are quite often contaminated with L. monocytogenes. However, when properly 
heated during cooking, this opposes no risk to the consumer. With the ready-to-eat 
smoked fish there is a higher risk of infection, in 41% of the cold-smoked fish samples 
Listeria monocytogenes was present. 
This raises the need for control of contamination of L. monocytogenes of fish 
and fish products, especially those products which do not undergo any final 
listericidal treatment, like cold-smoked salmon. In order to find measures to control 
61 
contamination during manufacturing of cold-smoked salmon, a survey on the 
presence of Listeria species was carried out in fish smoking plants. Critical points 
of contamination and control were investigated and evaluated. The results of this 
study, including typing, will be presented in this paper. 
Table 1. Incidence of L. monocytogenes in Dutch fish and fish products (1998-1999), obtained 
from supermarkets, fish markets. Isolation method according to ISO 1 1290-1: 1996 (5). 
Product 
Fresh fillets 
Atlantic salmon 
Plaice 
Pollack 
Rainbow trout 
Red fish 
Whiting 
Total 
Product 
Cold-smoked fillets 
Atlantic salmon 
Cod 
Halibut 
Herring 
Marlin 
Rainbow trout 
Trout 
Tuna 
Total 
Product 
Other 
Salmon cubes, smoked 
Salmon cuts, smoked 
Sprat, smoked 
Shrimps, fresh 
Total 
Number of 
1 
3 
I 
2 
1 
2 
10 
Number of 
54 
4 
8 
6 
1 
2 
3 
4 
82 
Number of ; 
1 
1 
5 
1 
8 
samples 
samples 
(amples 
Number and % 
L. spp 
1(100) 
2(67) 
0(0) 
2(100) 
1(100) 
2(100) 
8(80) 
Number (%) oi 
L. spp 
31(57) 
3(75) 
5(63) 
2(33) 
1(100) 
2(100) 
0(0) 
4(100) 
48(59) 
Number (%) ot 
L. spp 
0(0) 
1(100) 
2(40) 
0(0) 
2(25) 
of sam| 
L. 
I'sampl 
L. 
'sampli 
L. 
îles positive for 
monocytogenes 
1(100) 
2(67) 
0(0) 
2(100) 
1(100) 
2(100) 
8(80) 
es positive for 
monocytogenes 
23(43) 
1(25) 
4(50) 
1(17) 
1(100) 
1(50) 
0(0) 
3(75) 
34(41) 
3s positive for 
monocytogenes 
0(0) 
0(100) 
1(20) 
0(0) 
1(13) 
Materials and Methods 
Sampling method and microbiological analysis 
Swab samples (5 sterile swabs containing 3 ml peptone saline solution) taken from 
surfaces (10 cm2) and product samples (25 g) were incubated in 225 ml Half Fraser broth 
62 
for 1 day at 30°C as a pre-enrichment and further analysed according to ISO 11290-1 (5). 
This means enrichment in Fraser broth for 2 days at 37°C, isolation on PALCAM and 
Oxford media, confirmation of typical colonies with Gram staining, catalase, oxidase, 
glucose and xylose fermentation, and ß-haemolysis on sheep-blood agar. Indentification 
of L. spp isolates was carried out by using API-Listeria rM (6), enabling the distinction 
between species (Table 2). Differentiation on species level is useful, not only to identify 
the pathogenic L. monocytogenes, but also to use the other identified species for 
identifiing contamination. 
Table 2. Listeria species able lo identify with API-Listeria™ (6) 
Species 
Listeria monocytogenes 
Listeria innocua 
Listeria ivanovii 
Listeria grayi 
Listeria seeligeri 
Listeria welshimeri 
Typing 
Further identification of L monocytogenes isolates was carried out according to (7), 
using a typical set of reactions enabling to obtain 9 different biotypes based on a 
combination of serotyping and resistance / sensitivity tests to cadmium, arsenic, and 
tetracycline. 
Table 3. Differentiation of L. monocytogenes in nine biotypes, based on a combination of 
serotyping and resistance / sensitivity to cadmium, arsenic, and tetracycline (7) 
Biotype 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Serogroup 
1/2 
1/2 
1/2 
1/2 
4 
4 
4 
4 
4 
Tetracycline 
s 
s 
s 
s 
r 
s 
s 
s 
s 
Sensitivity to(l» 
Cadmium 
s 
r 
s 
r 
r 
r 
s 
s 
r 
Arsenic 
s 
s 
r 
r 
s 
s 
r 
s 
r 
1) s: sensitive, r: resistant 
Since typing as mentioned above will not provide enough information to 
differentiate between isolated strains (7, 8), experiments have started using AFLP, a 
method based on DNA restriction analysis with a high discriminatory power (9). 
63 
Sampling strategy 
Three different Dutch fish smoking plants have been visited. Samples were 
taken in the consecutive process areas, in the opposite direction starting with 
packaging area. Sampled sites are categorised as shown in Table 4, in order to 
compare results between the three fish smoking plants. In one plant the filleting and 
packaging took place in one room, but were separated from each other. 
Table 4. Sampling sites, ordered by category in four process areas 
Area Category Sampling sites 
fresh fish area floor personnel entrance, product entry 
product skins, flesh 
waste product heads, skins, bones 
waste water water from wash basin, brine 
work surface tables 
employee hands, aprons 
machines deboner, brine injector, knives 
containers trays 
smoking area floor 
product 
smoking cabinet 
entrance to smoking cabinet 
salmon fillets 
floor, racks 
processing of 
smoked product 
floor personnel entrance, product entry 
product salmon fillets 
waste product fish skins, smoke offcuts, pinbones 
work surface tables 
employee hands, aprons 
machines cutting blades, conveyor belts 
containers trays 
cutting and packaging floor 
product 
waste product 
work surface 
employee 
machines 
containers 
personnel entrance, product entry 
salmon fillets, cuts 
offcuts 
tables, scales 
hands, aprons 
cutting blades, conveyor belts 
trays 
Results and discussion 
Fish smoking plant survey 
Results of the first series of surveys of three fish smoking plants are presented in 
Table 5. In all three fish smoking plants, species of Listeria were found and in plant I and 
III L. monocytogenes was found. In plant IL . monocytogenes was found only in the fresh 
fish area, whereas in plant in L monocytogenes was found in all areas, except the 
smoking area. No L monocytogenes was found in the final products of all three plants. 
According to the sites of isolation in plant III, L. monocytogenes does not seem to 
inhabit any particular site. For example, L. monocytogenes was found in waste water 
64 
(deboner), but no L. monocytogenes has been identified in any product afterwards. 
Therefore no specific measures to eliminate L. monocytogenes can be put forward yet. 
The three plants are now improving their code of practice, cleaning and disinfection. At 
the end of this year, all plants will be sampled again. 
Table 5. Contamination of fish processing plants with Listeria species and Listeria 
monocytogenes in particular 
Area Sample Category Fish smoking plant 
I II III 
Fresh fish area Floor 
Product 
Waste product 
Waste water 
Work surface 
Employee 
Machines 
Containers 
L. monocytogenes L. innocua L. monocytogenes 
L. seeligeri 
n.t. 
n.t. 
L. monocytogenes 
L. monocytogenes 
L. monocytogenes 
L. welshimeri, 
L. innocua 
L. monocytogenes 
Smoking area Floor 
Product 
Smoking cabinet 
L. monocytogenes 
L. seeligeri 
L. grayi, L. innocua 
L. innocua 
Processing of Floor 
smoked product1' 
Product 
Waste product 
Work surface 
Employee 
Machines 
Containers 
L.seeligeri L. innocua, 
L. welshimeri 
L. monocytogenes, 
L. welshimeri 
L. innocua L. welshimeri, 
L. seeligeri 
L innocua -
L. innocua -
L. innocua -
Cutting and 
packaging area1' 
Floor L. seeligeri L.seeligeri L. welshimeii, L.innocua 
Product - - L.welshimeri, 
Waste product - L. innocua L. seeligeri 
Work surface 
Employee 
Machines 
L. monocytogenes 
L. welshimeri, 
M. L. monocytogenes 
Containers 
End product 
1): In factory I Filleting and Packing was carried out in one room. 
-: no Listeria spp isolated, n.t.: not tested 
65 
Typing 
Twelve isolates of L. monocytogenes of cold-smoked salmon from the market 
survey show that 7 biotypes are present in the Netherlands, in comparison with 
Iceland (1 biotype out of 21 samples of cold-smoked fish), France (8 out of 150), 
and Denmark (2 out of 9). The large variation in biotypes and number of different 
biotypes between countries has yet not been explained. 
Table 6. Differentiation of 12 isolates of L. monocytogenes from Duth fish samples (sec Table 3). 
Biotype Incidence 
j J 
2 4 
3 
4 1 
5 
6 3 
7 1 
8 I 
9 1 
Total Î2 " 
AFLP fingerprinting 
Six of the 12 isolates mentioned above are used for AFLP fingerprinting. Each 
isolate used is from a different biotype according to table 3. Fig. 1 shows that each 
different biotype can be discriminated from another by looking at the position and 
color of the bands (variation in concentration of molecules with the same number of 
base-pairs). This means that AFLP is at least as discriminating as the method of 
Duarte (7). Experiments are to be continued, analysing isolates from fish smoking 
plant surveys. 
66 
Base pc 1rs J 
800 
4S0-
400 
3 6 0 -
300 
2 5 0 -
2 0 0 -
160 
1 0 0 -
« 0 -
«•>., » 
» i ;•
 (; 4 >£ « v 
\ \ r ^ -
Ç^ çW 
^ IÉÉP*" * * " 
«ü^# W*ffM j^WiV * * * * » ffijfc 
52 62 83 88 92 94 Strains 
Fig. 1. Digital electrophoresis image of' AFLP fragments of Listeria monocytogenes run on an 
ALF-Expcress automatic DNA sequencer. Strain numbers do not refer directly to any strain 
mentioned in this article. 
67 
Conclusions 
The high incidence of Duch fish products contaminated with Listeria 
monocytogenes puts a pressure on smoking plants to comply with the Dutch 
legislation: Listeria monocytogenes should be absent in 25 g of product. Emphasis 
should be put on means to control contamination: e.g. the use of risk assessment for 
the evaluation of process safety, monitoring of processing steps instead of relying 
on end-product testing, as also recently has been advised a.o. by the FAO (10). 
Acknowledgements 
Technical assistance of Jos Buntsmaa-Hamers (RIVO) and Jack Speur (RIVO) 
is appreciated as well as Gabriela Duarte (ESB, Porto Portugal) for sero- and 
resistance typing, and Henk Aarts RIKILT, Wageningen The Netherlands) for 
assisting in setting up the AFLP analysis. 
The research is funded by the Netherlands Ministry of Agriculture, Nature 
Management and Fisheries and by the European Commission (FAIR CT95-I207: 
Spoilage and Safety of Cold-smoked Fish). 
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68 
8. Kcrouanton, A.A. Brisabois, E. Denoycr, F. Dilasscr, J. Grout, G. Salvat, B. Picard. 
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Contamination of cold-smoked fish with Listeria monocytogenes

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