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Applied and Environmental Microbiology, June 2001, p. 2586-2595, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2586-2595.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Elucidation of Listeria monocytogenes
Contamination Routes in Cold-Smoked Salmon Processing Plants
Detected by DNA-Based Typing Methods
Birte
Fonnesbech
Vogel,1,*
Hans Henrik
Huss,1
Bente
Ojeniyi,2
Peter
Ahrens,3 and
Lone
Gram1
Danish Institute for Fisheries
Research, Department of Seafood Research, Søltofts Plads,
Technical University of Denmark, DK-2800 Kgs.
Lyngby,1 Department of Veterinary
Microbiology. The Royal Veterinary and Agricultural University,
DK-1870 Frederiksberg,2 and Danish
Veterinary Laboratory, DK-1790 Copenhagen V,3
Denmark
Received 29 December 2000/Accepted 12 March 2001
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ABSTRACT |
The contamination routes of Listeria monocytogenes in
cold-smoked salmon processing plants were investigated by
analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in
plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In
plant II, the levels of both raw fish and product contamination varied
from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product
samples were positive for L. monocytogenes). A total
of 429 strains of L. monocytogenes were subsequently
compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the
products were identical to types found on the processing equipment and
in the processing environment, suggesting that contamination of the
final product (cold-smoked salmon) in both plants (but primarily in
plant I) was due to contamination during processing rather than to
contamination from raw fish. However, the possibility that raw fish was
an important source of contamination of the processing equipment and
environment could not be excluded. Contamination of the product
occurred in specific areas (the brining and slicing areas). In plant I,
the same RAPD type (RAPD type 12) was found over a 4-year period,
indicating that an established in-house flora persisted and was not
eliminated by routine hygienic procedures. In plant II, where the
prevalence of L. monocytogenes was much lower, no RAPD
type persisted over long periods of time, and several different
L. monocytogenes RAPD types were isolated. This
indicates that persistent strains may be avoided by rigorous cleaning
and sanitation; however, due to the ubiquitous nature of the organism,
sporadic contamination occurred. A subset of strains was also typed by
using pulsed-field gel electrophoresis and amplified fragment length
polymorphism profiling, and these methods confirmed the type division
obtained by RAPD profiling.
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INTRODUCTION |
Listeria monocytogenes is
a gram-positive, food-borne pathogen that is widely distributed in the
environment and occurs naturally in many raw foods. L. monocytogenes is psychro- and halotolerant (33) and
consequently can grow in many lightly salted and chilled food products,
which often have extended shelf lives (3, 11). Products
which do not receive any heat treatment by the consumer, so-called
ready-to-eat products like cheese, meat, and fish delicatessen products, may contain high levels of L. monocytogenes
when they are eaten. Ingestion of high numbers of L. monocytogenes cells is a significant health threat for people in
risk groups such as immunocompromised and elderly groups. In these
groups, the rate of mortality from listeriosis is high, typically 20 to
30% (13). Also, infection by L. monocytogenes in pregnant woman may cause abortion, stillbirth, or
delivery of an acutely ill baby (11). A diverse range of
foods has been associated with both outbreaks and sporadic cases of
listeriosis (25).
Ready-to-eat fish products like cold-smoked fish have been linked to
sporadic cases of listeriosis, and more recently, epidemiological evidence has suggested that listeriosis has been caused by smoked mussels (4), gravad trout (9), and smoked
trout (26). As such products support growth of
L. monocytogenes (20), it is crucial to
reduce the prevalence and level of this organism to an absolute
minimum. An important prerequisite for control of L. monocytogenes is knowledge concerning its niches during food
production. Cold-smoked salmon has become a major fish delicatessen commodity, and as the source of L. monocytogenes
contamination during cold-smoked salmon production is not known, we
focused on elucidating the contamination routes of this bacterium in
cold-smoked salmon processing environments.
L. monocytogenes occurs naturally in fish raw
materials, and Farber (10) reported the presence of
L. monocytogenes in salmon from the United States,
Chile, Norway, and Canada. The prevalence of L. monocytogenes in raw fish is quite low, ranging from 0 to 1%
(2, 21) to 10% (20), but a higher prevalence
can probably occur in fish from bodies of water that receive heavy
runoff from land (18). L. monocytogenes
may be detected in a large proportion of freshly produced cold-smoked
fish; typically, it is detected in 10 to 40% of samples (2,
22). Great plant-to-plant variation is seen; some production
plants are virtually free of L. monocytogenes, and
others have a prevalence close to 100% (20, 22).
A number of studies have shown that L. monocytogenes is
able to reside in food processing plants, including poultry production plants (24, 29, 40), meat processing plants (15,
28), ice cream plants (27), shrimp peeling plants
(5), and plants in which gravad (2) and
smoked trout (30) are produced. While some studies have
pointed to raw materials as the most likely sources of product
contamination with L. monocytogenes (7, 15), other studies have found that the major source of
contamination is the processing environment and equipment (2, 21,
30, 40).
Early studies of contamination routes depended solely on isolating and
counting the organism at different places along the processing line
(7), whereas recent studies have been greatly facilitated by the use of molecular typing methods with high
discriminatory power. These methods have included pulsed-field gel
electrophoresis, (PFGE) (2, 5, 27, 29) and randomly
amplified polymorphic DNA, (RAPD) profile analyses (5, 24,
39). The amplified fragment length polymorphism (AFLP)
technique, which is a very powerful molecular typing technique
(19), has not been used previously to trace contamination
routes of L. monocytogenes but has been used
successfully to fingerprint Pseudomonas isolates from a
poultry processing plant (14).
The purpose of the present study was to elucidate the L. monocytogenes contamination routes during the production and
processing of Danish cold-smoked salmon. L. monocytogenes was isolated from cold-smoked salmon from two
Danish smokehouses in 1995 and 1996, and the processing
environments of these plants, as well as the final products, were
sampled in 1998 and 1999. A total of 429 strains of L. monocytogenes were subsequently grouped by RAPD profiling.
Concerns about the reproducibility and stability of this method have
been raised (41); however, careful standardization allowed
us to reproducibly obtain a high level of discrimination with this
method (12). To confirm clonal separation, a subset of
strains (mainly the persistent types) was also characterized by PFGE
and AFLP profiling.
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MATERIALS AND METHODS |
Processing plants and product manufacturing.
The raw
material was farm (ocean)-raised salmon (Salmo salar)
from Norway and the Faroe Islands. The fish were gutted; they were then
transported to the smokehouses stored in ice, or occasionally they were
stored and transported frozen (less than 10% of the fish). Fish stored
in ice were used 1 to 5 days after slaughter. The ambient temperatures
in both plants were between 10 and 17°C.
In the raw fish processing areas of plant I, head cutting, filleting,
brining (injection of saturated brine), and removal of the skin were
done by commercial machines in one flow. The fillets were held in a
saturated salt solution from a few minutes to 2 h before cold
smoking (16 h at 22°C). The amount of brine and salting was adjusted
to obtain an NaCl level of approximately 4% (water phase salt) in the
final product. The smoked fillets were quick frozen and stored for a
few days at 
18°C. The fish were sliced and packed in a building
separate from the building in which raw fish were handled and smoking
took place. The fish were manually trimmed before slicing. Each of the
two slicing areas contained four commercial slicing machines (Mass)
coupled in pairs to two production lines. The sliced smoked fish were separated into 100-, 150-, or 200-g portions before vacuum packing.
In the raw fish processing area of plant II the head of each salmon
was cut off by hand, and the fish were washed in a commercial
fish
washer. The fish were mechanically filleted, brined (saturated
brine)
with commercial injection machines, and manually trimmed.
The brined
fillets were ripened for 18 h at 0°C with a cover layer
of salt.
The amount of brine and salting was adjusted to obtain
an NaCl level of
approximately 4% (water phase salt) in the final
product. After
cold-smoking (16 h at 22°C), the skin was mechanically
removed, and
the fillets were quick frozen and stored for a few
days at
18°C.
The fillets were sliced with three commercial slicing
machines (Mass)
in the slicing area of plant II and were manually
separated into 50- 100- or 200-g portions before vacuum packing.
The different processing
operations took place in different rooms
with a continuous process
flow, and there was negative airflow
in the slicing area. Thus, care
was taken to separate the raw
material from the processed material. A
strict procedure regarding
personal hygiene was followed. Disinfecting
foot baths were installed
at the entrance to each area, and all
employees wore gloves which
were changed every 1.5 h at each
break. The slicing machines were
cleaned and disinfected with ethanol
twice a
day.
A complete cleaning and disinfecting procedure was carried out in both
plants at the end of each production day, fulfilling
legal
requirements, and once a week a decalcification procedure
was
performed. All removable parts of the machines and conveyer
belts were
cleaned and disinfected separately. Cleaning was performed
by using low
pressure and foam cleaners. In plant I primarily
sodium hypochlorite
was used as the sanitizing agent, whereas
in plant II peracetic acid
was
used.
Sampling procedure.
A total of 944 and 869 samples were
collected during two visits at processing plants I and II, respectively
(Tables 1 and 2). The
plants were visited at times when there was high throughput (before
Christmas) and at times when there was less activity (during the
spring). The same sampling strategy was used at both plants. Samples
were taken at control points, which were contact surfaces during
different production steps, at the start and at the end of production
for 2 to 3 days. Cleaning control samples for equipment surfaces were
taken once during each visit after disinfection and just before the
start of production. Samples were also obtained before and after
interval cleaning of slicing machines in processing plant II. The
sampling areas varied depending on the sampling location. The smallest
areas were screw heads (area approximately 1 cm2); the
largest areas were surfaces of conveyor belts (area up to 1 m2). Other product samples from processing plants I and II
were also analyzed (Tables 1 and 2). During each visit a number of whole raw fish (S. salar) were selected, and samples of
these fish were taken from the raw fish during production; samples of the final products were also taken (Tables
3 and 4). In addition, we took swab samples from surfaces (equipment surfaces, conveyor belts,
gloves, etc.) that came into contact with these fish and of the
processing environment (walls, tables, below machines, trucks, drains,
etc.) when the fish were processed. At plant I, 18 and 12 fish were
monitored during the two visits, and at plant II 15 and 14 fish were
monitored (Tables 3 and 4).
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TABLE 1.
Numbers of samples, numbers of L. monocytogenes-positive samples, and distribution of RAPD types
for L. monocytogenes isolates from processing plant I
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TABLE 2.
Numbers of samples, numbers of L. monocytogenes-positive samples, and distribution of RAPD types for
L. monocytogenes isolates from processing plant II
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TABLE 3.
Incidence of RAPD types for L. monocytogenes strains from samples obtained at different
production stages for 18 fish monitored during production in plant
I in November 1998
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TABLE 4.
Incidence of L. monocytogenes RAPD types
from samples obtained at different production stages for 15 fish
monitored during production from plant II in October 1998
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All sampling sites in the production environment, machines, and aprons
of employees were swabbed with sterile cotton swabs
or cellulose
sampling sponges (Bio-spo CS-100; Solar Biologicals
Inc., Ogdensburg,
N.Y.) moistened with 0.1% peptone-0.85% saline.
Sampling of surface
areas after disinfection was done with sterile
sponges premoistened
with neutralizing buffer (Bio-spo BS-10NB;
Solar Biologicals Inc.).
After sampling the swabs and sponges
were soaked in 20 and 40 ml of
Listeria selective medium (UVM
1) [see below],
respectively, and kept at 10 to 15°C during transport
to the
laboratory. Samples of raw fish and samples after every
processing step
were collected by scraping the surface with a
sterile jagged knife, and
each of these samples was mixed with
20 ml of UVM 1. Gloves of the fish
handlers were placed in sterile
plastic bags containing 40 ml of UVM 1;
we made sure that only
the outer surfaces of the gloves were in contact
with the liquid.
Twenty milliliters of saturated brine was mixed with
100 ml of
brain heart infusion (CM225; Oxoid Ltd., Basingtake,
Hampshire,
England) to dilute the salt. After incubation at 30°C for
24 h,
1 ml was mixed with 20 ml of UVM 1. Samples of the final
product
that had been stored at 5°C for 1 and 21 days were examined
by
combining cross sections from two packages into one 25-g sample
that
was suspended in 225 ml of UVM
1.
As part of its quality assurance program, processing plant II has a
standard sampling regime for
L. monocytogenes. A total
of 1,712 samples were collected by the processor as part of routine
checks during a 10-month period (Table
5). Twenty-five-gram samples
were cut off
the bellies of raw fish and 25-g samples were also
collected from
products and suspended in 225 ml of UVM 1. Samples
taken from the
production environment during production and after
cleaning and
disinfection were swabbed with sterile cotton swabs
and soaked in UVM
1.
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TABLE 5.
Numbers of samples, numbers of L. monocytogenes-positive samples, and distribution of RAPD types
for L. monocytogenes isolates from processing plant
IIa
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Bacteriological analyses.
L. monocytogenes
strains were detected (and isolated) by a two-step enrichment
procedure. Samples were mixed with modified University of Vermont broth
1 (Oxoid Listeria enrichment broth base [CM863] with Oxoid
Listeria selective supplement UVM 1 [SR 142]) and
incubated for 24 h at 30°C. One milliliter was transferred to 10 ml of modified University of Vermont broth 2 (Oxoid Listeria enrichment broth base [CM863] with Oxoid Listeria
selective supplement UVM 2 [SR 143]) and incubated for 24 h at
30°C. If visible growth occurred with UVM 2, a loopful was plated on
Oxford selective agar (Listeria selective agar base
[CM856] with Listeria selective supplement Oxford [SR
140]; Oxoid) and incubated at 37°C for 48 h. Three presumptive
Listeria colonies per plate were identified as L. monocytogenes colonies, by beta-hemolysis, positive Gram and
catalase tests, motility (as determined by phase-contrast microscopy),
and the ability to ferment rhamnose and methyl mannoside but not
xylose. Isolates collected at processing plant II (Table 5) were
verified by the laboratory of the processor as L. monocytogenes isolates by using the Accuprobe L. monocytogenes culture identification test (Gen-Probe Inc., San
Diego, Calif.). Thirty strains from products from processing plant I
isolated in November 1995 or April 1996 (12) were also included.
RAPD typing.
RAPD analysis was performed as described
previously (12). Briefly, DNA was prepared with Dynabeads
DYNAL DIRECT system 1 (Dynal A/S & Nordic, Oslo, Norway). Two
microliters of the DNA preparation was transferred to a PCR tube
containing a PCR mixture (Ready-To-Go RAPD analysis beads [Amersham
Pharmacia Biotech, Inc., Piscataway, N.J.]) dissolved in 23 µl
of Milli Q water along with 4 µM primer HLWL85 (5'-ACAACTGCTC;
DNA Technology, Aarhus, Denmark). The PCR was performed with a
thermocycler (model 9600; Perkin-Elmer, Norwalk, Conn.) by using 45 cycles of 1 min at 95°C, 2 min at 35°C, and 1 min at 72°C,
followed by 10 min at 72°C. Amplification products were visualized
after electrophoresis in a 2% agarose gel by staining with ethidium
bromide. A 100-bp ladder (Amersham Pharmacia Biotech Inc.) was included
three times in each agarose gel as a standard. RAPD reaction mixtures
without bacterial DNA and DNA preparations from L. monocytogenes strains La22 and La150 (isolated from
cold-smoked salmon) and H4239 (isolated from a human case in
Denmark in 1998) were used as negative and positive controls,
respectively
PFGE typing.
PFGE was performed as described previously
(29), with a few modifications. The restriction enzyme
used was ApaI (Boehringer, Mannheim, Germany). The
restriction fragments were separated by using the polygonal
contour-clamped homogeneous electric field system (CHEF-DR III;
Bio-Rad, Richmond, Calif.). The initial pulse time was 1 s, and
the final time was 15 s. The running time was 20 h.
AFLP analysis.
All AFLP procedures were performed as
described by Kokotovic et al. (23); however, the enzyme
combination was changed, and consequently the primers were also
changed. Genomic DNA were extracted from strains of L. monocytogenes by using an Easy-DNA kit (Invitrogen, De Schelp, The
Netherlands) according to the manufacturer's instructions. Bacterial
DNA (100 to 500 ng) was digested with 10 U of BamHI and 10 U
of EcoRI at 37°C for 3 h in EcoRI buffer
(New England Biolabs, Beverly, Mass.). Double-stranded adapters were
prepared by mixing equimolar amounts of corresponding oligonucleotides (Table 6) and were incubated for 10 min
at 65°C and cooled for 15 min at room temperature. Adapters were
ligated to the restriction fragments by using a 20-µl (total volume)
reaction mixture containing 5 µl of the digested DNA, 2.6 pmol
of the BKO-RC adapter, 26 pmol of the BKO-FC adapter, 1 U of T4 DNA
ligase, 2 µl of 10× ligase buffer (Amersham Pharmacia, Cleveland,
Ohio), and 8 µl of TAC buffer (38). Ligation was carried
out overnight at room temperature. Two microliters of 10-fold-diluted
ligation product was transferred to a PCR tube containing 48 µl
of a PCR mixture containing (final concentrations) 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 2.5 mM MgCl2, each of the four
deoxynucleoside triphosphates (Perkin-Elmer) at a concentration of 200 µM, 130 ng of primer BamHI0 (DNA Technology), 130 ng of primer
EcoRIO-F (labelled at the 5' end with 6-carboxyfluorescein; Oswell DNA
Services, Ltd., Southampton, United Kingdom), and 0.3 U of
Taq polymerase (Perkin-Elmer). Fragments were amplified with a thermocycler (Biometra T3 thermocycler) by using 3 min at 94°C, followed by 23 cycles of 60 s at 94°C, 60 s at 54°C, and
90 s at 72°C and then 10 min at 72°C. The amplification
products (1-µl portions of PCR products and 0.25-µl portions of
internal-lane size standards labelled with ROX, [GeneScan-500 ROX size
standard; Perkin-Elmer Applied Biosystems, Warrington, England]) were
analyzed by electrophoresis in 5% denaturing polyacrylamide gels for
3.5 h using an ABI 377 automated sequencer (Perkin-Elmer). The
AFLP patterns were collected with the GeneScan software (Perkin-Elmer Applied Biosystems). DNA preparations obtained from L. monocytogenes strains La22 and La150 (isolated from
cold-smoked salmon) and H4239 (isolated from a human case in
Denmark in 1998) were included in each gel to control reproducibility.
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TABLE 6.
Adapter and primer oligonucleotides used for AFLP DNA
typing of L. monocytogenes from cold-smoked
salmon processing plants
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Numerical pattern analysis.
Photographs of RAPD patterns
were scanned with a Pharmacia DeskTop scanner (Pharmacia Biotech) and
transferred the PC Windows software package GelCompar (version 4.1;
Applied Maths, Kortrijk, Belgium) as tiff files. AFLP patterns were
transferred as densitometric values to GelCompar (version 4.1), and
gels were normalized by using the internal size standard ROX-500
(Perkin-Elmer Biosystems), which was added to each lane. RAPD gels were
normalized by using a 100-bp ladder (Pharmacia Biotech) that was
included three times in each agarose gel as a standard. The data
analyzed were transferred to the Bionumerics software (Applied Maths).
Photographs of PFGE patterns were scanned (Vista scan; Umas Data
Systems, Inc.) and transferred to Bionumerics as tiff files.
Normalization was done with a Low Range PFG marker (New England
Biolabs) that was included in every sixth lane as a standard. Grouping
was performed by using the Dice coefficient and cluster analysis by the
unweighted pair group method using arithmetic averages. One band
difference was used to differentiate between types of RAPD, PFGE, and
AFLP patterns. When at least two patterns were allocated to the same
type, the type was given a number designation. When a pattern was
obtained for only one strain, the type was designated x. Thus, x
indicates a multitude of different types, each represented by only one
strain. The band tolerances (maximum tolerance expressed as a
percentage of the curve to match bands) for the RAPD and PFGE patterns
were 3 and 1% of the band tolerance for the AFLP patterns.
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RESULTS |
Prevalence of L. monocytogenes.
A total of
3,585 samples were tested. Of these, 60 samples were obtained in 1995 and 1996 (22), while 944 and 869 samples were obtained in
1998 and 1999 from two visits to each of the two cold-smoked salmon
processing plants, respectively. During a 10-month routine inspection
1,712 samples were taken by the processor at plant II.
In November 1998, 602 samples were taken from plant I, and 189 of these
samples (approximately 31%) were positive for
L. monocytogenes (Table
1). For the second visit in March 1999, of
342 samples
(approximately 13%) were positive for
L. monocytogenes. At plant
II 52 of 528 samples (10%) were
L. monocytogenes positive in October
1998 (Table
2),
whereas 6 of 341 samples (2%) were positive in
March 1999. During
these four visits, a total of 59 raw fish were
sampled, and 1 was found
to contain
L. monocytogenes.
At plant II, a more frequent sampling regime was undertaken by the
processor (Table
5). During the period from June 1998
to March 1999, a
total of 1,712 samples were analyzed, and 123
of the samples were
positive for
L. monocytogenes. A total of
108 isolates
were obtained from these samples. The prevalence
varied from 1 to 25%
in the final product and from 0 to 25% in
raw
fish.
RAPD profiling of L. monocytogenes isolated at
plants I and II.
One L. monocytogenes isolate from
each of 429 of the 444 positive samples was analyzed by the RAPD
technique performed with primer HLWL85. The 429 strains were divided
into 55 different RAPD profiles, and 41 of these profiles included only
one strain (designated type x in Tables 1 to 3, 5, and
7), and RAPD type 12 included as many as
166 strains. The RAPD profiles of five strains isolated from processing
plant I and five strains isolated from processing plant II are shown in
Fig. 1.
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TABLE 7.
Comparison of RAPD, PFGE, and AFLP analysis results for
selected strains of L. monocytogenes isolated from
cold-smoked salmon and processing environments
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FIG. 1.
RAPD patterns of 10 isolates of L. monocytogenes from samples of cold-smoked salmon from
processing plants I and II generated with primer HLWL85. Lanes 1 and
12, 100-bp ladder (Pharmacia); lanes 2 to 6, strains from plant I (RAPD
types 2, 6, 12, 13, and 15, respectively); lanes 7 to 11, strains from
plant II (RAPD types 105, 107, 108, 110, and 127, respectively).
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During the visits to plant I in November 1998 and March 1999, RAPD type
12 dominated, and 118 of 233 strains were this type.
This type was also
dominant in products from plant I sampled in
1995 and 1996; 29 of the
30 strains examined were RAPD type 12
(Table
1). RAPD types 2, 6, 7, and 15 were typically isolated
from the raw fish processing area of
plant I (Table
1). RAPD
type 2 was the dominant type in plant II during
the visits in
1998 and 1999, representing 48 of 58 strains (Table
2).
(i) Contamination routes in plant I.
In processing plant I,
RAPD types 2, 7, 12, and 15 were found in final products that were
produced from salmon that were free of L. monocytogenes (in November 1998 and March 1999). These RAPD types
were also found in many samples obtained from the processing environment, whereas the raw fish area harbored different RAPD types.
RAPD type 6 (except for one strain) was found only in the raw fish
processing area of plant I was not found in the slicing area or in the
products. RAPD type 7 was found only in the raw fish processing area
and in the products. When 18 raw fish (all with no detectable
L. monocytogenes) were monitored throughout processing
(Table 3), they were contaminated with RAPD type 2, 6, and 15 strains
in the raw fish processing area. No L. monocytogenes could be detected after smoking, but RAPD type 12 was isolated from
slicing machines when the fish were sliced and subsequently from smoked
salmon produced from the 18 fish (Table 3). Six of seven
L. monocytogenes strains in products from slicing
machine 1 were RAPD type 2 (Table 3). This type was also isolated from three contact surfaces related to slicing machine 1. These surfaces included specific components of slicing machine 1 that were in direct
contact with the unfinished product and gloves from the worker who had
handled the product. RAPD type 2 was not found elsewhere in the slicing
areas but was isolated in the raw fish area from three samples obtained
from the filleting machine on the same day that the 18 fish were
processed (Table 3). RAPD type 15 was found in a product sample sliced
by slicing machine 2, and this type was found on different machines and
equipment in both the raw fish and slicing areas.
(ii) Contamination routes in plant II.
L.
monocytogenes RAPD type 2 was the dominant type during the visits
to plant II in October 1998 and March 1999, and only few sporadic
non-type 2 strains were obtained (Table 2). Following 15 specific fish
through plant II in October 1998, we found only one sample of raw fish
contaminated with L. monocytogenes, whereas 8 of 45 samples of the final product harbored this type (Table 4). Several RAPD
type 2 L. monocytogenes strains were found in the raw
fish area and on contact surfaces where processing occurred. In
particular, samples from the brine injector needles and the recirculated brine solution and some other samples from the brine injector harbored this type. RAPD type 2 could also be found in dried
and smoked fillets and in the final product.
Change in RAPD types over time at plant II.
During the routine
sampling regimen conducted by the processor at plant II from June 1998 to March 1999, several different RAPD types were detected (Table 5).
RAPD types 2, 7, and 12 were all found in the final products and in the
processing environment (Table 5). Thus, despite recurring isolation of
RAPD type 2 during visits in October 1998 and March 1999, other RAPD
types were present in plant II. Specifically, RAPD type 2, which was
detected during intensive sampling along the processing lines (Table
3), was not found by the processor on other days in October 1998 and
March 1999. RAPD types 7 and 12 were found in the raw fish. Seventeen strains (designated RAPD type X) with distinct, different RAPD profiles
were found in raw fish or in the final products.
Comparison of typing methods.
Ninety-five and 30 randomly
selected strains were also typed with the PFGE and AFLP techniques,
respectively (Table 7). A subset of 30 strains typed by the RAPD, PFGE,
and AFLP techniques is shown in Fig. 2.
PFGE and AFLP fingerprinting resulted in the same groups as RAPD
typing, with only two exceptions. Strain V190a was identified as RAPD
type 6, and the RAPD pattern of this strain was similar to the RAPD
patterns of strains V5a, V110a, and V607a; however, PFGE and AFLP data
separated V190a from the other three strains (Table 7). Similarly, the
RAPD pattern of strain 2V920b was identical to the RAPD patterns of
strains identified as RAPD type 12, whereas the PFGE and AFLP patterns
separated strain 2V920b from strains La22, V203a, V417a, V455a, V477a,
V517b, and 2V575a (PFGE type 36 and AFLP type 12) (Table 7).
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FIG. 2.
Combined dendrogram for 30 L. monocytogenes isolates analyzed by RAPD, AFLP, and PFGE typing.
The dendrogram was constructed with GelCompar and Bionumerics (Applied
Maths) software by using the Dice correlation and cluster analysis by
the unweighted pair group method using arithmetic averages. Percentages
of similarity are shown above the dendrogram. The molecular sizes of
RAPD, AFLP, and PFGE DNA ranged from 200 to 2,000 bp, from 40 to 500 bp, and from 50 to 400 kb, respectively. The origins of strains are
shown in Table 7.
|
|
| |
DISCUSSION |
Prevalence of L. monocytogenes.
The
prevalence of L. monocytogenes in cold-smoked
salmon has been shown to vary from 0 to 100% in the final product
(22). In the present study, we found that the prevalence
in a processing plant may vary significantly over time. The higher
number of positive samples found during the first visits than during
the second visits in March 1999 could reflect the fact that October and
November are very busy periods with both day and evening shifts.
Consequently, disinfection of the whole plant cannot take place
between shifts, and this could explain the higher level of
L. monocytogenes in November. In general, the
prevalence in the raw fish was low, which has been reported in other
studies (30).
RAPD profiling and L. monocytogenes contamination
routes.
In agreement with our earlier study (12), we
found that different L. monocytogenes RAPD types were
dominant in vacuum-packed cold-smoked salmon from different Danish
smokehouses. The present study showed that the RAPD types found in the
product from a particular plant are also associated with the specific
processing environment; i.e., RAPD type 12 appeared to have colonized
plant I, and RAPD type 2 was dominant in plant II. The presence of a
few dominant clones or closely related strains in food processing
plants has been reported for a Norwegian salmon smokehouse
(30), poultry abattoirs (29), and pork
slaughtering and cutting plants (15). It is not known if
RAPD type 2 and 12 strains are the most common L. monocytogenes strains in Denmark or if they are strains with a
special ability to adapt the processing environment. Rørvik et al.
(30) determined by using multilocus enzyme electrophoresis that the most common electrophoretic type in Norway seemed to have
colonized a smokehouse.
To reduce the number of
L. monocytogenes cells, the
source of contamination must be known. Products from plant I which were
produced from salmon free of
L. monocytogenes were
contaminated
with RAPD types identical to those found in the processing
environment,
specifically in the slicing area. Processes before slicing
did
not contribute to contamination with RAPD type 12. These results
indicate that the slicing machines can spread a certain type and
may be
a reservoir for
L. monocytogenes (Table
3). In plant
II,
the bringing process may have contributed to contamination of
the
product, as
L. monocytogenes was isolated from brine
and injection
needles. Autio et al. (
2) similarly found
that the predominant
L. monocytogenes pulsotypes in
cold-smoked trout final products
were associated with brining and
slicing. In general, our results
indicate that contamination occurs
downstream along the processing
line. Other studies dealing with
different found processing operations
have similarly concluded that the
plant and processing environment
rather than the raw material is the
source of product contamination
with
L. monocytogenes
(
2,
15,
24,
27,
29,
30). However,
this does not exclude
the possibility that the raw fish or material
is an important initial
source for contaminating the processing
equipment and environment. In
plant II RAPD types 2, 7, and 12,
which were all found in the final
product, were also detected
on the raw fish and in the raw fish
handling area, indicating
that raw fish may also have been a source of
contamination. Eklund
et al. (
7) found that the primary
source of contamination was
the surfaces of frozen or fresh raw fish
coming into a plant;
however, this conclusion was based on prevalence
studies, and
comparisons of the strains at the DNA level were not
conducted.
Certain RAPD types were found only in specific sections of the plants;
in particular, some RAPD types were found only in the
raw material area
and not in the slicing section. Two processes,
smoking and freezing,
separate the raw material from the sliced
material, and both processes
are known to reduce bacterial levels
(
8,
35,
36). The
shift in DNA types during processing could
be explained by different
abilities of different DNA types to
withstand freezing or smoking.
Differential susceptibility to
freezing was suggested previously by
Destro et al. (
5), who
found that only two PFGE profile
groups were present on a frozen
shrimp product even though nine
different groups were isolated
from shrimp in the processing area.
Guyer and Jemmi (
16) found
that the cold-smoking process
did not affect
L. monocytogenes,
although it has been
reported that smoke compounds are inhibitory
to
L. monocytogenes (
35,
36). Eklund et al.
(
7) observed
a decrease in
L. monocytogenes populations in surface-inoculated
portions treated
with smoke and an increase when the organism
was injected into the
injector of the flesh. Correspondingly,
RAPD types 6 and 7 may have
been located on the surfaces of the
fillets and exposed to the smoking
and drying process. To our
knowledge, no studies have evaluated the
possible differential
susceptibility of
L. monocytogenes genetic types to food processing
factors.
Change in RAPD types over time at plant II.
The routine check
performed at plant II over time showed that several different
L. monocytogenes RAPD types could be isolated. During
this period, a variety of different products (smoked salmon, gravad
salmon, gravad halibut, smoked tuna, etc.) were produced, and if
the different kinds of raw fish were contaminated, this could explain
the large variation in genetic types. The appearance of different
L. monocytogenes RAPD types over time could also be a
reflection of the lower prevalence. Thus, it is likely that rigorous
cleaning and disinfecting in plant II (in which the prevalence was
significantly lower than the prevalence in plant I) actually eliminate
the organism at the end of production but that sporadic recontamination
occurs due to the ubiquitous nature of L. monocytogenes. Based on the data available it was not possible to
point to specific sources of contamination in plant II.
Comparison of plants I and II.
The frequency of L. monocytogenes contamination was lower in plant II than in plant I. The buildings of plant II were specifically designed for production of
smoked fish, and the facilities were in a good state of repair. In
plant II different processing operations were located in different
rooms with a continuous process flow. For example, raw material was
received in one room and separated from the filleting machine, and fish
waste material and products from the filleting process were transported
on conveyor belts to a separate room. In comparison, these processes
were done in the same hall in plant I. Consequently, improper traffic
by trucks and use of wooden pallets from the outside in the processing
environment took place. These could potentially be high-risk sources;
however, samples obtained from trucks and wooden pallets did not
substantiate this hypothesis. Likewise, sampling did not allow us to
identify other potentially high-risk sources. In plant II more careful attention was paid to changing parts of machines, which were difficult to clean and sanitize. Also, attention was paid to removing smaller parts of different machines to allow separate and thorough cleaning. In
plant II the slicing machines were cleaned twice during production, and
this may have reduced the levels, as reported by Rørvik et al.
(31) The processing lines for cold-smoked salmon in
plants I and II consist of several very complex machines, such as
filleting, skinning, brining, and slicing machines, which can be
difficult to clean; thus, complete eradication of L. monocytogenes is difficult. Differences in the sanitizing
procedures in the two plants were observed, but as other factors varied
in the plants varied, the results did not allow us to determine if
different sanitation regimes contributed to the lower prevalence of
L. monocytogenes in plant II.
Comparison of typing methods.
The use of both RAPD and PFGE
for typing L. monocytogenes has previously been
reported to identify similar groups (12, 15); however,
using more than one method may increase the discriminatory ability
(5), which was the case in this study, albeit for a very
limited number of strains. Consequently, the two DNA typing approaches
should be nearly equally suitable and can efficiently differentiate
strains from well-defined habitats like cold-smoked salmon
processing plants I and II investigated in this study. Compared to PFGE
and AFLP typing, RAPD profiling is rapid and inexpensive, and we
therefore chose RAPD typing with primer HLWL85 as the sole method for
typing all the isolates of L. monocytogenes. AFLP
fingerprinting for typing L. monocytogenes has been
described previously (1), but the discriminatory power was
compared only with that of serotyping. AFLP fingerprinting is
universally applicable and has been used successfully for typing and
classifying a number of bacterial strains (6, 14, 32). For
the subset of 30 strains which we examined, the groups of strains
determined by AFLP typing correlated completely with the groups
resulting from PFGE typing. In our study, AFLP analysis was used for 30 L. monocytogenes strains isolated from well-defined
habitats. A more thorough evaluation of the discriminatory power of
AFLP typing of L. monocytogenes would require further
testing of both environmental and clinical strains of L. monocytogenes.
Concluding remarks.
RAPD types 2 and 12 were dominant among
429 L. monocytogenes isolates from Danish salmon
smokehouses and cold-smoked salmon. RAPD type 12 was typical of the
slicing environment and the products of plant I, whereas RAPD type 2 prevailed in all sections of plant II. RAPD typing of the isolates
indicated that contamination with L. monocytogenes was
mostly due to direct contact with contaminated processing equipment,
and it was also possible to identify specific areas (bringing and
slicing) at which contamination of the final product occurred. However,
raw materials may also have contributed to contamination, particularly
in one plant. Over time, different RAPD types appeared in plant II,
probably as a result of a general relatively low prevalence of
L. monocytogenes. In contrast, specific types of
L. monocytogenes became established in the factory
environment of plant I, and one RAPD type (RAPD type 12) was isolated
from products from processing plant I over a 4-year period. This
indicates that an established in-house flora was not eliminated by the
hygiene procedures used. Several investigations have suggested that
reservoirs of L. monocytogenes seem to be established
in processing plants (15, 27, 29, 30). This view is
supported by this study. Some strains may be adapted to specific
niches. Similarly, specific types have been found to persist for up to
7 years in a dairy industry plant by Unnestad et al. (37)
and in an ice cream plant by Miettinen et al. (27). In
several instances we isolated L. monocytogenes from
cleaned and sanitized surfaces. L. monocytogenes is
capable of adhering to food processing surfaces like stainless steel
(17, 34), and cells in the adherent state may be more resistant to cleaning and disinfecting procedures than cells in the
planktonic state (42). Such resistance may explain why the same L. monocytogenes DNA types can persist in a food
processing plant for years (27).
The ways in which
L. monocytogenes may be introduced
into cold-smoked salmon processing plants are numerous due to the
ubiquitous
nature of
L. monocytogenes, and raw fish
could be an important
source for contaminating the processing equipment
and environment.
Because
L. monocytogenes will continue
to be introduced into plant
environments, control must be directed
toward preventing establishment
and growth of this organism in these
environments. The control
options must rely primarily on a proper
cleaning and sanitation
programs. However, production of products
consistently free of
the organism may be
impossible.
| |
ACKNOWLEDGMENTS |
We are indebted to the smoked fish processors who participated in
this study. The excellent technical support provided by Anemone Bundvad
and Tina Holde is gratefully acknowledged. We also thank Lasse Vigel
Jørgensen for providing strains of L. monocytogenes isolated in 1995 and 1996.
This study was supported by the Danish Food Technology Program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Danish Institute
for Fisheries Research, Department of Seafood Research, Søltofts
Plads, Technical University of Denmark, Bldg. 221, DK-2800 Kgs. Lyngby, Denmark. Phone: 45 45 88 33 22. Fax: 45 45 88 47 74. E-mail:
bfv{at}dfu.min.dk.
| |
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Applied and Environmental Microbiology, June 2001, p. 2586-2595, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2586-2595.2001
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Abstract
Introduction
