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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. 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
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).
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
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
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.
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