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Journal of Clinical Microbiology, July 1999, p. 2358-2360, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Epidemiology of an Outbreak of Febrile Gastroenteritis
Caused by Listeria monocytogenes in Cold-Smoked
Rainbow Trout
M. K.
Miettinen,1,*
A.
Siitonen,2
P.
Heiskanen,3
H.
Haajanen,4
K. J.
Björkroth,1 and
H. J.
Korkeala1
Department of Food and Environmental Hygiene,
00014 University of Helsinki,1
Laboratory of Enteric Pathogens, National Public Health
Institute, 00300 Helsinki,2 Health
Center of City of Varkaus, 78300 Varkaus,3
and Varkaus Municipal Food and Environment Department, 78250 Varkaus,4 Finland
Received 7 December 1998/Returned for modification 29 January
1999/Accepted 26 March 1999
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ABSTRACT |
Febrile gastroenteritis in five healthy persons was associated with
the consumption of vacuum-packed cold-smoked rainbow trout containing
Listeria monocytogenes. L. monocytogenes isolates from the
incriminated fish product lot and the stool samples were all of
serotype 1/2a and were indistinguishable by pulsed-field gel electrophoresis employing AscI and SmaI.
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TEXT |
Listeria monocytogenes is
a food-borne pathogen causing listeriosis mainly in immunocompromised
patients (5, 8, 13). The predominant clinical forms of
listeriosis are infections of the central nervous system, sepsis,
abortion, and stillbirth. A diarrheal form of disease due to the
ingestion of foods contaminated with L. monocytogenes
in previously healthy persons has also been reported (3, 7, 11,
12). However, in only one previous case was L. monocytogenes cultured from the implicated food, in that case from
chocolate milk, characterized with pulsed-field gel electrophoresis
(PFGE), and found to be identical to the isolates from patients
(3).
Vacuum-packed salmon and rainbow trout have previously been associated
with cases of listeriosis, but not with noninvasive gastroenteritis
(4). We report febrile gastroenteritis in five previously healthy persons associated with the consumption
of vacuum-packed cold-smoked rainbow trout (Onchorhyncus
mykiss) containing high levels of L. monocytogenes.
Two couples, aged between 39 and 52 years, and a 3-year-old child dined
together, consuming a meal including cold-smoked rainbow trout. They
had no known underlying diseases. All of them fell ill with
gastroenteritis within the next 27 h, experiencing nausea, abdominal cramps, and diarrhea. Three of the adults had fever (38.6 to
39.2°C). Other reported symptoms were vomiting, headache, fatigue,
and arthralgia. The symptoms lasted 3 to 4 days. The child was admitted
to a hospital for one night because of vomiting, diarrhea, and fever.
Based on patient interviews and a questionnaire on the types and
quantities of the foods consumed, the cold-smoked rainbow trout seemed
to be a very likely vehicle of the pathogen that caused the food poisoning.
The patients had eaten the cold-smoked rainbow trout shortly after
buying it from a retail store. The fish product had been vacuum packed
17 days prior to consumption. This retail store was visited, and the
temperature of the display cabinet for smoked fish products was
measured. At the bottom of the cabinet the temperature was 4.6°C, but
at the top it was 11.6°C.
A vacuum-packed cold-smoked rainbow trout sample from the same
production lot was then obtained from the same retail store and
analyzed for L. monocytogenes. Analyses were performed
both by selective enrichment according to the method of The
Nordic Committee on Food Analyses (10) and by quantitative
analysis for L. monocytogenes. The quantitative
analysis was performed with Listeria enrichment broth,
before selective supplements were added in serial dilutions on
Listeria-selective Oxford agar.
Stool samples taken on the day after the onset of the symptoms were
analyzed only for Salmonella, Shigella,
Campylobacter, and Yersinia. Two additional
stool swabs were obtained from one of the couples a week after the
onset of the symptoms, and these were analyzed for L. monocytogenes by selective enrichment by using the medium
described above for the fish sample.
Serotyping of L. monocytogenes isolates was performed
according to the serotyping scheme of Seeliger and Höhne
(14). Commercial Listeria antisera (Denka Seiken,
Tokyo, Japan) were used according to the manufacturer's instructions
with the exception of the incubation temperature of the 0.2% brain
heart infusion agar tubes, which was lowered to 26°C instead of
30°C.
The L. monocytogenes isolates were grown overnight in 5 ml of brain heart infusion broth at 37°C. DNA isolation was performed as described by Maslow et al. (9) with the modifications
described previously (2). Two rare-cutting restriction
enzymes, AscI and SmaI (New England Biolabs,
Beverly, Mass.), were used according to the manufacturer's
recommendations. The samples were run through a 1.0% (wt/vol)
agarose gel (SeaKem Gold; FMC Bioproducts, Rockland, Maine) in
0.5× TBE (45 mM Tris, 4.5 mM boric acid [pH 8.3], and 1 mM sodium
EDTA) at 200 V at 10°C by using a Gene Navigator system with a
hexagonal electrode (Pharmacia, Uppsala, Sweden). Macrorestriction fragments were resolved with pulse times ramping linearly from 1 to 35 s over 18 h for AscI fragments and from 1 to
18 s over 18 h for SmaI fragments. Lambda ladder
PFG marker I and low-range PFG marker (New England Biolabs) were used
as fragment size markers.
The cold-smoked rainbow trout sample was found to be positive for
L. monocytogenes by selective enrichment and by
quantification. The fish sample contained 1.9 × 105
CFU of L. monocytogenes per g. The storage temperature
of the fish product at the retail outlet had been around 10°C, rather than the range of 0 to 3°C recommended by the manufacturer. The high
storage temperature of the fish product probably allowed L. monocytogenes to grow to such high levels.
Both of the additional stool swabs were positive for L. monocytogenes. Taken together with the fact that no other enteric pathogens could be found, this strongly suggests that this
outbreak of gastroenteritis among five persons was caused by
L. monocytogenes. Furthermore, L. monocytogenes isolates from the stool swabs and the
cold-smoked rainbow trout were of serotype 1/2a and showed indistinguishable macrorestriction enzyme patterns by PFGE with both
restriction enzymes, AscI and SmaI (Fig.
1 and 2).
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FIG. 1.
Macrorestriction patterns of L. monocytogenes with restriction enzyme AscI. Lane 1, lambda ladder PFG marker; lane 2, LMU1 (isolated from patient); lane 3, LMU10 (isolated from patient); lane 4, LMK55 (isolated without an
enrichment step from cold-smoked rainbow trout); lane 5, LMK65
(isolated through an enrichment step from cold-smoked rainbow trout);
lane 6, low-range PFG marker.
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View larger version (52K):
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[in a new window]
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FIG. 2.
Macrorestriction patterns of L. monocytogenes with restriction enzyme SmaI. Lane 1, lambda ladder PFG marker; lane 2, LMU1 (isolated from patient); lane 3, LMU10 (isolated from patient); lane 4, LMK55 (isolated without an
enrichment step from cold-smoked rainbow trout); lane 5, LMK65
(isolated through an enrichment step from cold-smoked rainbow trout);
lane 6, low-range PFG marker.
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|
These results provide additional supporting evidence for previous
findings that L. monocytogenes may cause febrile
gastroenteritis without invasiveness in healthy persons, if the
consumed food is heavily contaminated with L. monocytogenes. They also show that vacuum-packed cold-smoked
rainbow trout may contain large numbers of L. monocytogenes and must be considered as a potential source of
infection (4, 6). The cold-smoking process does not kill
L. monocytogenes (1), and despite the cold
storage of the product, growth may occur. The sell-by date is usually around 3 weeks from the packing date; this is clearly too long, especially during the summer months. This kind of product is usually eaten without heat treatment before consumption.
This is, to our knowledge, the first report of febrile gastroenteritis
associated with L. monocytogenes where a fish product acted as the vehicle of the pathogen. It also shows the value of
selective enrichment for L. monocytogenes in stool
samples. Without this enrichment, the Listeria might have
been missed. PFGE was a very useful tool in confirming the food-borne
origin of L. monocytogenes in the cases of febrile
gastroenteritis presented in this study. The study emphasizes the need
to keep the possibility of L. monocytogenes
gastroenteritis in mind, especially after the presence of other
enteropathogens has been ruled out.
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ACKNOWLEDGMENTS |
This work was supported by the Technology Development Center (TEKES).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Food and Environmental Hygiene, Faculty of Veterinary Medicine,
University of Helsinki, P.O. Box 57, FIN-00014 Helsinki University,
Finland. Phone: 358 0 708 49 706. Fax: 358 0 708 49 718. E-mail:
msjoman{at}vetmed.helsinki.fi.
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Journal of Clinical Microbiology, July 1999, p. 2358-2360, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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