ISOLATION OF STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI; THE IMPORTANT FOOD BORNE PATHOGENS FROM SEVERAL RESTAURANTS IN BASRAH CITY, IRAQ.

Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
74
ISOLATION OF STAPHYLOCOCCUS AUREUS AND ESCHERICHIA
COLI; THE IMPORTANT FOOD BORNE PATHOGENS FROM SEVERAL
RESTAURANTS IN BASRAH CITY, IRAQ.
Nidham M. Jamalludeen*
* Department of Microbiology,College of Medicine ,University of Basrah
Basrah, Iraq
Key words: Restaurant, Staphylococci, E. coli, Basrah
ABSTRACT
The goal of this research was to isolate, identify and characterize Staphylococcus aureus,
Escherichia coli and some other foodborne pathogens from randomly public distributed
restaurants in Basrah city, Iraq. Of the 134 bacterial isolates from restaurants samples, 36 were
confirmed as S. aureus and out of 141 isolates, 72 were confirmed as E. coli using different
selective and enrichment bacteriological media. Data results of other microorganisms have been
excluded from this study. However, result from this work indicates that preventive and
disinfectant plans should be considered to ensure contamination free restaurants for the better
health of all consumers.
INTRODUCTION
Bacterial food borne infections occur when food that is contaminated with bacteria is
eaten and the bacteria continues to grow in the intestines, setting up an infection which causes
illness. Salmonella, Campylobacter, hemorrhagic E. coli, Staphylococci and Listeria all cause
infections (1). Moreover, Contamination of food at restaurants, with pathogenic bacteria is
mostly due to processing, handling, and unhygienic conditions. For example, microorganisms
may spread to food by hands that are not washed after using the toilet. They also may spread to
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
75
raw meat during processing so that it is contaminated when brought into the kitchen. Because of
this, it is important to make sure hands and working surfaces are thoroughly washed after contact
with raw meat, fish and poultry and before working with foods that require no further cooking (1,
2).
The bacterium of Staphylococci, Escherichia, Candida and other food borne pathogens are wide
spread in nature and have been isolated separately from an enormous range of environmental
sources such as plant surfaces, meat, poultry, air, water, soil, and dairy products (3). These
bacteria are able to cause mild to life threatening diseases, which also includes food borne
illnesses. Multiple species in these microorganisms are having capability to produce diseases
with contaminated food. Of these, Staphylococcus aureus is reported to be a third most important
cause of foodborne diseases in the world among the foodborne pathogens (4, 5). E. coli
frequently contaminates food organism and it is a good indicator of fecal pollution (6, 7, 8).
Existence of E. coli in food indicates the presence of enteric pathogens, which constitute a public
health hazard. Enteropathogenic E. coli can cause severe diarrhoea and vomiting (9). There is
huge evidence that food handlers whose work involves touching unwrapped foods to be
consumed raw or without further cooking or other forms of treatment are those most commonly
implicated in foodborne outbreaks. The unhygienic handling of such foods constitutes a
particularly grave risk (1, 10). The risk of foodborne illness has clearly noticeable over the last
20 years, with nearly a quarter of the population at higher risk for illness today. Thus, preventing
illness and death associated with foodborne pathogens remains a major public health challenge.
Therefore, this study was aimed to isolate and identify some pathogenic microorganism that
capable to contaminate and transfer through food processing chain from several restaurants in
Basrah city, Iraq.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
76
MATERIALS AND METHODS
Collection of Samples:
Swab samples from food processing surfaces, food handlers, and portion of salads were collected
during the period of two months from different restaurants at the Basrah region, Iraq. The
collected samples were examined for the existing of several pathogenic microorganisms that can
be transmitted through food consumption. Samples were collected aseptically, transferred to
sterile plastic bags and then were directly transported to the laboratory under cold conditions.
Samples were processed within 1 hour at the microbiology laboratory of Basrah Medical
College.
Microbiological analysis:
The swabs and a portion of salad sample after (homogenized with 100 ml sterile normal saline)
were cultured into Blood Agar (BA), Mannitol Salt Agar (MSA), MacConkey Agar (MA), and
Eosin Methylen Blue agar (EMB) (Oxoid, UK). Cultured media were incubated at 37 OC for 24
hours, further investigation were done using biochemical tests. Bacteria from suspected colonies
were also stained with Gram stain for the primary identification; and a technique mentioned by
(11) was used for isolation and identification of Staphylococcus aureus. Sample was streaked on
MSA and BA and the plates were incubated at 37 OC for 24 hours. Appearance of golden yellow
colonies on MSA and hemolytic colonies on BA were considered to be presumed S. aureus. E.
coli was isolated by culturing samples into selective medium Eosin Methylene Blue (EMB) agar
and MacConkey Agar and incubated at 37 OC for 24 hours. Morphologically typical colonies
producing metallic sheen and lactose fermentor had been selected for further identification (11).
Biochemical examination:
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
77
Four colonies from pure culture of suspected bacteria were selected to be cultured and identified
by the various biochemical tests. Confirmation of Genus, Staphylococcus was done by Gram
staining and several biochemical tests including Catalase , Oxidase , Indole, Methyl red, Voges-
Proskauer tests. In addition, Nitrate reduction, acid from different sugars, and haemolysis on
blood agar were also used for identification following the method described by Forebs et al. (12).
The suspected species of S aureus was also confirmed by Coagulase test as mentioned by
Monica (13) (Table 1)
Table 1. Biochemical tests reaction for S. aureus
Biochemical test Reaction
Catalase +
Oxidase -
Indole Production -
Nitrate Reduction +
Methyl Red +
Voges- Proskauer +
Glucose +
Mannitol +
Maltose +
Lactose +
Raffinose -
Sucrose +
Haemolysis +
Coagulase +
On the other hand, Gram staining, Catalase , Indole, Methyl red, Voges- Proskauer tests, Nitrate
reduction, Urease production, Simon citrate agar, and various sugar fermentation tests (Table 2)
were used to confirm E.coli isolates
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
78
Table 2. Biochemical tests reaction for E. coli
Biochemical test Reaction
Lactose fermentation +
Catalase +
Simmon’s Citrate -
Indole Production +
Nitrate Reduction +
Methyl Red +
Voges- Proskauer -
Urease -
Glucose +
Mannitol +
Lactose +
Salicin +
Sucrose +
RESULTS
The research findings are related to isolate of S. aureus and E. coli from several samples
taken from restaurants around Basrah city. The results were summarized in Table 3 which
describes the sampling data that consists of various numbers of samples analyzed and confirmed
as S.aureus and E. coli. Out of 134 isolates, only 36 isolates were confirmed as S. aureus; out of
141 isolates 72 were confirmed as E. coli on the basis of morphological and biochemical
characterization (Table 1, 2). According to these results a high contamination with E. coli and S.
aureus was found in samples collected from different restaurants distributed at the Basrah area.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
79
Table 3. S. aureus and E. coli detected in examined samples
Organis
m
Number of sampling Number of bacterial
isolates taken
Number of positive
samples
Food
processin
g
surfaces
Food
handler
s
Salad
s
Food
processin
g
surfaces
Food
handler
s
Salad
s
Food
processin
g
surfaces
Food
handler
s
Sala
ds
S.
aureus
33
20 33 61 27 46 18 8 10
E. coli 33
20 33 54 34 53 21 14 37
DISCUSSION
The presence of foodborne pathogens in food processing area or in restaurant equipments
as well as food handler’s arm, nose or skin are considered a big problem for the public health.
Contaminated food plays an overwhelming role in transmission of infection. Results of this study
indicate contamination of several restaurants with important pathogenic bacteria together with
the normal flora which may lead to outbreak infection. E. coli and S. aureus were found to be the
most prevalent species among the bacterial species identified in the samples. Their accurate
identification is important and the pathogenicity of these species needs to be studied, as many
species of these bacteria are known for their enterotoxigenic potential (14, 15). Coagulase
positive S. aureus was previously isolated from nasal swabs of 10 apparently healthy humans
(16). S. aureus is a among the most important nasocomial pathogens because of both the
diversity and the severity of the infections. It causes superficial and deep skin and soft tissue
infections, endocarditis and bacteraemia and a variety of toxin mediated diseases such as
gastroenteritis, staphylococcus scalded-skin syndrome and toxic shock syndrome (17). On the
other hand, S. aureus releases a toxin such as enterotoxin. As less than 1.0 μg of this toxin
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
80
present in contaminated food produces symptoms of illness. It has been found that this level of
toxin can be released at 105 cells /g of food (18).The incidence of the species of E. coli itself in
restaurants, as a possible cause of food borne disease, is not significant if E. coli is normally a
ubiquitous organism (19), yet the pathogenic strains if present could be harmful to consumers.
The results of the existing study indicate that strict preventive and standards disinfectant should
be performed to ensure contamination free restaurants for the good health of all consumers. For
this care is required from the point of food preparing to the point of consumption.
In conclusion, S. aureus and E. coli were found to be most prevalent species identified in the
samples taken from different restaurants distributed randomly in the Basrah city. These
organisms are very important foodborne pathogens and should be eliminated from food chain.
Acknowledgments
The author would like to thank Ms. Mareim Nabeel for her kindly assist in samples processing.
عزل جراثیم المکورات الذھبیھ والاشریکیا القولونیھ الممرضھ التی تنتقل عن طریق الغذاء من عدة مطاعم
منتشره فی محافظة البصرة-العراق
نظام محمد جمال الدین
قسم الاحیاء المجھریھ ، کلیة الطب،جامعة البصره ،البصره ،العراق
الخلاصھ
تھدف ھذه الدراسھ الى عزل وتشخیص جراثیم المکورات الذھبیھ والاشریکیا القولونیھ الممرضھ والتی عادة ما تنتقل عن
طریق تناول الاغذیھ الملوثھ بھا من عدة مطاعم عامھ منتشره عشوائیا فی مدینة البصره- العراق. فقد تم تاکید عزل 36 عزلھ
من جراثیم المکورات الذھبیھ من مجموع 134 عزلھ جرثومیھ نامیھ على اوساط زرعیھ غنیھ، انتخابیھ ومفرقھ مستخدمھ لعزل
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
81
ھکذا جراثیم و 72 عزلھ من جراثیم الاشریکیا القولونیھ من مجموع 141 عزلھ نامیھ على الاوساط الزرعیھ.تم استبعاد نتائج
عزلات لمجموعھ من الکانات الدقیقھ الاخرى من ھذه الدراسھ. تشیر نتائج ھذا العمل الى ضرورة اجراء واتباع خطط الوقایھ
الصحیھ اللازمھ واستخدام المعقمات الضروریھ لاسطح اماکن تحضیر وجبات الاغذیھ من البدایھ وحتى وصولھا للمستھلک
لغرض الحصول على منتج نھائی خالی من الکانات الممرضھ للاستخدام الادمی.
REFERENCES
1. Kendall P. (2012). Bacterial Foodborne Illness. Colorado State University. Food and
Nutrition Series. Food science and human nutrition. 7.
2. Kaclikova E, Kuchta T, KayT, Gray D.(2001). Separation of Listeria from Cheese and
enrichment media using antibodycoated microbeads and centrifugation. J. microbial.
Methods, 46: 63–67.
3. Kloos WE and Schleifer KH. (1986). “Genus IV Staphylococcus,” In: P. H. A Sneath, et
al., Eds., Bergey’s Manual of Systematic Bacteriology, Vol. 2, Williams & Wilkins,
Baltimore, 1013-1035.
4. Normanno G, Firinu A, Virgilio S, Mula G, Dam-brosio A, Poggiu A, et al. (2005).
“Coagulase–Positive Staphylococci and Staphylococcus aureus in Food Products
Marketed in Italy,” International Journal of Food Microbiology, 98 (1): 73-79.
5. Boerema JA, Clemens R., and Brightwell G. (2006). “Evaluation of Molecular Methods
to Determine Enterotoxigenic Status and Molecular Genotype of Bovine, Ovine, Human
and Food Isolates of Staphylococcus aureus,”. International Journal of Food
Microbiology, 107 (2):192-201.
6. Dilielo LR. (1982). Methods in Food and Dairy Microbiology. AVI publishing Co. Inc.
Westport Connt. USA, 39.
7. Soomro AH, Arain MA, Khaskheli M, Bhutto B. (2002). Isolation of Escherichia coli
from raw milk and milk products in relation to public health sold under market condition
at Tandojam. Pak. J. Nutr, 1 (3): 151–152.
8. Benkerroum N, Bouhal Y, EI Attar A, Marhaben A. (2004). Occurrence of Shiga toxinproducing
E. coli 0157:H7 in selected diary and meat products marketed in the city of
Rabat, Morocco, J. Food. Prot, 67(6):1234–1237.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
82
9. Anonymous, (1975). E. coli Enteritis. Lancet, 1131–1132.
10. De Wit JC and Kampelmacher EH.(1988). Some aspects of bacterial contamination of
hands of workers in food service establishments. Zentralbl Bakteriol Mikrobiol Hyg
B.,186(1):45-54.
11. Brooks GF, Carroll KC, Butel JS, Morse SA. (2010). Jawetz, Melnick & adelberg’s
medical microbiology, 25th edition. Mc Graw Hill Lange.
12. Forbes BA, Sham DF, and Weissfeld AS. (2007). Bailey & Scott's Diagnostic Microbiology,
12th Edition. Mosby.
13. Monica, C. (1991). Medical Laboratory manual for Tropical countries. VOL 11. ELBS,
60–63.
14. Singh P and Prakash A.(2008). Isolation of Escherichia coli, Staphylococcus aureus and
Listeria monocytogenes from milk products sold under market conditions at Agra region.
Acta agriculturae Slovenica, 92(1): 83–88.
15. Rohinishree YS and Negi PS. (2011). Detection, Identification and Characterization of
Staphylococci in Street Vend Foods. Food and Nutrition Sciences, 2:304-313.
16. EL-Jakee J, Nagwa AS, El-Said G, Bakry MA, Samy AA, Khairy EA and Elgabry EA.
(2010). Diversity of Staphylococcus aureus isolated from human and bovine estimated by
PCR-Gene Analysis. J. Of American Science, 11: 487-498.
17. Lowy FD. (1988). Staphylococcus aureus infections. N Engl J Med, 339: 520-532.
18. Ananthanarayan R and Panikaran CKJ.(2001). Diagnostic value of mannitol for sugar
fermentation in S. aureus. Textbook of Microbiology, 6: 178–186.
19. Hahn, G. (1996).Pathogenic bacteria in raw milk situation and significance. In:
Bacteriological quality of raw milk. Brussels (Belgium), Int. Dairy Federation, 67–83.