Post on 03-Feb-2023
Advance Publication
The Journal of Veterinary Medical Science
Accepted Date: 28 Sep 2010
J-STAGE Advance Published Date: 12 Oct 2010
Public Health
Full paper
Isolation of Staphylococcus aureus from raw fish in relation with culture methods
Etsuko Saito1), Nanako Yoshida2), Junichi Kawano2), Akira Shimizu2) and Shizunobu
Igimi3)
1) Department of Bioresource and Agrobiosciences, Graduate School of Science and
Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501
2) Department of Microbiology and Immunology, Faculty of Agriculture, Kobe
University, Nada-ku, Kobe, Hyogo 657-8501
3) Division of Biochemical Food Research, National Institute of Health Science, 1-18-1
Kamiyoga, Setagaya-ku, Tokyo 185-8501
S. AUREUS FROM FISH AT DISTRIBUTION STAGES
Corresponding author
Kawano, J.
Department of Microbiology and Immunology, Faculty of Agriculture, Kobe University,
Nada-ku, Kobe, Hyogo 657-8501, Japan
Kawanoj@kobe-u.ac.jp
Fax number. 078-803-5817
Abstract
Five hundred and fifty fish samples from various stages in the course of
distributions in Hyogo prefecture (209 retailed in super markets, 173 obtained from
fishery cooperatives at the harbor, 91 caught by trawling, and 77 caught by rod fishing)
were examined for contamination with Staphylococcus aureus (S. aureus). S. aureus
was detected in 41 (19.6%) of the retail fish samples and 46 (26.6%) of the samples from
the fishery cooperatives. No S. aureus was isolated from the living fish (91 trawled and
77 fished by rod). With regard to the retail fish, the contamination rate of processed fish
(26.0%) was significantly higher than that of unprocessed (14.2%). For 88 samples, the
efficacy of the selective medium was compared using Baird-Parker agar and mannitol
salt agar supplemented with egg yolk (MSEY agar) by the direct plate and enrichment
culture methods. Using the direct culture method, the S. aureus positive rate with the
Baird-Parker agar (30.7%) was significantly higher (P < 0.01) than that with the MSEY
agar (6.8%). The enrichment culture method remarkably raised the S. aureus
detection rate. Seventy-eight (85.7%) of 91 isolates belonged to the Human ecovar.
Sixty-two (68.1%) of the 91 isolates had some enterotoxin genes, including 44 (48.4%)
with the sea gene. These data showed that fish was contaminated with S. aureus after
landing and Baird-Parker agar had an advantage in detecting S. aureus with a direct
plate culture.
Key words:
biotype, distribution, enterotoxin, raw fish, Staphylococcus aureus
Introduction
Staphylococcal food poisoning is caused by staphylococcal enterotoxins (SEs),
which is mostly produced by S. aureus, and is an important intradietetic intoxication in
the world. The number of staphylococcal food poisoning incidences in Japan has
decreased according to the improvement of equipments of food production, distribution
systems, and the conscious of food sanitary. Fifty to 100 cases caused by S. aureus were
annually reported during the period between 2002 and 2008 [21-27]. In Japan, main
causes of staphylococcal food poisoning are processed foods composed mainly of rice,
such as rice ball which are made by hands [12, 34], while those are dairy products, raw
meat and its products in foreign countries [10, 41].
To Japanese, fish has been an important protein source as foods for a long time,
and Japan is world’s leading consumers of fish. Fish is often processed with bear hands,
such as sashimi, and supposed to have frequent occasion for contamination with S.
aureus from humans and producing environment. Actually, served cases of
staphylococcal food poisoning by fish has been reported between 2002 to 2008, including
seven caused two by cooked eel, two by sashimi, three by flaked fish, and so on
[14-20]. This suggests the need for S. aureus contamination control on fish. Although
there have been many reports on S. aureus contamination of foregoing foods which are
main causes of staphylococcal food poisoning, there are a few reports dealing with S.
aureus contamination of retail fish [30, 33, 38] and no report on living fish. It is
necessary to investigate S. aureus contamination route of fish to prevent staphylococcal
food poisoning by fish.
In Japan, there is no standard operation procedure of detecting S. aureus from
foods and it varies depending on inspection agency. According to the food sanitation
inspection guidelines [39], most agencies adopted the direct culture method by mannitol
salt agar supplemented with 3% egg yolk emulsion (MSEY agar). In contrast, the
enriched culture method using Baird-Parker agar is widely used in foreign countries,
which is recommended by International Organization for Standardization (ISO) in
Switzerland [13] and Bacteriological Analytical Manual (BAM) [2] by Food and Drug
Administration (FDA) in the United States. In the modern era of expanding
globalization of food distribution, the detecting method for S. aureus in Japan needs to
harmonize internationally.
In this report, we examined S. aureus contamination of fish at the various stages
of commercial distributions in Hyogo prefecture, retailed in supermarkets, taken in
fishery cooperatives at fishing harbor, i.e. whole sale fish markets, and caught by
trawling and rod fishing and its characteristic by phenotypic and genotypic tests. In
addition, we evaluate the efficacy of MSEY and Baird-Parker agar in detecting S.
aureus with fish samples.
Material and method
Samples
From July 2005 to September 2007, a total of 550 samples in various species of
fish were examined, including 209 samples (51 prawns, 40 squids, 30 horse mackerels,
28 sauries, 24 sardines, 19 mackerels, 6 yellowtails, 4 sea breams, 2 spanish
mackerels, 1 tuna,1 salmon, 1 trevally, 1 sea bass, 1 oyster) obtained from 40 retail
shops, 173 samples (17 horse mackerels, 16 gobies, 16 sea breams, 13 sand borers, 9
soles, 9 globefishes, 9 lizardfishes, 7 squids, 6 lefteye flounders, 5 white croakers, 5
black rockfishes, 4 octopuses, 4 hairtails, 4 congers, 4 shrimps, 3 herrings, 3 black
scrapers, 2 righteye flounders, 2 rays, 2 barracudas, 2 threadsail flatfishes, 2 stone
fishes, 2 bartail flatfishes, 2 halfbeaks, 2 blend banded soles, 2 seabasses, 2 surfperches,
2 rivulatuses, 2 conger pikes, 2 gurnards, 1 greenling, 1 angler, 1 frogfish, 1 sardine, 1
rockfish, 1 bonito, 1 crab, 1 largescale blackfish, 1 mackerel, 1 shark, 1 sea cucumber, 1
wart perch, and 1 striped mullet) from 3 fish markets, 91 samples (9 horse mackerels, 8
shrimps, 7 congers, 7 soles, 6 octopuses, 5 gobies, 5 wart perchs, 5 squids, 3 sea breams,
3 sharks, 3 white croakers, 3 rays, 3 sand borers, 2 gurnards, 2 globe fishes, 2 squillas, 2
lefteye flounders, 2 sardines, 2 goat fishes, 2 barracudas, 2 conger pikes, 2 bartail
flatheads, 1 hairtail, 1 Japanese ice fish, 1 black crocker, 1 stone fish, 1 lizardfishes, 1
angler) caught by trawl fishing and 77 samples (44 horse mackerels, 14 mackerels, 6 sea
breams, 5 sardines, 3 pearl spot chromises, 2 filefishes, 1 rock fishes, 1 wrasse, 1
largescale blackfish) by rod fishing. Samples were purchased at retail shops in Hyogo
prefecture, taken at fish markets in Hyogo prefecture, caught by trawling and rod
fishing in Hyogo prefecture. Samples purchased at the retail shops included 96
processed fish, such as fillet, bony parts, and 113 unprocessed fish.
Isolation of S. aureus
Samples were taken by swabbing surface of fish with culture swabs (EZ II, Becton,
Dickinson and Company, USA) which was paired sterile cotton swabs. All samples were
subjected to the enrichment culture method. Namely, the swabs were inoculated in
trypticase soy broth (TSB, Nissui Pharmaceutical Co., LTD., Tokyo) supplemented with
1% sodium pyruvate, 0.5% dipotassium phosphate, 6.5% NaCl and incubated at 30°C for
24hr. A drop of these broth cultures was plated onto mannitol salt agar (Nissui)
supplemented with 3.0% egg-yolk emulsion (MSEY agar), smeared with a spreader and
incubated at 30°C for 48hr.
As for 88 samples obtained from fish markets from January to March in 2006, the
efficacy of the selective media was compared with Baird-Parker agar (OXOID Co., LTD.,
England) supplemented with 1.5% egg-yolk emulsion and 0.01% potassium tellurite as
directed by the manufacturer and MSEY agar by the direct plate culture method and
the enrichment culture method. The samples were taken with EZ II culture swabs, one
was streaked on MSEY and Baird-Parker agar. Another swab was inoculated onto the
modified TSB mentioned above. A drop of these broth cultures was plated onto MSEY
and Baird-Parker agar and incubated at 30°C for 48hr. Typical colonies were black or
gray in colors surrounded by clear zone while yellow and egg-yolk positive in MSEY
agar.
Suspect colonies, which revealed acidification of mannitol and positive results for
egg-yolk reaction, were subjected to identification procedures. Colonies with
coagulase-positive, Gram-positive cocci and catalase-positive were picked as possible S.
aureus and identified by PCR for the Sa442 gene specific for S. aureus using a primer
set of SAU-1 and SAU-2 [11]. Following PCR program was used: denaturing at 94°C for
45 sec, annealing at 61°C 1 min, extension at 72°C for 2 min for 30 cycles to completion;
the final cycles consisted of extension at 72°C for 7min.
Biotyping
S.aureus isolates were subjected to biotypes with the method of Devries [7] using 4
characteristics; production of staphylokinase and β-hemolysin, coagulation of bovine
plasma and the type of growth on crystal violet agar.
Detection of staphylococcal enterotoxin genes
The multiplex PCR technique was used to detect enterotoxin genes (sea, seb, sec,
sed, see, seg, seh and sei) with Takara Taq (Takara Bio, Inc., Shiga) with using primer
sets designed by Becker et al [3] and Omoe et al [36]. Following PCR program was
used in this study: denaturing at 94°C for 30 sec, annealing at 55°C 1 min, extension at
72°C for 1 min for 30 cycles to completion; the final cycles consisted of extension at 72°C
for 5 min.
As positive controls, five strains were used: strain 243 (seb); strain FRI-326 (see);
strain FRI-361 (sec, seg and sei); strain No.18 (sea); strain No. 5 (sed and seh). Strain
No. 18 and Strain No. 5 were isolated from chicken meat [29].
Productions of staphylococcal enterotoxins (SEA, SEB, SEC and SED) were
confirmed by reverse-passive latex agglutination (RPLA) method using SET-RPLA
(Denka Seiken Co., LTD., Tokyo).
Statistical analysis
Fisher’s exact test was used to determine the significance of differences between
the S. aureus isolation rates in fish species and S. aureus isolation rate with two
selective mediums.
Results
Isolation of S. aureus
S. aureus was detected in a total of 87 (15.8%) of 550 samples examined in this
study (Table 1). According to the sample sources, 41 (19.6%) of the 209 retailed fish
samples and 46 (26.6%) of the 173 samples obtained in fish markets were positive for S.
aureus. None of the 91 trawled or 77 rod-caught fish were positive for S. aureus. Of the
209 retailed fishes, 25 (26.0 %) of the 96 processed fish were positive for S. aureus, while
the rate of isolation is significantly lower (P < 0.05) for unprocessed fish (14.2%, 16/113).
In terms of species, retail prawns were highly contaminated (39.2%, 20/51), followed by
yellowtails (33.3%, 2/6), mackerel (31.6%, 6/19). Of note, samples obtained either by
trawling or rod fishing were free of S. aureus contamination.
In retailed fish, 2 S. aureus colonies with different characteristics were isolated
from 4 samples, and a total of 91 isolates were subjected to the following tests.
Efficacy of the selective medium
By the direct plate culture isolation method for 88 samples from fish markets, S.
aureus was detected in 6 (6.8%) and 27 (30.7%) samples using MSEY and Baird-Parker
agar, respectively (Table 2), and the differences was significant (P < 0.01). By the
enrichment culture method, S. aureus was detected in 36 (40.9%) samples using MSEY
agar and 36 (40.9%) samples using Baird-Parker agar, with no significant differences (P
= 1.0). With both MSEY and Baird-Parker agar, the enrichment culture method
remarkably increased the detection rate.
Biotyping
A total of 91 isolates of 87 S. aureus-positive samples were subjected to biotyping.
Of these, 78 (85.7%) belonged to the K+β-CV:A biotype (Human ecovar) , 7 (7.7%) to the
K-β+CV:A biotype (Bovine ecovar) , and 6 (6.6%) to the K-β+CV:A biotype (Poultry
ecovar) (Table 3) . The human ecovar was the biotype most frequently detected among
isolates from retail fish and those obtained from fish markets.
Detection of staphylococcal enterotoxin genes
Ninety-one isolates were examined for enterotoxigenicity. Of these, 62 isolates
(68.1%) had some enterotoxin genes (Table 3). Forty-four isolates (48.4%) had the sea
gene, followed by seg + sei (5 isolates, 5.5%), seb + seg + sei (4, 4.4%), seb (3, 3.3%), sec
(2, 2.2%), sec + seh (2, 2.2%), seh (1, 1.1%), and sea + seg + sei (1, 1.1%) genes. In total,
10 isolates harbored seg + sei with or without other genes. Isolates from the retail fish
showed various type of enterotoxin, while most isolates from wholesale fish markets
possessed the sea gene. Production of SEA, SEB and SEC from isolates harboring the
sea, seb and sec genes, respectively, was confirmed by the RPLA analysis.
Discussion
This paper is the first report on S. aureus contamination during the commercial
distribution of fish. No S. aureus was detected in live fish, but contamination in fish
from fish markets was evident from the beginning of the distribution process. The
contamination rate increased during the course of distribution, as is shown by the level
of contamination in fish available for retail, particularly when these were processed.
Given the fact that the human ecovar was the most prevalent biotype, the fish appear to
have been contaminated with S. aureus from humans engaged in processing operations.
The isolation rate of S. aureus in retail fish samples in the present study (19.6%,
41/209) is relatively low compared to previous reports, which ranged from 29.3%–61.7%
[30, 33, 38]. Hygienic practices in fish-processing facilities have recently improved, with
some facilities adopting measures such as the use of ozone water and ultraviolet
sterilizers. The procedures above may have resulted in the lower incidence of S. aureus
in the present study. The contamination rate of processed fish (26.6%) was significantly
higher than that of unprocessed fish (14.3%). In terms of fish species, the highest rate of
contamination was 39.2% in prawns (20/51). Prawns examined in this study were all
imported and processed using a method similar to that usually performed in Japanese
markets. This might be the reason for high S. aureus prevalence in prawns. The rate of
isolation in unprocessed Pacific saury (25.0%, 7/28) was significantly higher (P < 0.01)
than that in squid (3.1%, 1/32) and horse mackerel (0%, 0/24). In addition to the fact
that S. aureus was not detected in live fish (0%, 0/168), it appears that S. aureus
contamination in fish occurs during handling and processing through contact with
humans or processing equipment contaminated with S. aureus. The differences in the
contamination rates between unprocessed fish may be attributed to sanitary conditions
during their transportation.
We obtained fish samples from 3 fish markets, and the S. aureus-positive rate
differed for each market. This may be because of differences in hygiene control at
individual fish markets. Samples obtained in the fish market A in January 2006 were
all S. aureus-positive. All 39 isolates belonged to the same ecovar (Human) and had the
same enterotoxin gene (sea), indicating that they were derived from the same
contamination source. This suggested the existence of highly contaminated source of S.
aureus in the market at that time.
The simplified Devriese biotyping system for S. aureus has been useful for tracing
the origin of this organism in animal food and the food industry [7, 8]. The predominant
biotype determined in this study was the human biotype (85.7%), which
characteristically originates from humans. The fact that no living fish was S.
aureus-positive in this study indicates that the fish were contaminated with S. aureus
by humans after landing and during the course of distribution.
In the present study, 68.1% of the isolates possessed enterotoxin genes (sea–sei),
and sea was the most frequently found gene. Japanese staphylococcal food poisoning is
mainly caused by SEA [12, 34]: therefore, it is notable that fish were contaminated with
SEA producers.
S. aureus possessing seg and sei genes are frequently found in healthy human and
foods [5, 6, 31, 35, 37]. However, there are food poisoning incidences that might have
been caused by S. aureus possessing both seg and sei genes [4, 5, 28, 36]. On the other
side, it has been reported that when SEG and SEI are co-expressed, they interact
differently with MHC class II and stimulate completely different subsets of human and
mouse T cells, indicating complementary superantigenic activities [9]. Although such
type of S. aureus may be derived from healthy humans, its growth and production of
SEG and SEI in foods could lead to intoxication. From a public health perspective, it is
notable that fish isolates had the seg and sei genes.
In Japan, microorganism tests, including S. aureus detection in food are conducted
according to the food sanitation inspection guidelines [39]. These guidelines have been
revised several times without sufficient examination of their effects, so they have been
reviewed by the exploratory committee of standard methods of microorganism tests of
food. The committee indicated that Baird-Parker agar, which is widely used in many
countries outside Japan to detect S. aureus and MSEY agar were equally effective in
detecting S. aureus [40]. Using a direct plate culture method, the S. aureus-positive rate
with Baird-Parker agar (30.7%) was found to be higher than that with MSEY agar
(6.8%), indicating the advantage of Baird-Parker agar when isolating S. aureus from
fish. This may reflect the efficacy of sodium pyruvate present in Baird-Parker agar for
isolating the viable but non-culturable (VBNC) S. aureus [13]. It has been suggested
that fish are often contaminated with S. aureus in this state. Sodium pyruvate has been
reported to recover VBNC cells with damaged metabolic pathways [1]. Introducing an
enrichment culture method remarkably increased the S. aureus detection rate in agar
(Table 2), which is consistent with previous reports [32, 33, 38]. In this context, there
was no difference in the isolation rate between MSEY (40.9%) and Baird-Parker (40.9%)
agar. This suggests that the enrichment culture method using enrichment broth
containing sodium pyruvate is useful for the detection of S. aureus in fish, regardless of
which selective media is used.
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Table 1. Isolation of S.aureus from fish samples
Origin Sampling dates Sample type No. of samplesRetail shops Jul. -Aug. 2005 processed 96 25 (26.0%)
Jul. -Aug. 2005 unprocessed 113 16 (14.2%)Fish market A Jan. 2006 unprocessed 39 39 (100.0%) A Mar. 2007 unprocessed 35 3 (8.6%) B Mar.2006 unprocessed 21 1 (4.8%) B Mar.2006 unprocessed 28 0 C Feb. 2007 unprocessed 50 3 (6.0%)
Caught by trawling Oct.-Jul. 2007 live 91 0Caught by rod fishing Aug.-Sep.2007 live 77 0
Total 550 87 (15.8%)
No. of S. aureus -positive samples
Table 2. Isolation results of S. aureus from fish samples by different culture methods
Direct culture 88 6 (6.8%) 27 (30.7%)Enrichment culture 88 36 (40.9%) 36 (40.9%)
Isolation No. of samples Baird-Parker agarb)MSEY agara)
No. of samples with isolates on:
a) MSEY agar, mannnitol salt agar supplemented with 3.0% egg-yolk emulsion.
b) Baird-Parker agar, Baird-Parker agar supplemented with 1.5% egg-yolk emulsion and 0.01% potassium tellurite.
Table 3. Biotypes and posession of the enterotoxin genes among S. aureus isolates obtained from fish samples
Fish marketsprocessed unprocessed unprocessed
n=28 n=17 n=46 n=91Biotypea)
Human ecovar 20 14 44 78 (85.7%) Bovine ecovar 4 2 1 7 (7.7%) Poultry ecovar 4 1 1 6 (6.6%)Enterotoxin geneb)
sea 2 1 41 44 (48.4%)seb 1 1 1 3 (3.3%)sec 1 1 2 (2.2%)seh 1 1 (1.1%)
seg +sei 3 2 5 (5.5%)sec +seh 2 2 (2.2%)
sea +seg +sei 1 1 (1.1%)seb +seg +sei 3 1 4 (4.4%)
nonec) 16 10 3 29 (31.7%)
c) Negative for enterotoxin examined in this study, sea , seb , sec , sed , see , seg , seh and sei .
b) Enterotoxin genes were determined by PCR according to Becker et [3] and Omal oe et al [36]. Of the 91 isolates, 62 (68.1%) were positive for enterotoxin genes tested.
CharacteristicsRetail shops
Total
a) Biotype was determined with 4 characteristics of staphylokinase, β-hemolysin, bovine plasma coagulation and growth on crystal violet agar according to Devries, L..A [7].