DETECTION OF Staphylococcus aureus FROM LOCAL AND IMPORTED CHICKEN IN DUHOK PROVINCE/KURDISTAN REGION OF IRAQ USING CONVENTIONAL AND MOLECULAR METHODS

This study aimed to detect Staphylococcus aureus in local fresh whole chicken and frozen imported chicken using conventional and PCR assay by targeting specific S . aureus thermonuclease gene ( nuc ). A total of 200 whole chicken carcasses were examined and samples include 100 of local chickens from the Duhok chicken abattoir and 100 of imported chicken from supermarkets in Duhok city. The samples were cultured on mannitol salt agar and the confirmation is done by colony morphology, Gram stain, biochemical test including catalase test and coagulase test. The results showed that 28 (28%) of 100 local chicken and 80 (80%) of 100 imported frozen chicken carcasses were found to be positive with S . aureus using conventional methods. Amplification of nuc gene specific for S . aureus is used to confirm the isolates of S . aureus . The results showed that only 18 of 22 coagulase positive isolates from local chicken and 57 of 68 coagulase positive isolates from imported chicken were confirmed as S . aureus . The results indicate that PCR assay seem to be more specific for detection of S . aureus in food sample and appear to be more reliable than conventional methods for assessing bacteriological safety of food. These results showed high prevalence of S . aureus in imported chicken than in local chicken meat and thus may happen as a result of processing and storage conditions.


INTRODUCTION
In developing countries, food-borne diseases (FBD) are one of the major concerns causing annually several deaths along with economic losses. According to World Health Organization (WHO), annually an estimated 600 million people are infected and about 420,000 die worldwide due to ingestion of food contaminated with foodborne pathogens.
Many pathogens, including E. coli, Salmonella, Campylobacter, Listeria, Vibrio cholera and S. aureus have been reported to be responsible for food-borne outbreaks around the world (1).
Worldwide S. aureus food-borne is one of the major concerns in public health (2,3,4,5) . S. aureus causes various infections including skin infections to sever invasive disease (4,6). S. aureus can grow in a wide range of temperatures but the optimum is 30 to 37 ºC, optimum pH 7 to 7.5 and at NaCl concentrations as high as 16% (7). There are some characteristics of the S. aureus that favour its growth in many food products (8) . These features include surviving in dry and stressful environments. For example, nose, on skin, and inanimate surfaces (9) . Therefore, it can remain persist for extended duration after initial contact on hands and environmental surfaces (10) . Food poisoning by S. aureus occurs rapidly following consumption of contaminated food and it usually takes 3 to 5 hours. This is due to the production of toxins by S. aureus as they grow at optimum temperature (8). For S. aureus, variety of foods provides as an optimum growth medium. It has been reported that variety of foods including meat and meat products, poultry meat and poultry products and, milk and dairy products are frequently involved in S. aureus FBD (3,8,11,12,13,14,15) . Other major factors that are involved in increasing the number of FBDs are absences of sanitary conditions and incorrect microbiological checking of food (5) .
Furthermore, other conditions are involved to a high number of FBD outbreaks especially in retail food industry such as improper handling processes of food (16) . Poultry meat is one of the popular foods with constant increase in consumption worldwide and commonly contaminated by antibiotic resistant S. aureus strains (5,17) . S. aureus is found normally on the skin and in the intestinal epithelium of animals and therefore, during slaughtering process this may contaminate meat. Possibly contamination of meat may also happen during meat processing and storage time because this pathogen is widely distributed in environment. Consequently, one of the main cause of FBD in human around the world is ingestion of contaminated raw poultry meat (5,17) because of above information that emphasises the importance of detection of S. aureus in whole raw chicken as major foodborne pathogen in human. Therefore, the aim of this study was to detect S. aureus from local whole raw chicken obtained from Duhok chicken slaughter house in a Summel and whole raw imported chicken from supermarkets in Duhok city using both conventional and molecular methods.

MATERIALS AND METHODS
Sample collection:In this study, a total of 200 whole chickens were examined for the detection of S. aureus. The samples include 100 of local chickens and 100 of imported chickens. Local raw chicken carcasses were collected from the Duhok chicken abattoir in Summel. The whole imported chicken carcasses were collected from supermarkets in Duhok city. Both local and imported chicken were collected and placed in a cold box and transported immediately to Microbiology Laboratory at College of Veterinary Medicine for microbiological analysis.

Identification of bacteria using conventional cultural methods:
The whole chicken including both local and imported was transferred to a sterile plastic bag and 400 ml buffer peptone water (BPW) was added to each bag. The whole chicken was then rinsed inside and out for 2 min with a rocking motion. Aseptically the whole chicken was removed from the bag, and the rinse was collected. For isolation 10 ml of chicken rinse was collected aseptically from fresh local chicken and pipetted into 90 ml of BPW. The broths were incubated for 24 h at 37°C. After 24 h incubation, a loop from the broth was cultured onto the mannitol salt agar (MSA). Plates were incubated for 18-24 h at 37°C. S. aureus colonies were first identified according to Bergeys Manual of systematic Bacteriology (18) based on colonial morphology, Gram-stain and biochemical tests. The suspected S. aureus colonies were selected then streaked onto MSA to obtain pure colonies of S. aureus. Colonies with typical morphology were selected and subjected to Gram-staining and catalase test. These isolates were further examined using coagulase test and molecular method. Phenotypically suspected S. aureus isolates were purified and maintained in 50% glycerol and brain heart infusion broth (BHIB) stocks at -20°C for further processing.

Molecular detection:
Phenotypically suspected S. aureus isolates were further confirmed by PCR.
DNA extraction: S. aureus isolates were plated out onto MSA from -20°C stock cultures and incubated overnight at 37°C for 24 h. Chromosomal DNA was prepared according (19).
Briefly, DNA was prepared by resuspending 3 to 4 bacterial colonies in 50 µl of deionized doubled distilled water and mixed very well. Bacterial suspension was boiled in water bath for 5 to 10 min. The suspension was then centrifuged at 10,000 x g for 1 min. The  Table 1.

Prevalence of S. aureus:
A total of 200 whole chicken carcasses were examined and these samples include 100 of local chickens from the Duhok chicken abattoir and 100 of imported chicken carcasses from supermarkets in Duhok city. The prevalence of S. aureus on the local and imported chicken carcasses using conventional method was found to be 108/200 (54%). The results showed that the prevalence S. aureus in local chicken was lower than in imported chicken.

Isolation of S. aureus using conventional culture methods
The overall results using conventional culture methods from both local and imported chicken carcasses are presented in Table 2 (Table 2). Mannitol fermenter colonies on MSA were further sub-cultured in order to be characterized by microscope and biochemical tests.

Identification of S. aureus:
Mannitol fermenter colonies were exposed to Gram stain and biochemical tests such as catalase test, coagulase test for the characterization of isolates. All the 108 mannitol fermenter isolates including 28 from local chicken and 80 from imported frozen chicken carcasses appeared as Gram positive grape-like cocci and catalase positive. Bacterial isolates were further analysed using coagulase test. In this study, 22 of 28 (78.5%) of local chicken carcasses isolates and 68 of 80 (85%) of imported chicken carcasses isolates were found to be coagulase positive. However, 6 of 28 (21.5%) of local chicken isolates and 12 of 80 (15%) of imported chicken isolates were found to be coagulase negative using both tube and slide coagulase test (Table 2).

based on nuc gene by PCR
Two sets of specific primers were synthesized for this study and these primers were S. aureus specific) as shown already in material and methods.
Previously, it has been revealed that rapid diagnosis of S. aureus gene (20). Identification of S. aureus strain was based on and 281 bp of nuc-F2 and nuc-R2 as shown in Fig. 1

One of the major public health concerns nowadays is microbial food safety because
Poultry meat consumption is increasing worldwide and this result protein and nutrient contents (5,17) . For that, it is very important to ensure proper hygiene of both poultry meat in order to prevent that may be dangerous to consumers (4,17,21,22) . ingestion of contaminated food (23,24). The source of outbreaks can be detected through isolation of S. aureus in poultry meat (25) . Outbreaks of S. aureus FBD is related with the consumption of poultry meat and mostly improper cooked meat, poor microbiological safety and storage conditions of meat and meat products. Generally, cultural methods are used for the isolation and identification of S. aureus from chicken meat as mentioned above in materials and methods. In this study, colony morphology on MSA, Gram stain, catalase and coagulase test are used for the primary identification of S. aureus in chicken sample (26,27,28,29,30) .
In this study, a total of 200 whole chicken carcasses including both local and imported chicken were examined, 108 of them showed unique characteristic of S. aureus on MSA which appears as yellow colonies. Primary confirmation was performed based on some biochemical test. All 108 isolates were appeared to be grape-like Gram positive cocci by Gram staining and they were catalase positive (31) . Generally, coagulase test is one of the tests which is used to confirm identification of S. aureus. Therefore, 108 bacterial isolates were further tested by coagulase test. In this study, 22 of 28 (78.5%) of local chicken isolates and 68 of 80 (85%) of imported chicken isolates were found to be coagulase positive.
However, 6 of 28 (21.5%) of local chicken isolates and 12 of 80 (15%) of imported chicken isolates were found to be coagulase negative using both tube and slide coagulase test.
hyicus and S. intermedius, respectively (34) . the percentage of S. aureus infection in chicken meat suggests that contamination of meat could be due to handling, processing, transportation and storage (35). As mentioned above that other Staphylococcus species including S. intermedius and S. hyicus are coagulase positive (33) . Therefore, molecular diagnostic methods such as PCR assay are used for better differentiation of pathogens in terms of species level (15,36,37) . Previously, it has been shown that rapid diagnosis of S. aureus infections can be done through PCR amplification of the nuc gene (20) . Amplification of nuc gene fragment by PCR showed that only 81.8% ( The data of this study showed the differences between conventional and PCR for the identification of S. aureus from chicken meat. This confirms that PCR assay is specific to diagnose and to understand the mechanisms involved in pathogenesis, resistance and survival of the strains in raw meat (37). Overall, the presence of S. aureus is normally not grounds for rejecting carcasses. However, it is often considered an indicator of the overall hygienic status of slaughter plants (38). Proper management, hygiene and microbial safety of poultry meat are highly recommended especially in supermarkets in order to minimize the occurrence of foodborne diseases including S. aureus food-borne from either fresh or frozen chicken meat to human. Generally, contamination of carcasses, cuts, and processed meat products occur through bacteria from animal intestine, environment of slaughterhouse and the equipment that are used during and after slaughtering (17) . Some of these bacterial contaminants are resistant and are capable to grow and survive especially during processing and storage of food (17) . Subsequently, proper management, hygiene and microbial safety of poultry meat is highly recommended either fresh or frozen chicken meat in order to minimize the occurrence of food-borne diseases including S. aureus food-borne in human.