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AASCIT Journal of Health 2018; 5(2): 34-38 http://www.aascit.org/journal/health ISSN: 2381-1277 (Print); ISSN: 2381-1285 (Online) Isolation of Staphylococcal Enterotoxins Causing Gastroenteritis Saly M. E. Toubar 1 , Abdllah A. Elbialy 2 , Mahmoud M. M. Zaky 1 , Ahmed S. El-Shafey 3, * 1 Botany Department, Faculty of Science, Port-Said University, Port-Said, Egypt 2 Microbiology and Immunology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt 3 Microbiology Section, Faculty of Science, Tanta University, Tanta, Egypt Email address * Corresponding author Citation Saly M. E. Toubar, Abdllah A. Elbialy, Mahmoud M. M. Zaky, Ahmed S. El-Shafey. Isolation of Staphylococcal Enterotoxins Causing Gastroenteritis. AASCIT Journal of Health. Vol. 5, No. 2, 2018, pp. 34-38. Received: February 26, 2018; Accepted: March 21, 2018; Published: May 16, 2018 Abstract: Staphylococcal enterotoxins (SE) A and B are two of the most important gastroenteritis causing agents. In some areas, more than 50% of food poisoning cases are caused by staphylococcal enterotoxin A (SEA). In USA and England, Staphylococcal enterotoxins A and B (SEA and SEB) are the most food poisoning causing agents (> 60%). Staphylococcal nasal carriage is constantly established in 20-40 of healthy human population and intermittently in 60% and only 10-20% of people are non-carries. If food providers don't obey the rules of hygiene, they can transfer the contamination to food. A concentration of 10 bacteria per gram of food is sufficient for toxin production and induction of disease Staphylococcal enterotoxins are low molecular weight proteins (MW 26.900–29,600). They are encoded by genes embedded in mobile genetic elements such as phags and pathogenicity islands. There are several methods for detection of enterotoxigeneic bacteria. The phenotypical methods are not reliable in specificity, because staphylococcal enterotoxins serotypes are antigenically similar. On the other hand, commercial serological kits can not detect all the serotypes and are limited in serotypes (A-B). Therefore, molecular techniques such as multiplex PCR and real-time PCR are recommended for detection of Staphylococcus aureus enterotoxins genes. Keywords: Staphylococcal Enterotoxins (SE), Serological Kits, Molecular Techniques, Polymerase Chain Reaction (PCR) 1. Introduction Several studies have shown that 15% to 80% of the Staphylococcus aureus isolated from various sources (dairy products, ice cream, meat products) is able to produce enterotoxin [1-3]. Staphylococcal enterotoxins (SE) A and B are two of the most important gastroenteritis causing agents. In some areas, more than 50% of food poisoning is caused by SEA. In USA and England Staphylococcal enterotoxins A & B are the most Food poisoning causing agents (> 60%) [4]. Staphylococcal enterotoxins are low molecular weight proteins MW 26.900-29.600 KD). These are encoded by genes embedded in mobile genetic elements such as phages and pathogenicity islands [5]. There are several methods for detection of enterotoxigenic bacteria. The phenotypical methods (agglutination, SRID) are not reliable in specificity, because SE serotypes are antigenically similar [6]. On the other hand, commercial serologic kits can not detect all the serotypes and is limited to serotypes (A-E) [7]. Therefore, molecular techniques such as PCR and real- time- PCR are recommended for detection of Staphylococcus aureus enterotoxin genes [7]. In this study, genotypic method is utilized to detect Staphylococcal enterotoxins A and B genes. Furthermore, we used these methods to examine the contamination rate of traditional dairy products by Staphylococcus aureus. 2. Materials and Methods 2.1. Samples One hundred samples of Milk and Milk products were

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Page 1: Isolation of Staphylococcal Enterotoxins Causing ...article.aascit.org/file/pdf/9720791.pdfAbstract: Staphylococcal enterotoxins (SE) A and B are two of the most important gastroenteritis

AASCIT Journal of Health

2018; 5(2): 34-38

http://www.aascit.org/journal/health

ISSN: 2381-1277 (Print); ISSN: 2381-1285 (Online)

Isolation of Staphylococcal Enterotoxins Causing Gastroenteritis

Saly M. E. Toubar1, Abdllah A. Elbialy

2, Mahmoud M. M. Zaky

1, Ahmed S. El-Shafey

3, *

1Botany Department, Faculty of Science, Port-Said University, Port-Said, Egypt 2Microbiology and Immunology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt 3Microbiology Section, Faculty of Science, Tanta University, Tanta, Egypt

Email address

*Corresponding author

Citation Saly M. E. Toubar, Abdllah A. Elbialy, Mahmoud M. M. Zaky, Ahmed S. El-Shafey. Isolation of Staphylococcal Enterotoxins Causing

Gastroenteritis. AASCIT Journal of Health. Vol. 5, No. 2, 2018, pp. 34-38.

Received: February 26, 2018; Accepted: March 21, 2018; Published: May 16, 2018

Abstract: Staphylococcal enterotoxins (SE) A and B are two of the most important gastroenteritis causing agents. In some

areas, more than 50% of food poisoning cases are caused by staphylococcal enterotoxin A (SEA). In USA and England,

Staphylococcal enterotoxins A and B (SEA and SEB) are the most food poisoning causing agents (> 60%). Staphylococcal

nasal carriage is constantly established in 20-40 of healthy human population and intermittently in 60% and only 10-20% of

people are non-carries. If food providers don't obey the rules of hygiene, they can transfer the contamination to food. A

concentration of 10 bacteria per gram of food is sufficient for toxin production and induction of disease Staphylococcal

enterotoxins are low molecular weight proteins (MW 26.900–29,600). They are encoded by genes embedded in mobile genetic

elements such as phags and pathogenicity islands. There are several methods for detection of enterotoxigeneic bacteria. The

phenotypical methods are not reliable in specificity, because staphylococcal enterotoxins serotypes are antigenically similar.

On the other hand, commercial serological kits can not detect all the serotypes and are limited in serotypes (A-B). Therefore,

molecular techniques such as multiplex PCR and real-time PCR are recommended for detection of Staphylococcus aureus

enterotoxins genes.

Keywords: Staphylococcal Enterotoxins (SE), Serological Kits, Molecular Techniques, Polymerase Chain Reaction (PCR)

1. Introduction

Several studies have shown that 15% to 80% of the

Staphylococcus aureus isolated from various sources (dairy

products, ice cream, meat products) is able to produce

enterotoxin [1-3].

Staphylococcal enterotoxins (SE) A and B are two of the

most important gastroenteritis causing agents. In some areas,

more than 50% of food poisoning is caused by SEA. In USA

and England Staphylococcal enterotoxins A & B are the most

Food poisoning causing agents (> 60%) [4].

Staphylococcal enterotoxins are low molecular weight

proteins MW 26.900-29.600 KD). These are encoded by

genes embedded in mobile genetic elements such as phages

and pathogenicity islands [5].

There are several methods for detection of enterotoxigenic

bacteria. The phenotypical methods (agglutination, SRID) are

not reliable in specificity, because SE serotypes are

antigenically similar [6].

On the other hand, commercial serologic kits can not

detect all the serotypes and is limited to serotypes (A-E) [7].

Therefore, molecular techniques such as PCR and real-

time- PCR are recommended for detection of Staphylococcus

aureus enterotoxin genes [7].

In this study, genotypic method is utilized to detect

Staphylococcal enterotoxins A and B genes. Furthermore, we

used these methods to examine the contamination rate of

traditional dairy products by Staphylococcus aureus.

2. Materials and Methods

2.1. Samples

One hundred samples of Milk and Milk products were

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35 Saly M. E. Toubar et al.: Isolation of Staphylococcal Enterotoxins Causing Gastroenteritis

collected randomly from different areas in Prot Said governorate.

Table 1. Different samples of milk and milk products used in the study.

Sample type Number of samples

Raw Milk 20

Pasteurized Milk 6

Powdered Milk 4

Hand-made Yogurt 10

Canned Yogurt 10

Newziland Butter 10

Cream 10

Damitta Cheese 5

Karish Cheese 10

Rommy Cheese 5

Cheedar Cheese 5

Cooked Cheese 5

2.2. Processing of Samples:[8].

Figure 1. The stomacher.

2.3. Bacteriological Identification

Media used in cultivation:

1- Cooked meat with 9% NaCl (Oxoid, UK)

Figure 2. Cooked meat medium 9% NaCl.

2- Baird Parker agar with Egg Yolk Emulsion (Oxoid, UK)

a- Baird-Parker agar

b- Egg Yolk Tellurite Emulsion

3- Mannitol salt agar (Oxoid, UK)

Preparation:

Cultivation of samples: [9].

Colonial morphology

2.4. Identification of the Suspected Colonies

1- A smear from the suspected colonies was stained by

Gram stain and examined under microscope.

Gram Stain Kit

2- Catalase reagent

3- Tube coagulase test [10].

Figure 3. Coagulase positive test.

Preparation:

Meathod of DNase test [11].

Figure 4. DNase positive test shows a clear halo around the organism.

2.5. Mainteenance of the Selected Isolates

The selected isolates that fulfilled the criteria of being

Staphylococcus aureus were inoculated on nutrient agar

slopes. After an overnight incubation at 37°C, the slopes

were kept at 4°C. Passage of the isolates was done every 2-3

weeks. Also, before starting any experiment, subculture was

done twice to allow the cells to restore its viability. The

isolate also inoculated on deep soft agar by direct stabbing

and inoculated overnight at 37°C, then kept at freezer for one

to three months.

2.6. Multiplex PCR for Detection of Sea &

Seb Genes

2.6.1. DNA Extraction

1. DNA extraction kit: i-genomic BYF DNA Extraction

Mini Kit, (Cat. No. 17361) (iNtRON Biotechnology,

Korea).

2. Additional required equipment and reagent:

2.6.2. DNA Amplification

Material for DNA amplification: PCR Premix: 2x PCR

Master mix Solution (i-Taq™) tubes (Cat. No. 25027 "1ml",

25028 "5 ml") (iNtRON Biotechnology, Korea).

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AASCIT Journal of Health 2018; 5(2): 34-38 36

2.6.3. DNA Detection by Gel Electrophoresis

All the following are used

1. 0.5 M disodium ethylenediamine tetraacetate (EDTA).

2. Electrophoresis buffer: Tri acetate EDTA (TAE) (1X)

buffer.

3. Agarose gel powder (Boehringer Mannheim, Germany):

Molecular screening agarose for separation of small

DNA fragments. It was stored at room temperature.

4. Ethidium bromide (Sigma, USA).

5. A DNA Molecular weight marker, SiZer™-100 DNA

Marker (iNtRON Biotechnology, Korea).

2.7. Statistical Analysis of the Results

The results were calculated, tabulated and statistically

analyzed. The collected data were entered, checked and

analyzed using chi square (x2) according to knapp and Odds

ratio Using statistical computer program SPSS version II

under windows 7 as follow:

Chi square x 2:

Used to compare between more than 2 percentages and

determine whether there is a significant difference between

the expected frequencies and the observed frequencies in one

or more categories.

∑−=E

EOx

22 )(

3. Results

The present work was carried out on 100 samples of milk

and milk products collected randomly from different areas in

Prot Said Governorate.

Milk samples were 30; they were 20 samples of raw milk,

6 samples of pasteurized milk and 4 samples of powdered

milk.

Cheese samples were 30; they were 10 samples of karish

cheese, 5 samples of damitta cheese, 5 samples of rommy cheese,

5 samples of cheedar cheese and 5 samples of cooked cheese.

Yoghurt samples were 20; they were 10 samples of canned

yoghurt and 10 samples of hand-made yoghurt.

Also 10 samples of cream and 10 samples of butter were

examined.

Figure 5. Staphylococcal enterotoxin genes isolated by PCR in milk and milk products.

Table 2. Type of Staphylococcal enterotoxin genes isolated by PCR in milk and milk products.

Sample type % Positive samples

for S. aureus

Positive samples for

enterotoxin by PCR No. (%)

Negative samples

No. (%) Odds ratio 95% CI X2 P. value

Cheese 15 4 (33.3) 11 (66.7) 0.25 (004-1.43) 3.35 0.078

Milk 16 8 (50.0) 8 (50.0) 1 (0.2-5.01) 0.0 1.0

Cream 4 2 (50.0) 2 (50.0) 1 (0.0-39.77) 0.0 1.0

Butter 4 1 (25.0) 3 (75.0) 0.11 (0.0-5.15) 2.1 0.457

Yogurt 7 3 (42.8) 4 (57.2) 0.56 (0.04-7.46) 0.29 0.593

Total 46 18 (41.3) 28 (58.7)

X2 1.54

P. value 0.843

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37 Saly M. E. Toubar et al.: Isolation of Staphylococcal Enterotoxins Causing Gastroenteritis

Figure 6. Showing agarose gel electrophoresis for PCR of 270 bp sea gene and 165 bp seb gene. (Lane M) is the (100 bp ladder marker), (Lane 2) is for sea,

(Lane 3) is for seb, (Lane 4) is for both genes (sea& seb).

4. Discussion

Among food stuffs implicated in food poisoning; milk,

dairy products, meat, poultry and eggs, specially handled

food, play an important role since enterotoxigenic strains

have been frequently isolated from food handelars [12, 13].

Milk products can cause sever health hazards to people as

they are highly susceptible to variety of microorganisms

because of their high nutritive value [14]. They are

responsible of many outbreaks of food poisoning [15]. The

importance of the enterotoxins comes from their heat stability

and their resistance to inactivation by gastrointestinal

proteases like pepsin. Although Staphylococcus can be killed

at normal cooking temperature, the toxins remain active [16].

Also they are potent even in very small amount ranging from

20 ng to < 1µg can produce symptoms to human beings [17].

These results agreed with Niskanen and Aalto [18] who.

detected Staphylococcus growth on Baird-Parker agar in 83%

of samples versus 59.4% by Mannitol Salt agar, on the other

hand AlKhafaji [19] detected equal growth of S. aureus on

both media.

All enterotoxins are superantigens which are encoded by

mobile genetic elements including phages, plasmids and

pathogenicity islands [20-22]. Detection of SE gene does not

indicate presence of biologically active molecules or

production of enough amount of toxin to cause disease. But it

allows the determination of potentially enterotoxigenic

strains of S. aureus [23]. In the present study enterotoxin

genes were detected in 19 isolated strains of S. aureus out of

46 strains positive for S. aureus in a percent of (41.3%). This

result is higher than that detected by Imanifooladi et al [9]

who detected enterotoxin gene in (31.1%) in isolated S.

aureus colonies from milk and milk products While S. aureus

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AASCIT Journal of Health 2018; 5(2): 34-38 38

organism is heat labile, its produced enterotoxin is heat stable.

Hence the importance of Multiplex PCR technique in

detecting genes encoding enterotoxigenic strains especially in

food poisoning outbreak.

5. Conclusion

The phenotypical methods are not reliable in specificity,

because staphylococcal enterotoxins serotypes are

antigenically similar. On the other hand, commercial

serological kits can not detect all the serotypes and are

limited in serotypes (A-B). Therefore, molecular techniques

such as multiplex PCR and real-time PCR are recommended

for detection of Staphylococcus aureus enterotoxins genes.

References

[1] 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, 246: 191-198.

[2] Fueyo, J. M.; Mendoza, M. C; Rodicio, M. R.; Muniz, J.; Aivarez, M. A. and Martin, M. C. (2005): Cytotbxin and pyrogenic. toxin superantigen gene profiles of Staphylococcus aureus associated with subclinical mastitis in dairy cows and relationships with macrorestriction genomic profiles. J. Clin. Microbiol., 43: 1278-1284.

[3] Bania, J.; Dabrowska, A.; Bystron, J.; Korzekwa, K.; Chrzanowska, J. and Molenda, J. (2006): Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food. Int. J. Food Microbiol., 108: 36-41.

[4] Kluytmans, J. A. And Wertheim, H. F. L. (2005): Nasal carriage of Staphylococcus aureus and prevention of nosocomial infections. Infect., 33: 3-7.

[5] Martin, M. C.; Fueyo, J. M.; González-Hevia, M. A, and Mendoza, M. C. (2004): Genetic procedures for identification of enterotoxigenic strains of Staphylococcus aureus from three food poisoning outbreaks. Int. J. Food Microbiol., 94: 279-286.

[6] Edwin, C.; Tatini, S. R. and Maheswaran, S. K. (2011): Specificity and cros reactivity of Staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, Cl, D, and E. Appl. Environ. Microbiol., 52: 1253 - 1257.

[7] Van Belkum, A. (2003): Molecular diagnostics in medical microbiology: Yesterday, today and tomorrow. Curr. Opin. Pharmacol., 3: 497-501.

[8] Duncan S. E., Yaun B. R. & Sumner S. S. (2004): microbiological methods for dairy products, standard methods for the examination of dairy products, 17th edition, Washington, Amer. Pub. Heal. Associ., pp, 249-268.

[9] ImaniFooladi A. A.; Tavakoli, H. R. and Naderi, A. (2010): Detection of enterotbxigenic Staphylococcus aureus isolates in. domestic dairy products. Iranian J. Microbiol., 2: 135-140.

[10] Barry, A. L.; Lachica, R. V. and Atchison, F. W. (2013): Identification of Staphylococcus aureus by simultaneous use of tube coagulase and thermo-nuclease tests. Appl. Microbiol., 25: 496-497.

[11] Smith, P. B.; Hancock, G. A. and Rhoden, D. L. (2013): Improved medium for detecting deoxyribonuclease-producing bacteria. Appl. Microbiol., 18: 991-993.

[12] De Buyser, M. L.; Dufour, B.; Maire, M. and Lafarge, V. (2001): Implication of milk and milk products in. food-borne diseases in France and in different industrialized countries. Int. J. Food Microbiol.; 67: 1-17.

[13] Normanno, G.; La Salandra, G.; Dambrosio, A.; Quaglia, N. C.; Corrente, M.; Parisi, A.; Santagada, G.; Firinu, A.; Crisetti, E. and Ceiano, G. V. (2007): Occurrence, characterization and antimicrobial resistanc of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products. Int. J. Food Microbiol., 115: 290-296.

[14] Soomro, A. H.; Arain, M. A.; Khaskheli, M. and Bhutto, B. (2003): Isolation of Staphylococcus aureus from milk products sold at sweet meat shops of Hyderabad. Online J. Biol. Sci., 3: 91-94.

[15] Thaker, H. C; Brahmbhatt, M. N. and Nayak, J, B. (2013): Isolation and identification of Staphylococcus aureus from milk and milk products and their drug resistance patterns in Anand, Gujarat. Vet. World, 6: 10-13.

[16] Presscott, L. M.; Harley, J. P. and Klein, D. A. (2002): Text book of Microbiology. Brown Publishers. 5th ed: 441-442.

[17] Bergdoll, M. S.; Borja, C. R. and Avena, R. M. (2008): Identification of a new enterotoxin as enterotoxin C. J. Bacteriol., 90:. 1481-1485.

[18] Niskanen, A., and Aalto. M.(1978): Comparison of Selective Media for CoagulasePositive Enterotoxigenic Staphylococcus aureus. Appl. Environ. Microbiol. 35, 1233—1236.

[19] Al-Khafaji, M. H. M (2013): Detection of enterotoxins genes in Staphylococci isolated from milk and cheese. Thesis Ph.D., Coll. Sci., Univ. Baghdad.

[20] Alouf, J. E. and Muller-Alouf, H. (2003): Staphylococcal and Streptococcal superantigens: molecular, biological and clinical aspects. Int. J. Med. Microbiol., 292: 240-429.

[21] Mempel, M,; Una, G.; Hojka, M,; Sclmopp, C.; Seidl, H, P.; Schafer, T.; Ring, J.; Vandenesch, F. and Abeck, D. (2003): High prevalence of superantigens associated with the egc locus in Staphylococcus aureus isolates from patients with atopic eczema. Eur. J. Clin. Microbiol. Infect. Dis., 22: 306-309.

[22] Orwin, P. M.; Fitzgerald, J. R.; Leung, D. Y.; Gutierrez, J. A.; Bohach, G. A. and Schlievert, P. M. (2003): Characterization of Staphylococcus aureus enterotoxin L. Infect. Immun., 71: 2916-2919.

[23] Ercolini D, Blaiotta G, Fusco V, Coppola S (2004). PCR-based of enterotoxigenic Staphylococcus aureus in the early stages of raw milk cheese making, J. Appl. Microbiol., 96: 1090-1096.