staphylococcus aureus is an important food - borne pathogen involved in a variety of invasive diseases ( 1 ) .
it produces many toxins as well as non - toxic enzymes and can facilitate bacterial attack and proliferation in the body of host ( 2 ) .
some strains produce staphylococcal enterotoxins ( ses ) that can cause food poisoning if food containing preformed se is ingested
. symptoms of staphylococcal food poisoning ( sfp ) have a rapid onset ( 2 to 6 h ) and may include vomiting , stomach pain and diarrhea ( 3 ) .
enterotoxins from s. aureus strains can be classified into 18 serological types : a - u ( except s , f and t)(2,4 - 9 ) .
the ses are a group of heat stable and pepsin resistant exotoxins encoded by genes in the chromosome , pathogenicity island , phages or plasmids ( 2 ) .
for production of sufficient amount of toxin to cause intoxication symptoms , 10 cfu / ml or cfu / g enterotoxigenic strains of bacteria are needed ( 10 ) .
ses are low molecular weight proteins ( mw 26.900  29600 kd ) ( 11 ) .
sea is a leading cause of food intoxication ( 12 ) and is an extremely potent gastrointestinal toxin , as little as 100 ng is sufficient to cause symptoms of intoxication ( 13 ) .
milk and milk products have frequently been implicated in staphylococcal food poisoning and contaminated raw milk is often involved ( 2 , 3 ) .
s. aureus mastitis is a serious problem in dairy production and infected animals may contaminate bulk milk .
human handlers , milking equipment , the environment and the udder and teat skin of dairy animals are other possible sources of bulk milk contamination ( 3 , 14 ) .
surveys to detect classical enterotoxins and to identify enterotoxin genes in s. aureus from milk and milk products have been conducted in many counties including italy , norway , turkey , brazil and iran ( tehran ) ( 16 - 20 ) .
however , there are no published reports about presence of sea gene in milk and its products in tabriz - iran .
therefore , this study was conducted to investigate the presence of sea gene of s. aureus in organic milk and cheese using pcr method in tabriz - iran .
a total of 200 samples ( 100 milk samples , 100 cheese samples ) were collected from milk collection points and food stores in tabriz , iran .
the samples were collected in sterile containers , immediately kept in an ice box and transported to the laboratory .
the samples were investigated microbiologically for the presence of s. aureus , according to the standard method no .
6806 - 1 of the institute of standards and industrial research of iran ( isiri ) .
the milk and cheese samples were diluted in ringer and pepton water , respectively . from each diluted solutions produced , 1 ml was transferred to brain- heart infusion broth and incubated at 37c for 24 h. baird- parker agar supplemented with potassium tellurite and egg yolk emulsion was used for isolation of s. aureus . after 24 - 48 h incubation at 37c , the bpa plates were analyzed for the presence of s. aureus , which appeared as black colonies with transparent zone .
biochemical confirmation tests ( gram staining , coagulase , catalase , oxidase , vp and growth on mannitol salt agar ) were carried out for final identification of the suspected s. aureus isolates . for dna isolation ,
biochemically confirmed single colonies of s. aureus isolates on bpa were cultured on lb broth .
dna extraction procedure was done by a commercial kit ( cinna pure kit ) , according to the supplier s instructions on the bacterial cells pellet from an overnight culture in lb broth .
the primers used for the detection of nuc and sea genes are listed in table 1 .
pcr reactions were performed in reaction buffer ( 10x ) , mgcl2 ( 50 mm ) in a total volume of 25 l , containing 5 l of template dna , 0.75 l of each primers ( 10 pm ) , 0.25 l of dntp ( 10 mm ) , 0.2 l of taq dna polymerase ( 5 unit/l ) and 14.05 l of ddh2o .
pcr was performed under the following conditions : initial denaturation at 94c for 5 min , subsequently followed by 30 cycles of 94c for 1 min , 55c for 1 min and 72c for 1 min with a final extention of 5 min at 72c .
pcr reaction was conducted like the previous test but with some changes in annealing temperature ( 50c for 1 min ) .
the presence of 552 bp amplicon indicates the positive samples for sea gene in these isolates .
a total of 200 samples ( 100 milk samples , 100 cheese samples ) were collected from milk collection points and food stores in tabriz , iran .
the samples were collected in sterile containers , immediately kept in an ice box and transported to the laboratory .
the samples were investigated microbiologically for the presence of s. aureus , according to the standard method no .
6806 - 1 of the institute of standards and industrial research of iran ( isiri ) .
the milk and cheese samples were diluted in ringer and pepton water , respectively . from each diluted solutions produced , 1 ml was transferred to brain- heart infusion broth and incubated at 37c for 24 h. baird- parker agar supplemented with potassium tellurite and egg yolk emulsion was used for isolation of s. aureus .
after 24 - 48 h incubation at 37c , the bpa plates were analyzed for the presence of s. aureus , which appeared as black colonies with transparent zone .
biochemical confirmation tests ( gram staining , coagulase , catalase , oxidase , vp and growth on mannitol salt agar ) were carried out for final identification of the suspected s. aureus isolates .
for dna isolation , biochemically confirmed single colonies of s. aureus isolates on bpa were cultured on lb broth .
dna extraction procedure was done by a commercial kit ( cinna pure kit ) , according to the supplier s instructions on the bacterial cells pellet from an overnight culture in lb broth .
the primers used for the detection of nuc and sea genes are listed in table 1 .
pcr reactions were performed in reaction buffer ( 10x ) , mgcl2 ( 50 mm ) in a total volume of 25 l , containing 5 l of template dna , 0.75 l of each primers ( 10 pm ) , 0.25 l of dntp ( 10 mm ) , 0.2 l of taq dna polymerase ( 5 unit/l ) and 14.05 l of ddh2o .
pcr was performed under the following conditions : initial denaturation at 94c for 5 min , subsequently followed by 30 cycles of 94c for 1 min , 55c for 1 min and 72c for 1 min with a final extention of 5 min at 72c .
pcr reaction was conducted like the previous test but with some changes in annealing temperature ( 50c for 1 min ) .
the presence of 552 bp amplicon indicates the positive samples for sea gene in these isolates .
in the present study , s. aureus was observed in 54 ( 27 % ) out of 200 samples : 9 from milk and 45 from cheese samples .
the number of bacteria in contaminated samples by cfu / ml is shown in table 2 .
for further molecular confirmation of s. aureus colonies , pcr reaction for nuc gene was performed .
results showed that 7 out of 54 s. aureus isolates harbored sea gene ( table 2 ) .
several researchers have reported different results of the number of bacteria in contaminated dairy products by cfu / ml ( 10 , 20 , 21 ) .
variations in s. aureus count in dairy products may depend on sanitary precautions during milk processing chain .
the existence of s. aureus in food and dairy products was previously confirmed by imani fooladi in iran ( 20 ) .
sea is the most common enterotoxin recovered from food poisoning outbreaks in the usa ( 77.8% of all outbreaks)(24 ) . in the current study
considering the findings of brakstad et al.(22 ) , and comparing them to our study , it can be stated that the pcr for amplification of the nuc gene has potential for rapid diagnosis and confirmation of s. aureus isolates . the present study aimed to evaluate the prevalence rate of sea gene in organic milk and cheese in tabriz , iran . outside of iran
, various results have been reported on the presence of s. aureus and its enterotoxins in milk and its products ( 28 - 31 ) . in the current study
, we found that 27% of the organic milk and cheese samples were contaminated by s. aureus .
pcr results for sea gene , showed that 12.96% of the isolates possessed sea gene .
similar level of incidence was reported by holeckova et al . , ( 32 ) , imani fooladi et al . , ( 20 ) and el - jakee et al .
this discrepancy could be attributed to the improvements in the handling and sanitary procedures during milking and its processing .
it was concluded that even though the level of microorganisms in dairy products was not sufficient to cause disease , the presence of toxins could be considered a potential risk for public health .
several conditions such as delay in processing , inadequate refrigeration , poor personal hygiene and post process contamination are associated with staphylococcal growth and enterotoxin production ( 34 ) .
therefore it is essential to ensure high safety standards for preventing staphylococcal food poisoning . in conclusion ,
the results of this study provides some important preliminary data about the prevalence of enterotoxigenic s. aureus in organic milk and cheese in tabriz , iran .
consumption of organic milk and specially cheese is still widespread and could cause a potential risk to public health , so an effective reduction of contamination levels could be achieved by improving sanitation and hygiene procedures .
further investigations are needed to evaluate the production of other toxins in milk and cheese .
 background and objectivesstaphylococcal food poisoning is a gastrointestinal disease , which is caused by consumption of contaminated food with enterotoxins produced by staphylococcus aureus ( ses ) . 
 milk and its products are known sources of food borne diseases . 
 this study was carried out to evaluate the prevalence of enterotoxigenic s. aureus strains in organic milk and cheese in tabriz - iran.materials and methodsa total of 200 samples ( 100 milk samples and 100 cheese samples ) were collected from farms and milk collection points in tabriz - iran . 
 the samples were cultured and identified by standard bacteriological methods , then pcr was performed to detect sea gene.results and conclusionstaphylococcus aureus was found in 27% of all samples ( milk and cheese ) . 
 results of pcr showed that 12.96% of s. aureus isolates possessed sea gene . 
 it suggested the potential public health threat of s. aureus resulting from contamination of dairy products . 
 so , efforts are required to improve safety standards for preventing staphylococcal food poisoning . 
INTRODUCTION
MATERIALS AND METHODS
Specimen collection
Microbiological analysis
DNA extraction and PCR experiments
RESULTS AND CONCLUSION