Open Journal of Safety Science and Technology, 2011, 1, 75 -78
doi:10.4236/ojsst.2011.12007 Published Online September 2011 (
Copyright © 2011 SciRes. OJSST
Enterotoxigenicity of Staphylococcus aure us Isolated
from Food H a n d le r s d u r i n g Ha j j Season
in Saudi Arabia
Anas Serag Dablool1*, Saeed Saeed Al-Ghamdi2
1Makkah Public Health Laboratory, Ministry of Health, Makkah, Kingdom of Saudi Arabia
2Department of Pha rmacology and Toxicology, Faculty of Medicine, Umm Al-Qura University,
Makkah, Kingdom of Saudi Arabia
E-mail: *dablool@
Received July 21, 2011; revised August 29, 2011; accepted September 6, 2011
Food poisoning during Hajj season is one of the main hazardous issues where most of the health services in
Saudi Arabia are targeting to minimize every year during Hajj seasons. Ordinarily, food handlers are sub-
jected to medical examination before assignment to work. However, they are mostly lacking proper training
in food handling operations, mass feeding, and sanitary practices. This situation may encourage causing food
poisoning especially with staphylococcus enterotoxins. 1516 clinical specimens from food handlers of dif-
ferent nationalities in Makkah were microbiologically investigated for bacterial pathogens during the hajj
seasons of 2001-2002 in Makkah, Saudi Arabia. 129 Staphylococcus aureus isolates were isolated. Of which,
35% produced enterotoxins A, B, C and D singly or in pairs, when such enterotoxins were evaluated by Re-
versed Passive Latex Agglutination test (RPLA). Enterotoxins C and A, elaborated by 15.5% and 12.4%,
isolates respectively, which showed the highest percentage. They were mostly isolated from nasal swabs than
throat swabs. All isolates were resistant to Penicillin G. On the other hand, they were sensitive to Clindamy-
cin, Oxacillin and Gentamicin when tested by Kirby-Bauer method. The (RPLA) method yielded satisfactory
Keywords: Staphylococcus aureus, Makkah, Hajj, Enterotoxin
1. Introduction
Staphylococcal intoxication worldwide stands out as one
of the main food-borne diseases [1]. Ten Staphylococcal
enterotoxins, enterotoxin ASEA [2], SEB [3] SEC 1 [4],
SEC 2 [5], SEC 3 [6], SED [7], SEE [8], SEH [9], SEG
(10), SEI [11], SEJ [12], SEK [13], SEL [1], SEM, SEN,
SEO [14] have been identified and there is a possibility
to identify more SEs [15]. The majority of those entero-
toxin-positive strains most probably belong to human
staphylococci [16]. Characterization of those SEs has
been hindered because of the lack of reliable and rapid
testing method .On the other hand , most foods impli-
cated in staphylococcal food poisoning outbreaks contain
low levels of enterotoxins; often less than 1 μg/100 g of
food [6].
Food safety is one of the most important concern of
health authorities in the Kingdom of Saudi Arabia during
Hajj. Hajj (pilgrimage season in Makkah) is a period
when more than 2.5 million people gather in this Holy
city for at least five days to perform religious rituals of
worship. Previous unpublished data has been shown that
Staphylococcal food intoxication is the most common
food poisoning, followed by Salmonella gastroenteritis.
On account of large public gathering with huge amounts
of food prepared and consumed, food handlers may ne-
glect hygienic practice and may be considered as a major
source of food contamination; they may contaminate raw
materials, equipments, and expired products [17] and
often do not have visible lesions.
The primary aim of this study is to screen food han-
dlers for coagulase positive staphylococci and to deter-
mine the entreotoxigenicity of these isolates by the
RPLA test. Before the identifications of SEH, SEG and
SEI about 5% of food-borne staphylococcal outbreaks
are caused by unidentified SEs [8]. Therefore. The
RPLA test still the most reliable and rapid testing method
especially when the time is an important factor. Also to
use this isolated strains in further studies [18].
2. Materials and Methods
2.1. Organisms
A total of 1516 clinical specimens were isolated from
different nationality food handlers during the Hajj sea-
sons (2001-2002) in Makkah, Saudi Arabia including
428 nasal swabs, 428 throat swabs , 428 nail swabs, 228
stool samples and 4 wounds swabs, were examined for
presence of staphylococcus aureus. Organisms were iso-
lated on sheep blood agar and mannitol salt agar for stool
2.2. Characterization of Strains
Colonies resembling staphylococci and consisting of
catalase-positive, gram-positive cocci in clusters were
tested for clumping factor and staphylococcal protein A
by Staphaurex kit purchased from Murex Diagnostics
2.3. Enterotoxin Detection
The strains were cultured in Brain Heart Infusion (BHI)
at 37˚C for 18 – 24 h, after centrifuging at 3000 rpm at
4˚C for 20 min. The supernatant was used for enterotoxin
evaluation. The staphylococcal enterotoxins A, B, C, and
D were detected by using an RPLA diagnostic Kit from
DENKA SEIKEN CO. Because these kits showed high
specificity and sensitivity with a detection limit of 0.75
ng, the test was completed within 24 h and neither re-
quired complicated procedures nor expensive equipments
2.4. Antimicrobial Sensitivity Determinations
A total of 45 Staphylococcus aureus enterotoxin produc-
ers cultures were tested for sensitivity to the following
Antimicrobial agents :Penicillin G (PG), Oxytetracycline
(OT), Gentamicin (GM), Erythromycin (E), Cotrimoxa-
zole (TS), Oxacillin (OX), Clindamycin (CD), and
Cephalothin (KF) by Kirby-Bauer method (12). (Sensi-
tivity multi.Disk for Gram (+) microorganism were ob-
tained from Mast Group Ltd.).
3. Results
Of the 129 Staphylococcus aureus isolates from 1516
clinical specimens from different nationalities foodhan-
dlers in Makkah, 45 produced one or more enterotoxins.
Most strains produced only one type of enterotoxin but
few produced two enterotoxins simultaneously. The in-
cidence of enterotoxins A, B, C, and D production in
strains of Staphylococcus aureus is shown in Table 1.
Thirty eight strains (29.5%) produced only a single en-
terotoxin; of these, 16 strains produced enterotoxin A,
one produced B, 20 produced C and only one produced
enterotoxin D. Seven strains (5.4%) produced > 1 en-
terotoxin; 3strains produced A with B, 3 strains produced
A with C and only one strain produced C and D
The Sensitivity results of 45 Staphylococcus aureus
enterotoxins producers tested in this study fell in to 7
groups. Every group has the same results as shown in
Table 2.
4. Discussion
Enterotoxins were shown to be produced by 35% of the
strains isolated from 29% of the total working food han-
dlers who look healthy. These results are in agreement
with those obtained by Wei and Chiou [18].
Enterotoxin C showed the highest incidence 15.5%.
Some researchers have reported that the source of these
enterotoxin C producing strains is unclear (6). On the
other hand, some researchers have related Enterotoxins C
to strains of animal origin [19]. But the interchange of
the staphylococci between animals and man might ex-
plain the high incidence of SEC. Enterotoxins A, B and
Table 1. The incidence of enterotoxins A, B, C, and D production in isolated strains of Staphylococcus aureus.
Enterotoxins produced according to source
Source of strains No. of strains producing enterotoxins
Nasal swabs 29 11 1 11 1 2 2 1
Throat swabs 9 2 - 5 - - - -
Nail swabs 6 3 - 3 - - - -
Stool samples 1 - - 1 - - - -
Total 45 16 1 20 1 3 3 1
Copyright © 2011 SciRes. OJSST
Table 2. Staphylococcus aureus isolates classified according to antimicrobial sensitivity test.
PG OT GM E TS OX CD KF Type of EnterotoxinS. aureus strains No. with same sensitivity
11 Strains from No. 1 to 11
14 Strains from No. 12 to 25
3 Strains from No. 26 to 28
2Strains No. 29 & No. 30
Strain No. 31
Strain No. 32
5 Strains from No. 33to37
Strain No. 38
Strain No. 39
R S S S R S S R C Strain No. 40
R S S R S S S S A Strain No. 41
Strain No. 42
Strain No. 43
R S S S S S S R A Strain No. 44
R R S S R S S S C Strain No. 45
Penicillin G (PG), Oxytetracycline (OT), Gentamicin (GM), Erythromycin (E), Cotrimoxazole (TS), Oxacillin (OX), Clindamycin (CD), and Cephalothin (KF).
AB elaborated by 12.2%, 0.7% and 2.3% respectively
which agreed with the findings of other researchers [6].
An interesting observation is that we isolated some
strains from different locations of three food handlers
(Nasal, Throat, Stool) (Nasal, Throat, nails) (Nasal,
nails), type C, A and A respectively with the same sensi-
tivity results. On the other hand, we isolated three dif-
ferent strains from different locations) (Nasal, Throat,
nails) of one female food handler.
This figure of 35% may be on the lower side of the
scale as other enterotoxins SEE, SHE, SEG and SEI were
omitted from the study because simple detection method
was not available. Also, coagulase negative staphylo-
cocci were not subjected to enterotoxin evaluation and
they have been reported that few of those strains are en-
terotoxigenic [16].
So, it may be concluded that 50% of the isolates could
be enterotoxigenic. Also, during Hajj periods food han-
dlers become under pressure to prepare large quantities
of food. Thus may neglect the instruction on hygienic
standards. In addition, those with poor hygienic practices
can contaminate the food which they handle [17] and
some of visitors buy food to be consumed later after sev-
eral hours allowing small numbers of staphylococci to
multiply and thus produce enterotoxins. All that indicate
that in the case of food poisoning, it’s necessary to detect
the food and food handlers for enterotoxigenicity of
staphylococci because their presence does not necessary
imply that enterotoxin was produced. Many strains are
not enterotoxin producer .Similarly, the absence of viable
staphylococci in food does not mean that toxin is also
absent. Because enterotoxins are more heat stable than
producing cells and can be present in heated foods, the
organisms isolated were sensitive to most of the Antim-
icrobial agents except Penicillin G (PG). No MRSA nor
Oxacillin (OX) resistant strains were isolated.
5. Conclusions
This study shows that the total ratio of both Arabs and
Asians carriers are nearly the same (8.7 and 8.9 respec-
tively). This indicates that bad habits such as; picking
nose (fingering the nose), nasal secretions and spiting on
the ground could be the reason for increasing the ratio of
staphylococcal intoxication. However, the predominance
of specific Enterotoxins type among S. aureus isolates
from human carriers is variable. Most of enterotoxigenic
S. aureus were isolated from the nose which could fa-
cilitate the transmission of this organism from nose to
hand during unhygienic cleaning habits. The sensitivity
results indicate the susceptibility of one person to harbor
or be infected with more than one strain of S. aureus
which reflects the importance of proper microbiological
investigation to achieve proper and successful treatment.
This study emphasizes the importance of implementing a
health certification process for food handlers supported
with a proper training and educational agenda.
6. Acknowledgments
This work was supported by: General Administration for
Research Grant 37921-A-22 in10/6/2001 from king Ab-
dul-Aziz City for Sciences and Technology.
7. References
[1] P. M. Orwin, J. R. Fitzgerald, D. Y. M. Leung, J. A.
Copyright © 2011 SciRes. OJSST
Gutierrez, G. A. Bohach and P. M. Schlievert, “Charac-
terization of Staphylococcus aureus Enterotoxin L,” Infec-
tion and Immunity, Vol. 71, No. 5, 2003, pp. 2916-2919.
[2] M. Ellis, A. Serreli, P. Colque-Navarro, U. Hedstrom, A.
Chacko, E. Siemkowicz and R. Mollby, “Role of Staphy-
lococcal Enterotoxin A in a Fatal Case of Endocarditis,”
Journal of Medical Microbiology, Vol. 52, No. 2, 2003,
pp. 109-112. doi:10.1099/jmm.0.05003-0
[3] T. O. Yarovinsky, M. P. Mohning, M. A. Bradford, M. M.
Monick and G. W. Hunninghake, “Increased Sensitivity
to Staphylococcal Enterotoxin B Following Adenoviral
Infection,” Infection and Immunity, Vol. 73, No. 6, 2005,
pp. 3375-3384. doi:10.1128/IAI.73.6.3375-3384.2005
[4] D. L. Hu, J.-C. Cui, K. Omoe, H. Sashinami, Y. Yoko-
mizo, K. Shinagawa and A. A. Nakane, “Mutant of
Staphylococcal Enterotoxin C Devoid of Bacterial Su-
perantigenic Activity Elicits a Th2 Immune Response for
Protection against Staphylococcus aureus Infection,” In-
fection and Immunity, Vol.73, No. 1, 2005, pp. 174-180.
[5] X. Wang, H. Zhang, M. Xu, Y. Cai, C. Liu, Z. Su and C.
Zhang, “Biological Characterization of the Zinc Site Co-
ordinating Histidine Residues of Staphylococcal Entero-
toxin C2,” Microbiology, Vol. 155, No. 3, 2009, pp.
680-686. doi:10.1099/mic.0.025254-0
[6] L. Jodi, “Staphylococcus: Molecular Genetics Caister,”
Academic Press, Cambridge, 2008, pp. 155-161.
[7] T. Tollersrud, A. H. Kampen and K. Kenny, “Staphylo-
coccus aureus Enterotoxin D Is Secreted in Milk and
Stimulates Specific Antibody Responses in Cows in the
Course of Experimental Intramammary Infection,” Infec-
tion and Immunity, Vol. 74, No. 6, 2006, pp. 3507-3512.
[8] N. Balaban and A. Rasooly, “Staphylococcal Enterotox-
ins,” International Journal of Food Microbiology, Vol.
61, No. 1, 2000, pp. 1-10.
[9] K. Omoe, M. Ishikawa, Y. Shimoda, D.-L. Hu, S. Ueda
and K. Shinagawa, “Detection of seg, seh, and sei Genes
in Staphylococcus aureus Isolates and Determination of
the Enterotoxin Productivities of S. aureus Isolates Har-
boring seg, seh, or sei Genes,” Journal of Clinical Micro-
biology, Vol. 40, No. 3, 2002, pp. 857-862.
[10] O. Dauwalder, D. Thomas, T. Ferry, A.-L. Debard, C.
Badiou, F. Vandenesch, J. Etienne, G. Lina and G. Mon-
neret, “Comparative Inflammatory Properties of Staphy-
lococcal Superantigenic Enterotoxins SEA and SEG:
Implications for Septic Shock,” Journal of Leukocyte Bi-
ology, Vol. 80, No. 4, 2006, pp. 753-758.
[11] M. M. Fernandez, R. Guan, C. P. Swaminathan, E. L.
Malchiodi and R. A. Mariuzza, “Crystal Structure of
Staphylococcal Enterotoxin I (SEI) in Complex with a
Human,” The Journal of Biological Chemistry, Vol. 281,
No. 35, 2006, pp. 25356-25364.
[12] H. Nakaminami, N. Noguchi, M. Ikeda, M. Hasui, M. Sato,
S. Yamamoto, T. Yoshida, T. Asano, M. Senoue and M.
Sasatsu, “Molecular Epidemiology and Antimicrobial
Susceptibilities of 273 Exfoliative to Xin-Encoding-
Gene-Positive Staphylococcus aureus Isolates from Pa-
tients with Impetigo in Japan,” Journal of Medical Micro-
biology, Vol. 57, No. 10, 2008, pp. 1251-1257.
[13] P. M. Orwin, D. Y. M. Leung, H. L. Donahue, R. P. No-
vick and P. M. Schlievert, “Biochemical and Biological
Properties of Staphylococcal Enterotoxin K,” Infection
and Immunity, Vol. 69, No. 1, 2001, pp. 360-366.
[14] L. L. Yves, F. Baron and M. Gautier “Staphylococcus
aureus and Food Poisoning,” Genetics and Molecular
Research, Vol. 2, No. 1, 2003, pp. 63-76.
[15] K. Omoe, K. Imanishi, D.-L. Hu, H. Kato, H. Takaha-
shi-Omoe, A. Nakane, T. Uchiyama and K. Shinagawa,
“Biological Properties of Staphylococcal Enterotoxin-
Like Toxin Type R,” Infection and Immunity, Vol. 72, No.
6, 2004, 3664-3667.
[16] K. Becker, B. Keller, C. von Eiff, M. Bruck,, G. Lubritz,
J. Etienne and G. Peters, “Enterotoxigenic Potential of
Staphylococcus intermedius,” Applied and Environmental
Microbiology, Vol. 67, No. 12, 2001, pp. 5551-5557.
[17] S. J. M. Bidawid and S. A. Sattar, “Contamination of
Foods by Food Handler: Experiments on Hepatitis A Vi-
rus Transfer to Food and Its Interruption,” Applied and
Environmental Microbiology, Vol. 66, No. 7, 2001, pp.
[18] H. L. Wei and C. S. Chiou, “Molecular Subtyping of
Staphylococcus aureus from an Outbreak Associated with
a Food Handler,” New York Press, New York, 2002, pp.
[19] O. Akineden, C. Annemuller, A. A. Hassan, C. Lammler,
W. Wolter and M. Zschock, “Toxin Genes and Other
Characteristics of Staphylococcus aureus Isolates from
Milk of Cows with Mastitis,” Clinical and Diagnostic
Laboratory Immunology, Vol. 8, No. 5, 2001, pp. 959-964.
Copyright © 2011 SciRes. OJSST