The Journal of Veterinary Medical Science - J-Stage

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The Journal of Veterinary Medical Science

Accepted Date: 28 Sep 2010

J-STAGE Advance Published Date: 12 Oct 2010

Public Health

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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

[email protected]

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.

References

1. Baird-Parker, A., and E. Daenport. 1965. The effective of recovery medium on

the isolation of Staphylococcus aureus after heat-treatment and after frozen or

dried cells. J. Appl. Bacteriol. 28282828: 390-420.

2. Bannett, R. W. and Lancette, G. A. 2001. Bacteriological Analytical Manual

online, Chapter 12, Staphylococcus aureus.

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalA

nalyticalManualBAM/UCM071429

3. Becker, K., Roth, R. and Peters, G. 1998. Rapid and specific detection of

toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme

immunoassays for amplification and hybridization of staphylococcal enterotoxin

genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene. J. Clin.

Microbiol.36363636: 2548-2553.

4. Cha, J. O., Lee, J. K., Jung, Y. H., Yoo, J. I., Park, Y. K., Kim, B. S. and Lee, T. S.

2006. Molecular analysis of Staphylococcus aureus isolates associated with

staphylococcal food poisoning in South Korea. J. Appl. Miclobiol.101101101101: 864-871.

5. Chen, T. R., Chiou, C. S. and Tsen, H. Y. 2004. Use of novel PCR primers specific

to the genes of staphylococcal enterotoxin G, H, I for the survey of

Staphylococcus aureus strains isolated from food-poisoning cases and food

samples in Taiwan. Int. J. Food Microbiol. 92929292: 189-197.

6. Collery, M. M., Smyth, D. S., Twohig, J. M., Shore, A. C., Coleman, D. C. and

Smyth C. J. 2007. Molecular typing of nasal carriage isolates of Staphylococcus

aureus from an Irish university student population based on toxin gene PCR,

agr locus types and multiple locus, variable number tandem repeat analysis. J.

Med. Microbiol. 57575757: 348-358.

7. Devriese, L. A. 1984. A simplified system for biotyping Staphylococcus aureus

strains isolated from different animal species. J. Appl. Bacteriol. 56565656: 215-220.

8. Devriese, L. A., Yde, M., Godard, C. and Isigidi, B. K. 1985. Use of biotyoing to

trace the origin of Staphylococcus aureus in foods. Int. J. Food Microbial. 9999:

365-369.

9. Fernández, M. M., De Marzi M. C., Berguer, P.,,,, Burzyn. D., Langley R. J.,

Piazzon, I., Mariuzza, R. A and Malchiodi, E. L. 2006. Binding of natural

variants of staphylococcal superantigens SEG and SEI to TCR and MHC class

II molecule. Molecul. Immunol. 43434343: 927-938.

10. Genigeorgins, C. A. 1989. Present state of knowledge on staphylococcal

intoxication. Int. J. Food Microbiol. 9999: 327-360.

11. Hata, E., Katsuda, K., Kobayashi, H., Ogawa, T., Endo, T. and Eguchi, M. 2006.

Characteristics and epidemiologic of Staphylococcus aureus isolates from

bovine mastitic milk in Hokkaido, Japan. J. Vet. Med. Sci. 68686868: 165-170.

12. Igarashi, H. 1997. Staphylococcal food poisoning. J. Antibact. Antifung.

Agents.25252525: 549-557 (in Japanese).

13. ISO 6888-1. 1999. Microbiology of food and animal feeding stuffs－Horizontal

method for the enumeration of coagulase-positive staphylococci (Staphylococcus

aureus and other species)－Part 1: Technique using Baird-Parker agar medium.

14. Japanese Ministry of Health, Labor, and Welfare. Food poisoning cases in 2002.

http://www.mhlw.go.jp/topics/syokuchu/xls/h14sokuho.xls






http://www.mhlw.go.jp/topics/syokuchu/05hassei/xls/jirei.xls


http://www.mhlw.go.jp/topics/syokuchu/06hassei/xls/jirei.xls


http://www.mhlw.go.jp/topics/syokuchu/07hassei/xls/jirei19.xls



21. Japanese Ministry of Health, Labor, and Welfare. Food poisoning outbreaks in

2002. http://www.mhlw.go.jp/topics/syokuchu/02hassei/xls/02hassei.xls






2005. http://www.mhlw.go.jp/topics/syokuchu/05hassei/xls/joukyou.xls


2006. http://www.mhlw.go.jp/topics/syokuchu/07hassei/xls/H18joukyou.xls





28. Kérouanton, A., Hennekinne, J. A., Lterte, C., Petit, L., Chesneau, O., Brisabois,

A. and De Buyser, M. L. 2007. Characterization of Staphylococcus aureus

strains associated with food poisoning outbreaks in France. Int. J. Food

Microbiol.115115115115: 369-375.



29. Kitai, S., Shimizu A., Kawano, J., Sato, E., Nakano, C., Kitagawa, H., Fujio, K.,

Matsumura, K., Yasuda, R. and Inamoto, T. 2004. Prevalence and

characterization of Staphylococcus aureus and enterotoxigenic Staphylococcus

aureus in retail raw chicken meat throughout Japan. J. Vet. Med. Sci. 67676767:

269-274.

30. Kusunoki, K., Ushioda, H., Jin, M., Arai, T., Iwaya, M., Ishikami, T. and Ymada

S. 1998. Results of bacterial contamination of commercial raw fish and shellfish

in Tama, Tokyo (1986-1996). Jpn. J. Food Microbiol. 15151515: 161-165 (in Japanese).

31. Lawrynowicz-Paciorek, M., Kochman, M., Piekarska, K., Grochowska, A. and

Windyga, B. 2007. The distribution of enterotoxin-like genes in Staphylococcus

aureus strains isolated from nasal carries and food samples. Int. J. Food

Microbiol. 117117117117: 319-323.

32. Matsumura, K., Shimizu, A., Kawano, J. and Igimi, S. 2009. Selective media for

detection of Staphylococcus aureus in commercial raw meat and fish. Jpn. J.

Food Microbiol. 26262626: 23-27 (in Japanese).

33. Nomura, S., Haraga, I., Hanaki, H., Nagayama, A. 2002. Staphylococcus aureus

contamination of commercial slices of raw fish－comparison of direct plate

culture and enrichment culture － . Jpn. J. Food Microbiol. 19191919: 17-20 (in

Japanese).

34. Oda, T. 1998. A review of staphylococcal food poisoning in Japan. J. Food Hyg.

Soc. Jpn. 39393939: J179-185.

35. Omoe, K., Hu, D.-L., Takahashi-Omoe, H., Nakane, A. and Shinagawa, K. 2005.

Comprehensive analysis of classical and newly described staphylococcal

superantigenic toxin genes in Staphylococcus aureus isolates. FEMS Microbiol.

Lett. 246246246246: 191-198.

36. Omoe, K., Ishikawa, M., Shimoda, Y., Hu, D.-L., Ueda, S and Shinagawa, K.

2002. Detection of seg, seh and sei genes in Staphylococcus aureus isolates

harboring seg, seh, or sei genes. J. Clin. Microbiol. 40404040: 857-862.

37. Rosec, J. P. and Gigaud, O. 2002. Staphylococcal enterotoxin genes of classical

and new types detected by PCR in France. Int. J. Food Microbiol. 77777777: 61-70.

38. Shimizu, A., Ozaki, J., Kawano, J. and Kimura, S. 1991. Isolation and

characterization of Staphylococcus aureus from raw fish and meat. Jpn. J. Food

Microbiol.8888: 135-141 (in Japanese with English summary).

39. Shinagawa, K. 2004. Staphylococcus aureus. pp. 236-248. In: Food Sanitation

Inspection Guidelines, Microbiology Volume (Japanese Ministry of Health,

Labor, and Welfare. ed.), Japan Food Hygiene Association, Tokyo (in Japanese).

40. The exploratory committee of standard method of microorganism tests from

food. The minutes of the ninth meeting.

http://www.nihs.go.jp/fhm/9kaigijiroku.html

41. Wieneke, A. A., Roberts, D. and Gilbert, R.J. 1993. Staphylococcal food

poisoning in the United Kingdom, 1969-90. Epidemiol. Infect. 110110110110: 519-531.

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].

The Journal of Veterinary Medical Science - J-Stage

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Transcript of The Journal of Veterinary Medical Science - J-Stage