A Duplex PCR for detection of S. aureus and Staphylococcus spp. from culture and bovine milk samples

Mastitis süt sığırcılığı endüstrisinin en fazla ekonomik kayba neden olan problemidir. Stafilokok türleri en sık izole edilen etkenler olup, Staphylococcus aureus ise süt sığırlarında en önemli kontagiyöz etkendir. Bu çalışmanın amacı stafilokok türleri ve S. aureus’un kültür ve süt örneklerinden teşhisi için ikili bir PZR tekniğinin geliştirilmesidir. Çalışmada pozitif kontrol olarak, S. aureus (ATCC 25923) ve S. epidermidis (ATCC 12228) DNA’sı kullanıldı. Geliştirilen tekniğin denenmesi amacıyla Veteriner Hekimlerce laboratuvarımıza gönderilen örneklerden bir adet koagulaz negatif stafilokok bir adet de S. aureus pozitif süt örneği klinik örnek olarak kullanıldı. Çalışmada sütten ve kültür örneklerinden stafilokok ve S. aureus’un teşhisi ve ayrımı için ikili ve hızlı bir PZR tekniği geliştirildi. Geliştirilen bu tekniğin stafilokok türleri ve S. aureus’un bir kaç saat içinde hem süt hem de kültür örneklerinden tanısı ve ayrımı için alternatif, güvenilir ve hızlı bir tanı yöntemi olarak özellikle sonuçların çiftlik bazında kullanılmak üzere klinik ve subklinik olgularda ineğin tedavi edilmesi ya da kesime gönderilmesine karar verilmesi amacıyla kullanılabileceği ortaya konuldu.

Sığır mastitis süt örneklerinden stafilokok türlerinin ve S. aureus’un teşhisine yönelik ikili bir PZR tekniği

Mastitis is the most costly disease in dairy industry. Staphylococcus spp. is the most frequently isolated microorganisms and Staphylococcus aureus is one of the most important contagious mastitis agents in dairy cattle. The aim of this study is developing a duplex PCR technique for detection of Staphylococcus spp. and S. aureus from culture and milk samples. S. aureus (ATCC 25923) and S. epidermidis (ATCC 12228) DNAs were used as positive control. For the purpose of testing the developed technique, one coagulase-negative Staphylococcus and one S. aureus positive milk sample was used as a clinical sample that they were sent by Veterinary Practitioners. In this study, a duplex and rapid PCR protocol is developed for detecting and separating these organisms from culture and from milk samples. This procedure can be used as an alternative, reliable and fast detection and identification method for Staphylococcus spp. and S. aureus in few hours for deciding treating or culling the cows in farm base in clinical mastitis cases and can be a useful diagnostic method for subclinical mastitis cases too.

___

  • Ahmadi M, Rohani SMR, Ayremlou N (2010). Detection of Staphylococcus aureus in milk by PCR. Comp Clin Pathol, 19, 91–94.
  • Amin A S, Hamouda R H, Abdel-All AAA (2011). PCR Assays for Detecting Major Pathogens of Mastitis in Milk Samples. World J Dairy Food Sci, 6 (2), 199-206.
  • Ardic N, Sareyyupoglu B, Ozyurt M, Haznedaroglu T, Ilga U (2006). Investigation of aminoglycoside modifying enzyme genes in methicillin-resistant staphylococci. Microbiol Res, 161(1), 49-54.
  • Baştan A (2013). İneklerde Meme Hastalıkları, Hatiboğlu Basımevi, Ankara,
  • Belschner A P, Hallberg JW, Nickerson SC, Owens WE (1996). Staphylococcus aureus mastitis therapy revisited. Proceedings, National Mastitis Council annual meeting, Madison, Wis., 116-22.
  • Bone FJ, Bogie D, Morgan-Jones SC (1989). Staphylococcal food poisoning from sheep milk cheese. Epidemiol Infect, 103,449-458.
  • Fan Y, Pan F, Paoli G, Xiao Y, Sheng H, Shi X (2008). Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus. J Rapid Meth Aut Mic,16(4), 394-411.
  • Friedman S, Shoshani E, Ezra E (2004). Economical loses from clinical mastitis in 4 dairy herds in Israel. Israel J Vet Med, 59,1-2.
  • Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997). Multiplex PCR-critical parameters and step-by-step protocol. Biotechniques, 23,504–511.
  • Hookey JV, Richardson JF, Cookson BD (1998). Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequenceanalysis of the coagulase gene. J Clin Microbiol, 36, 1083–1089.
  • Huijps K, Lam TJ, Hogeveen H (2008). Costs of mastitis: facts and perception, J Dairy Res, 75,113-120.
  • Karahan M, Cetinkaya B (2006). Coagulase gene polymorphisms detected by PCR in Staphylococcus aureus isolated from subclinical bovine mastitis in Turkey. Vet J, 174, 428–431.
  • Maes N, Magdalena J, Rottiers S, De-Gheldre Y, Struelens M J (2002). Evaluation of a Triplex PCR Assay To Discriminate Staphylococcus aureus from Coagulase-Negative Staphylococci and Determine Methicillin Resistance from Blood Cultures. J Clin Microbiol, 40(4), 1514–1517.
  • NMC (1996). Current Concept of Bovine Mastitis. The National Mastitis Council. West Medison. WI.
  • Phuektes P, Mansell, P D, Browning G F (2001). Multiplex Polymerase Chain Reaction Assay for Simultaneous Detection of Staphylococcus aureus and Streptococcal Causes of Bovine Mastitis. J Dairy Sci, 84, 1140–1148.
  • Pradhan P, Gopinath SM, Reddy GR, Dechamma HJ, Suryanarayana VVS (2011). Detection of major pathogens in bovine sub-clinical mastitis by multiplex PCR directly from milk samples in presence of an internal control. Ind. J. Fund. Appl Life Sci, 1(4) ,209-218.
  • Quinn PJ, Carter ME, Markey BK, Carter GR (1994). Clinical Veterinary Microbiology. Mosby-Year Book Europe Limited, Lynton House, London WC1H9LB, England. p.: 209-236.
  • Riffon R, Sayasith K, Khalıl H, Dubreuil P, Drolet M, Lagace A (2001). Development of a Rapid and Sensitive Test for Identification of Major Pathogens in Bovine Mastitis by PCR. J Clin Microbiol, 39 (7), 2584– 2589.
  • Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, New York.
  • Schmitz FJ, MacKenzie CR, Hofmann B, Verhoef J, Finken-Eigen M, Heinz HP, Kohrer K (1997). Specific information concerning taxonomy, pathogenicity and methicillin resistance of staphylococci obtained by a multiplex PCR. J Med Microbiol, 46,773-778.
  • Strommenger B, Kettlitz C, Werner G, Witte W (2003). Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol, 41, 4089- 4094.
  • Taponen S, Simojoki H, Haveri M, Larsen HD, Pyorala S (2006). Clinical characteristics and persistence of bovine mastitis caused by different species of coagulase-negative staphylococci identified with API or AFLP. Vet Microbiol, 115, 199–207.
  • Tiwari HK, Sapkota D, Sen MR (2008). Evaluation of different tests for detection of Staphylococcus aureus using coagulase (coa) gene PCR as the gold standard. Nepal Med Coll J, 10(2),129-13.
  • Wieneke AA, Roberts D, Gilbert RJ (1993). Staphylococcal food poisoning in United Kingdom, 1969-90. Epidemiol Infect, 110, 519-531.