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.
Keywords: Bovine subclinical mastitis, Staphylococcus aureus, Antibiotic susceptibility, Coagulase gene polymorphism
Subklinik Mastitli İneklerden İzole Edilen Staphylococcus aureus İzolatlarinin Antibiyotik Duyarlılık Profillerinin Çıkarılması ve Koagulaz Geni Polimorfizmine Göre Tiplendirilmesi
ÖzetBu 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 görülm...
Staphylococci are pathogenic microorganisms that have various virulence factors. One of the most important of these virulence factors is biofilm production. Some staphylococci become resistant to antibiotics by producing biofilms and treatment of infections becomes more difficult. The aim of this study was to determine the effect of vancomycin, erythromycin, oxacillin antibiotics on planktonic and biofilm forming forms of staphylococci isolates which were isolated from mastitis cow's milk in a previous study. Minimum inhibitory concentration (MIC), minimum biofilm inhibitory concentration (MBIC), minimum biofilm eradication concentration (MBEC) values of antibiotics were determined. When the MIC, MBIC and MBEC values of antibiotics were compared; the highest range of MIC values was determined for erythromycin (32-64 µg ml -1 ). MBIC and MBEC values were similar for all test antibiotics. As a result of this study, staphylococci isolates were found to be more resistant to antibiotics by producing biofilm and were affected by higher doses of antibiotics than their free-living forms.
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