BEYTULLAH KENAR, Arzu Funda BAĞCIGİL, YAHYA KUYUCUOĞLU, BEREN BAŞARAN KAHRAMAN, SELAHATTİN KONAK

Antimicrobial Susceptibility Profiles and Coagulase Gene Polymorphism of Staphylococcus aureus Isolated from Bovine Subclinical Mastitis

Bu çalışmanın amacı subklinik mastitisli sığırlardan Staphylococcus aureus'u izole etmek, bunların antibiyotiklere duyarlılığını belirlemek ve bir PCR tabanlı restriksiyon fragment lengh polimorfizmi (RFLP) yöntemi kullanarak koagulaz gen polimorfizmi araştırmaktır. 463 sığırdan CMT pozitif olan 237 sığır memesinden süt örnekleri alınarak ekim yapılmıştır. İzolatların antimikrobiyal duyarlılığı disk difüzyon yöntemi ile belirlenmiştir. Toplam 83 izolatın 82'si (%98.8) uygulanan 16 antibiyotikten en az bir antibiyotiğe dirençli bulundu. Bu çalışmada 53 izolat (%63.8) penisiline, 52 izolat (%62.67 trimethoprim/sulphamethoxazole, 51 izolat (%61.5) ampisiline, 40 izolat (%48.2) eritromisine, 29 izolat (%35.0) tetrasikline, 18 izolat (%21.7) siprofloksasine, 16 izolat (%19.3) klindamisine, 13 izolat (%15.6) kloramfenikole, 8 izolat (%9.6) gentamisine, 5 izolat (%6.0) sefoksitine, 4 izolat (%4.9) vankomisine, 3 izolat (%3.6) sefalotine, 2 izolat (%2.4) nafsiline ve 1'er (%1.2) izolat ise oksasilin ve furazolidona dirençli bulundu. S. aureus izolatlarında imipenem dirençliliği görülmedi. Koagulaz gen polimorfizmi koagulaz genin tekrarlanan 81 bp DNA dizisinin AluI sindirimini müteakiben koagulaz genin amplifikasyonu ile incelenmiştir. Nested PCR'den sonra izolatların 8'inde çift bant görülmüş kalan 75 izolatta ise tek band vardı. AluI sindirimini müteakiben yaklaşık 300 bp uzunluğunda tek bant oluşturan izolatlar 3 farklı grup göstermiştir

Subklinik Mastitli İneklerden İzole Edilen Staphylococcus aureus İzolatlarinin Antibiyotik Duyarlılık Profillerinin Çıkarılması ve Koagulaz Geni Polimorfizmine Göre Tiplendirilmesi

The purpose of the study was to isolate Staphylococcus aureus from bovine subclinical mastitis, determine their antibiotic susceptibilities and investigate the coagulase gene polymorphism by using a PCR-based restriction fragment length polymorphism (RFLP) method. Milk samples from 463 CMT positive udders from 237 cows cultured. The antimicrobial susceptibility of the isolates were determined by disc diffusion method. A total of 82 out of the 83 isolates (98.8%) were found to be resistant at least one out of the 16 antibiotics studied. In this experiment 53 isolates (63.8%) were found to be resistant to penicillin; 52 (62.67%) to trimethoprim/sulphamethoxazole; 51 (61.5%) to ampicillin; 40 (48.2%) to erytromycin; 29 (34.9%) to tetracycline; 18 (21.6%) to ciprofloxacin, 16 (19.3%) to clindamycin, 13 (15.6%) to chloramphenicol; 8 (9.6%) to gentamicin; 5 (6.0%) to cefoxitin; 4 (4.9%) to vancomycin; 3 (3.6%) to cephalotin; 2 (2.4%) nafcillin; one (1.2%) to oxacillin and one to (1.2%) furazolidon. No imipenem resistance was seen in the S. aureus isolates. The coagulase gen polymorphism were examined by PCR amplification of coagulase gene followed by AluI digestion of repeating 81 bp DNA sequences. After nested PCR, double bands were produced in 8 of the isolates while there were single band in remaining 75 isolates. Following AluI digestion, isolates that formed single band in length of approximately 300 bp showed 3 different groups

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1. Aarestrup FM, Wegener HC, Rosdahl VT: Evaluation of phenotypic and genotypic methods for epidemiological typing of Staphylococcus aureus isolates from bovine mastitis in Denmark. Vet Microbiol, 45, 139- 150, 1995. DOI: 10.1016/0378-1135(95)00043-A
2. Güler L, Ok U, Gündüz K, Gülcü Y, Hadimli HH: Antimicrobial susceptibility and coagulase gene typing of Staphylococcus aureus isolated from bovine clinical mastitis cases in Turkey. J Dairy Sci, 88, 3149- 3154, 2005. DOI: 10.3168/jds.S0022-0302(05)72998-2
3. Su C, Herbelin C, Frieze N, Skardova O, Sordillo LM: Coagulase gene polymorphism of Staphylococcus aureus isolates from dairy cattle in different geographical areas. Epidemiol Infec, 122, 329-336, 1999.
4. Zadoks RN, van Leeuwen WB, Kreft D, Fox LK, Barkema HW, Schukken YH, van Belkum A: Comparison of Staphylococcus aureus isolates from bovine and human skin, milking equipment, and bovine milk by phage typing, pulsed-field gel electrophoresis, and binary typing. J Clin Microbiol, 40, 3894-3902, 2002. DOI: 10.1128/JCM.40.11.3894-3902.2002
5. Vintov J, Aarestrup FM, Zinn CE, Olsen JE: Association between phage types and antimicrobial resistance among bovine Staphylococcus aureus from 10 countries. Vet Microbiol, 95, 133-147, 2003. DOI: 10.1016/ S0378-1135(03)00156-1
6. Myllys V, Asplund K, Brofeldt E, Hirvela-Koski V, HonkanenBuzalski T, Junttila J, Kulkas L, Myllykangas O, Niskanen M, Saloniemi H, Sandholm M, Saranpaa T: Bovine mastitis in Finland in 1988 and 1995-Changes in prevalence and antimicrobial resistance. Acta Vet Scand, 39, 119-126, 1998.
7. Fitzgerald JR, Hartigan PJ, Meaney WJ, Smyth CJ: Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection. J Appl Microbiol, 88, 1028-1037, 2000. DOI: 10.1046/j.1365-2672.2000.01071.x
8. Saidi R, Cantekin Z, Khelef D, Ergün Y, Solmaz H, Kaidi R: Antibiotic susceptibility and molecular identification of antibiotic resistance genes of Staphylococci isolated from bovine mastitis in Algeria. Kafkas Univ Vet Fak Derg, 21, 513-520, 2015. DOI: 10.9775/kvfd.2014.12836
9. Aslantas Ö, Demir C, Türütoğlu H, Cantekin Z, Ergün Y, Doğruer G: Cagulase gene polymorphism of Staphylococcus aureus isolated form subclinical bovine mastitis. Turk J Vet Anim Sci, 31, 253-257, 2007.
10. Ünal N, İstanbulluoğlu E: İnsan ve sığır kökenli Staphylococcus aureus izolatlarının fenotipik ve genotipik özelliklerin araştırılması. Ankara Üniv Vet Fak Derg, 56, 119-126, 2009.
11. Karahan M, Acik MN, Cetinkaya B: Investigation of virulence genes by PCR in Stapylococcus aureus isolates originated from subclinical bovine mastitis in Turkey. Pak Vet J, 31, 249-253, 2011.
12. Türkyılmaz S, Yıldız Ö, Oryaşın E, Kaynarca S, Bozdoğan B: Molecular identification of bacteria isolated from dairy herds with mastitis. Kafkas Univ Vet Fak Derg, 16, 1025-1032, 2010. DOI: 10.9775/ kvfd.2010.2300
13. Ünal N, Yıldırım M: Antibiotic resistance profiles of staphylococci species isolated from milks, teat skins and noses mucous of cows. Kafkas Univ Vet Fak Derg, 16, 389-396, 2010. DOI: 10.9775/kvfd.2009.833
14. Bisping, W, Gunter A: Colour Atlas for the Diagnosis of Bacterial Pathogens in Animals. Paul Parey Scientific Publishers Berlin and Hamburg. Germany, 1998.
15. Quinn PJ, Carter ME, Markey BK, Carter GR: Clinical Veterinary Microbiology. 2nd edn., Mosby, London, UK. 1998.
16. National Committee for Clinical Laboratory Standards-NCCLS: Performance Standards for Antimicrobial Disk Susceptibility Tests. Approved Standard, 8th edn., NCCLS document M2-A8, Wayne, PA 2003.
17. Maes N, Magdalena J, Rottiers S, de Gheldre Y, Struelens MJ: 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, 1514-1517, 2002. DOI: 10.1128/JCM.40.4.1514-1517.2002
18. Goh SH, Byrne SK, Zhang JL, Chow AW: Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorhisms. J Clin Microbiol, 30, 1642-1645, 1992.
19. Moon JS, Lee AR, Kang HM, Lee ES, Joo YS, Park YH, Kim MN, Koo HC: Antibiogram and coagulase diversity in staphylococcal enterotoxinproducing Staphylococcus aureus from bovine mastitis. J Dairy Sci, 90, 1716-1724, 2007. DOI: 10.3168/jds.2006-512
20. Sori T, Hussien J, Bitew M: Prevalence and susceptibility assay of Staphylococcus aureus isolated from bovine mastitis in dairy farms of Jimma Town, South West Ethiopia. J Animal Vet Adv, 10, 745-749, 2011. DOI: 10.3923/javaa.2011.745.749
21. Kirkan S, Goksoy EO, Kaya O: Identification and antimicrobial susceptibility of Staphylococcus aureus and coagulase negative staphylococci from bovine mastitis in the Aydın region of Turkey. Turk J Vet Anim Sci, 29, 791-796, 2005.
22. da Silva ER, da Silva N: Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southern Brazil. Can J Vet Res, 69, 260-264, 2005.
23. Raimundo O, Deighton M, Capstick J, Gerraty N: Molecular typing of Staphylococcus aureus of bovine origin by polymorphisms of the coagulase gene. Vet Microbiol, 66, 275-284, 1999. DOI: 10.1016/S0378- 1135(99)00020-6
24. Karahan M, Çetinkaya B: Coagulase gen polymorphism detected by PCR in Staphylococcus aureus isolated form subclinical bovine mastitis in Turkey. Vet J, 174, 428-431, 2007. DOI: 10.1016/j.tvjl.2006.05.016