ثبت و شناسایی ژن های توکسیک S.aureus جدا شده از ماهی تیلاپیا با تکنیک Multi plex PCR
محورهای موضوعی :
میکروبیولوژی مواد غذایی
نازنین نیاخلیلی
1
,
حامد اهری
2
,
بهاره نوروزی
3
1 - دانش آموخته کارشناسی ارشد علوم و صنایع غذایی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
2 - استاد گروه مهندسی کشاورزی- صنایع غذایی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران.
3 - استادیار گروه زیست شناسی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران.
تاریخ دریافت : 1401/04/30
تاریخ پذیرش : 1401/06/28
تاریخ انتشار : 1402/01/01
کلید واژه:
ژن,
استافیلوکوکوس اورئوس,
ماهی تیلاپیا,
Multiplex PCR,
چکیده مقاله :
مقدمه: آبزیان به دلیل کالری و پروتئین بالا و همچنین وجود چربی غیراشباع امگا ۳ از اهمیت بسزایی در سبد غذایی برخوردارند. در مراحل مختلف از زمان صید تا رسیدن به دست مصرف‌کننده، همواره ممکن است بسیاری از آبزیان از جمله ماهی به برخی از میکروارگانیسم‌های بیماری‌زا آلوده شوند و در نهایت مسمومیت شدید را برای افراد ایجاد نمایند. در پژوهش حاضر به بررسی ثبت و شناسایی ژن‌های توکسیک استافیلوکوکوس اورئوس جدا شده از ماهی تیلاپیا با تکنیک Multi plex PCR پرداخته شده است.مواد و روش‌ها: تعداد کل نمونه‌ها 42 عدد (21 عدد نمونه ماهی تازه و 21 عدد نمونه ماهی منجمد) مدنظر قرار گرفته شد. ابتدا پس از آماده‌سازی نمونه‌های مورد بررسی به ترتیب استافیلوکوکوس اورئوس جدا شد و به صورت کشت سطحی در محیط برد پارکر مورد بررسی قرار گرفت، سپس تست کوآگولاز انجام شد و در مرحله بعد با استخراج DNA ژن انتروتوکسیک شناسایی شد. در نهایت توالی‌یابی ژن به صورت اتوماتیک و دستگاهی انجام گرفت.یافته‌ها: یافته‌های به دست آمده نشان داد، 9/92 درصد از کل نمونه‌های مورد بررسی (21 عدد ماهی تیلاپیا تازه و 18 عدد ماهی منجمد) فاقد باکتری استافیلوکوکوس اورئوس بودند و 1/7 درصد از کل نمونه‌ها آلوده به باکتری استافیلوکوکوس اورئوس شدند. نتایج بررسی تست کوآگولاز نیز نشان داد، هر سه نمونه ماهی منجمد مورد بررسی، کوآگولاز مثبت بودند. پس از بررسی ژن‌های تولید‌کننده انتروتوکسیک (SEA، SEB، SEC، SED، SEE و SEG) در بین سه نمونه منجمد آلوده به باکتری استافیلوکوکوس مشاهده شد که تنها در یک نمونه منجمد، ژن انتروتوکسیک SEA وجود داشت.نتیجه گیری: نتایج نشان داد با توجه به هزینه کم و زمان بسیار کوتاه‌تر مورد نیاز برای شناسایی ژن های استافیلوکوکوس اورئوس با روش Multiplex PCR، این روش ابزار قدرتمندی برای مطالعه ژنوتیپ‌های جدایه‌های استافیلوکوک می‌باشد. بنابراین با استفاده از این روش می‌توان به ارتقا سطح سلامت غذایی در جامعه به خوبی پرداخت.
چکیده انگلیسی:
Introduction: Aquatic animal products are considered very important food items in the food basket regarding their high calorie, protein, and omega-3 unsaturated fat. Many aquatic animals, including fish, may always be infected with pathogenic microorganisms at various stages, from hunting to purchasing. Eventually, these factors would cause severe poisoning in humans. The present study investigates the registration and identification of Staphylococcus aureus’s toxic genes isolated from tilapia using the Multiplex PCR technique.Materials and Methods: The sample size included 42 subjects (21 fresh fish samples and 21 frozen fish samples). First, after preparing the samples, Staphylococcus aureus was isolated and examined as surface culture in Baird-Parker medium. Next, a Coagulase test was performed, and the enterotoxin gene was identified by DNA extraction. Finally, gene sequencing was carried out automatically and systematically.Results: The results suggested that 92.9% of the total samples (21 fresh tilapia and 18 frozen fish) had no Staphylococcus aureus, and 7.1% of the samples were infected with Staphylococcus aureus. The coagulase test results also suggested that all three frozen fish samples were coagulase positive. Examining enterotoxin-producing genes (SEA, SEB, SEC, SED, SEE, and SEG) among three frozen samples infected with Staphylococcus bacteria revealed SEA enterotoxin gene only in one frozen sample. Conclusion: The results showed that due to the low cost and much shorter time required to identify toxic Staphylococcus aureus genes using Multiplex PCR, this method is highly effective in studying the genotypes of Staphylococcus isolates. Therefore, by using this method, one can improve the food health level in the society.
منابع و مأخذ:
Arfatahery, N., Davoodabadi, A. & Abedimohtaseb, T. (2016). Characterization of toxin Genes and antimicrobial susceptibility of staphylococcus aureus isolates in fishery products in iran. Science Reports, 6, 34216,1-7.
Ayulo, A.M.R., Machado, R.A. & Scussel, V.M. (1994). Enterotoxigenic Escherichia coli and Staphylococcus aureus in fish and seafood from the southern region of Brazil. International Journal of Food Microbiology, 24(1-2), 171-178.
Arslan, S. & Ȍzdemir, F. (2017). Molecular characterization and detection of enterotoxins, methicillin resistance genes and antimicrobial resistance of Staphylococcus aureus from fish and ground beef. Polish Journal of Veterinary, 20, 1, 85-94.
Argudín, M.Á., Mendoza, M.C. & Rodicio, M.R. (2010). Food poisoning and Staphylococcus aureus enterotoxins. Toxins, 2(7), 1751-1773.
Adib Far, P. (2014). Medical Microbiology. Noor Danesh Publications, 63-74. [In Persian]
Azizkhani, M. & Turian, F. (2019). Evaluation of the frequency of Staphylococcus aureus enterotoxinogenic genes isolated from minced meat samples in foodstores in Mazandaran province. Journal of Veterinary Research, 74 (1), 65-72. [In Persian]
Asgarpour, D. & Zeyghami, H. (2015). The role of Staphylococcus aureus enterotoxins (SEs) in staphylococcal food poisoning: A systematic review article. Quarterly Journal of Laboratory and Diagnosis, 28, 63-73. [In Persian]
Dinges, M.M., Orwin, P.M. Schlievert, P.M. (2000). Exotoxins of Staphylococcus aureus. Clinical Microbiology Reviews, 13(1), 16-34.
Ertas Onmaz, N., Abay, S., Karadal, F., Hizlisoy, H., Telli, N. & Al, S. (2014). Occurrence and antimicrobial resistance of staphylococcus aureus and salmonella spp in retail fish sample in turkey. Marine Pollution Bulletin (Elsevier), 1-5.
El-Sayed, A.F.M. (2006). Tilapia culture in salt water: environmental requirements, nutritional implications and economic potentials. Avances en Nutricion Acuicola.
Giannatale, E.D., Prencipe, V., Tonelli, A. & Marfoglia, C. (2011). Characterization of Staphylococcus aureus trains isolated from food human consumption. Veterinaria Italiana, 47(2), 165- 173.
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Holah, J.T., Higgs, C., Robinson, S., Worthington, D. & Spenceley, H. (1990). A conductance‐based surface disinfection test for food hygiene. Letters in Applied Microbiology, 11(5), 255-259.
Jawetz, E., Melnik, J. L., Adelberg, E., Brooks, G., Bottle, J. & Stephen, M. (2008) Medical Microbiology. Translated by: Rahimi, Mohammad Karim Vatahri, Amid, Ayizh Publications, Tehran, Iran, 269. [In Persian].
Ishraqi, S., Salehipour, Z., Pourmand, M.R., Rahimi Darushani, A., Salehi, Z., Taghi, M. & Aghamiri, S. (2009). Evaluation of frequency distribution of tst gene with entC, ent A, ent A/C genes in Staphylococcus aureus isolates isolated from different food products. Journal of Tehran University of Medical Sciences, 67(7), 470-476. [In Persian]
Isna-ashari, M., Shayeq, J. & Nasrollahi, I. (2012). A study on prevalence of common enterotoxin genes of Staphylococcus aureus isolated from buffalo milk in Tabriz province using Multiplex PCR. Journal of Food Hygiene in Tabriz, 2, (2), 61-68. [In Persian]
Kitai, S., Shimizu, A., Kawano, J., Sato, E., Nakano, C., Kitagawa, H., Fujio, K., Matsumura, K., Yasuda, R. & Inamoto, T. (2005). Prevalence and characterization of Staphylococcus aureus and enterotoxigenic Staphylococcus aureus in retail raw chicken meat throughout Japan. Journal of Veterinary Medical Science, 67(3), 269-274.
Kluytmans, J.A.J.W. & Wertheim, H.F.L. (2005). Nasal carriage of Staphylococcus aureus and prevention of nosocomial infections. Infection, 33(1), 3-8.
Loir, Y.L., Baron, F. & Guatier, M. (2003). Staphylococcus aureus and food poisoning. Genetics and Molecular Research, 2(1), 63-76.
Nakane, A. (2010). Protective effect of glutathione Stransferase-fused mutant staphylococcal enterotoxin C against Staphylococcus aureus-induced bovine mastitis. Vet Immunol Immunopathology, 135, 64-70.
Normanno, G., La Salandra, G., Dambrosio, A., Quaglia, N.C., Corrente, M., Parisi, A., Santagada, G., Firinu, A., Crisetti, E. & Celano, G.V. (2007). Occurrence, characterization and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products. International Journal of Food Microbiology, 115(3), 290-296.
Pelisser, M.R., Klein, C.S., Ascoli, K.R., Zotti, T.R. & Arisi, A.C.M. (2009). Ocurrence of Staphylococcus aureus and multiplex PCR detection of classic enterotoxin genes in cheese and meat products. Brazilian Journal of Microbiology, 40(1), 145-148.
Pepe, O., Blaiotta, G., Bucci, F., Anastasio, M., Aponte, M. & Villani, F. (2006). Staphylococcus aureus and Staphylococcal Enterotoxin A in Breaded Chicken Products: Detection and Behavior during the Cooking Process. Applied and Environmental Microbiology, 72(11), 7057–7062.
Redmond, E.C. & Griffith, C.J. (2003). Consumer food handling in the home: a review of food safety studies. Journal of food protection, 66(1), 130-161.
Rahimi, E., Mommtaz, H., Shakerian, A. & Kavyani, H.R. (2012). The detection of classical enterotoxins of Staphylococcus aureus in raw cow milk using the ELISA method. Turkish Journal of Veterinary and Animal Sciences, 36(3), 319-322.
Sanjeev, S. (2003). Bacteria of public health significance in seafood.
Sokari, T. (1991). Distribution of enterotoxigenic Staphylococcus aureus in ready-to-eat foods in eastern Nigeria. International Journal of Food Microbiology, 12(2-3), 275-279.
Tamasoki, M. S., Pahlavani, S., Pouria, M. & Shah Mahmoudi, S. (2011). Overview of tilapia breeding in the world, Regional Symposium on Applied Fisheries and Environmental Studies, Kermanshah, https://civilica.com/doc/159702. [In Persian]
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Arfatahery, N., Davoodabadi, A. & Abedimohtaseb, T. (2016). Characterization of toxin Genes and antimicrobial susceptibility of staphylococcus aureus isolates in fishery products in iran. Science Reports, 6, 34216,1-7.
Ayulo, A.M.R., Machado, R.A. & Scussel, V.M. (1994). Enterotoxigenic Escherichia coli and Staphylococcus aureus in fish and seafood from the southern region of Brazil. International Journal of Food Microbiology, 24(1-2), 171-178.
Arslan, S. & Ȍzdemir, F. (2017). Molecular characterization and detection of enterotoxins, methicillin resistance genes and antimicrobial resistance of Staphylococcus aureus from fish and ground beef. Polish Journal of Veterinary, 20, 1, 85-94.
Argudín, M.Á., Mendoza, M.C. & Rodicio, M.R. (2010). Food poisoning and Staphylococcus aureus enterotoxins. Toxins, 2(7), 1751-1773.
Adib Far, P. (2014). Medical Microbiology. Noor Danesh Publications, 63-74. [In Persian]
Azizkhani, M. & Turian, F. (2019). Evaluation of the frequency of Staphylococcus aureus enterotoxinogenic genes isolated from minced meat samples in foodstores in Mazandaran province. Journal of Veterinary Research, 74 (1), 65-72. [In Persian]
Asgarpour, D. & Zeyghami, H. (2015). The role of Staphylococcus aureus enterotoxins (SEs) in staphylococcal food poisoning: A systematic review article. Quarterly Journal of Laboratory and Diagnosis, 28, 63-73. [In Persian]
Dinges, M.M., Orwin, P.M. Schlievert, P.M. (2000). Exotoxins of Staphylococcus aureus. Clinical Microbiology Reviews, 13(1), 16-34.
Ertas Onmaz, N., Abay, S., Karadal, F., Hizlisoy, H., Telli, N. & Al, S. (2014). Occurrence and antimicrobial resistance of staphylococcus aureus and salmonella spp in retail fish sample in turkey. Marine Pollution Bulletin (Elsevier), 1-5.
El-Sayed, A.F.M. (2006). Tilapia culture in salt water: environmental requirements, nutritional implications and economic potentials. Avances en Nutricion Acuicola.
Giannatale, E.D., Prencipe, V., Tonelli, A. & Marfoglia, C. (2011). Characterization of Staphylococcus aureus trains isolated from food human consumption. Veterinaria Italiana, 47(2), 165- 173.
Gencay, Y.E., Ayaz, N.D. & Dogru, A.K. (2010). Enterotoxin Gene Profiles of Staphylococcus aureus and Other Staphylococcal Isolates from Various Foods and Food Ingredients. Journal of Faculty of Veterinary Medicine Erciyes University, 7(2), 75-80.
Holah, J.T., Higgs, C., Robinson, S., Worthington, D. & Spenceley, H. (1990). A conductance‐based surface disinfection test for food hygiene. Letters in Applied Microbiology, 11(5), 255-259.
Jawetz, E., Melnik, J. L., Adelberg, E., Brooks, G., Bottle, J. & Stephen, M. (2008) Medical Microbiology. Translated by: Rahimi, Mohammad Karim Vatahri, Amid, Ayizh Publications, Tehran, Iran, 269. [In Persian].
Ishraqi, S., Salehipour, Z., Pourmand, M.R., Rahimi Darushani, A., Salehi, Z., Taghi, M. & Aghamiri, S. (2009). Evaluation of frequency distribution of tst gene with entC, ent A, ent A/C genes in Staphylococcus aureus isolates isolated from different food products. Journal of Tehran University of Medical Sciences, 67(7), 470-476. [In Persian]
Isna-ashari, M., Shayeq, J. & Nasrollahi, I. (2012). A study on prevalence of common enterotoxin genes of Staphylococcus aureus isolated from buffalo milk in Tabriz province using Multiplex PCR. Journal of Food Hygiene in Tabriz, 2, (2), 61-68. [In Persian]
Kitai, S., Shimizu, A., Kawano, J., Sato, E., Nakano, C., Kitagawa, H., Fujio, K., Matsumura, K., Yasuda, R. & Inamoto, T. (2005). Prevalence and characterization of Staphylococcus aureus and enterotoxigenic Staphylococcus aureus in retail raw chicken meat throughout Japan. Journal of Veterinary Medical Science, 67(3), 269-274.
Kluytmans, J.A.J.W. & Wertheim, H.F.L. (2005). Nasal carriage of Staphylococcus aureus and prevention of nosocomial infections. Infection, 33(1), 3-8.
Loir, Y.L., Baron, F. & Guatier, M. (2003). Staphylococcus aureus and food poisoning. Genetics and Molecular Research, 2(1), 63-76.
Nakane, A. (2010). Protective effect of glutathione Stransferase-fused mutant staphylococcal enterotoxin C against Staphylococcus aureus-induced bovine mastitis. Vet Immunol Immunopathology, 135, 64-70.
Normanno, G., La Salandra, G., Dambrosio, A., Quaglia, N.C., Corrente, M., Parisi, A., Santagada, G., Firinu, A., Crisetti, E. & Celano, G.V. (2007). Occurrence, characterization and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products. International Journal of Food Microbiology, 115(3), 290-296.
Pelisser, M.R., Klein, C.S., Ascoli, K.R., Zotti, T.R. & Arisi, A.C.M. (2009). Ocurrence of Staphylococcus aureus and multiplex PCR detection of classic enterotoxin genes in cheese and meat products. Brazilian Journal of Microbiology, 40(1), 145-148.
Pepe, O., Blaiotta, G., Bucci, F., Anastasio, M., Aponte, M. & Villani, F. (2006). Staphylococcus aureus and Staphylococcal Enterotoxin A in Breaded Chicken Products: Detection and Behavior during the Cooking Process. Applied and Environmental Microbiology, 72(11), 7057–7062.
Redmond, E.C. & Griffith, C.J. (2003). Consumer food handling in the home: a review of food safety studies. Journal of food protection, 66(1), 130-161.
Rahimi, E., Mommtaz, H., Shakerian, A. & Kavyani, H.R. (2012). The detection of classical enterotoxins of Staphylococcus aureus in raw cow milk using the ELISA method. Turkish Journal of Veterinary and Animal Sciences, 36(3), 319-322.
Sanjeev, S. (2003). Bacteria of public health significance in seafood.
Sokari, T. (1991). Distribution of enterotoxigenic Staphylococcus aureus in ready-to-eat foods in eastern Nigeria. International Journal of Food Microbiology, 12(2-3), 275-279.
Tamasoki, M. S., Pahlavani, S., Pouria, M. & Shah Mahmoudi, S. (2011). Overview of tilapia breeding in the world, Regional Symposium on Applied Fisheries and Environmental Studies, Kermanshah, https://civilica.com/doc/159702. [In Persian]
