Murat ARAL, Esra KAYA, Zerife ORHAN, Arzu KAYIŞ

Staphylococcus aureus Klinik İzolatlarında Gentamisin, Penisilin, Metisilin, Vankomisin, Linezolid ve Tetrasiklin Direncinin Fenotipik ve Genotipik Olarak Araştırılması

Bu çalışmada, Staphylococcus aureus izolatlarında gentamisin, penisilin, metisilin, vankomisin, linezolid ve tetrasiklin direnç oranlarının araştırılması ve plazmid içeriğinin belirlenmesi amaçlanmıştır. 2015 yılı Ocak-Eylül ayları arasında yara, kan, idrar gibi farklı örneklerden 100 tane S. aureus izolatı elde edilmiştir. Antibiyotik duyarlılıklarını belirlemek için otomatik bakteri identifikasyon ve antibiyotik duyarlılık sistemi (BD PhoenixTM, Sparks, MD, ABD) kullanıldı. Ayrıca, metisilin direnci 30 μg sefoksitin disk kullanılarak Kirby-Bauer disk difüzyon yöntemi ile de araştırıldı. Antibiyotik direncine bağlı aac (6 ')/aph (2' '), blaZ, mecA, femA, vanA, vanB, cfr, tetK ve tetM genlerinin varlığı tüm izolatlarda PCR amplifikasyonu ile araştırıldı. Plazmid DNA'ları Thermo Scientific GeneJET Plazmid Miniprep Kiti kullanılarak izole edildi. Disk difüzyon ve otomatik sistem sonuçlarına göre belirlenen S.aureus izolatlarının sefoksitin direnci %19 olarak belirlendi. Vankomisin ve linezolid direnci izolatlarda görülmezken, otomotize sistem ile %2 gentamisin, %100 penisilin, %19 metisilin, %8 tetrasiklin direnci belirlendi. Moleküler analiz sonuçlarına göre aac (6 ')/aph (2' '), blaZ, mecA, femA, tetK ve tetM genleri sırasıyla % 2, % 100, % 19, % 100, % 17 ve %3 olarak belirlendi. Fakat vanA, vanB ve cfr genleri PCR ile amplifiye edilmedi. Antibiyotik direnci ve plazmid varlığı arasındaki ilişkiyi belirlemek için plazmidler, tanımlanmış bakteriyel izolatlardan izole edildi. Bakteriyel izolatların çoğunun (% 79) farklı sayılarda plazmid içerdiği bulundu. Antibiyotik duyarlılığı için hızlı ve güvenilir bir yöntem, uygun tedavi kararını belirlemek için önemlidir. Otomatik sistem ile elde edilen sonuçların doğrulanması için PCR kullanılabilir veya MRSA ile ilişkili antibiyotik direnç genlerinin hızlı, hassas ve spesifik saptanması için rutin tanıda alternatif bir tanı yöntemi olarak kullanılabilir.

Phenotypic and Genotypic Analysis of Gentamicin, Penicillin, Methicillin, Vancomycin, Linezolid and Tetracycline Resistance in Clinical Isolates of Staphylococcus aureus

In this study, it was aimed to investigate the resistance rates ofgentamicin, penicillin, methicillin, vancomycin, linezolid andtetracycline by phenotypic and genotypic methods in Staphylococcusaureus isolates and to determine plasmid content. Between themonths of January and September in 2015, 100 clinical isolates of S.aureus were obtained from different samples such as wound, blood,urine. The automated bacteria identification and antibioticsusceptibility system (BD PhoenixTM, Sparks, MD, USA) was used todetermine of antibiotic sensitivities. The resistance to methicillin wasalso investigated by Kirby-Bauer disc diffusion method using a 30 μgcefoxitin disc. The presence of aac(6’)/aph(2’’), blaZ, mecA, femA, vanA,vanB, cfr, tetK and tetM genes related to antibiotic resistance wasinvestigated by PCR amplification in all isolates. Plasmid DNAs wereisolated by using a Thermo Scientific GeneJET Plasmid Miniprep Kit.The cefoxitin resistance of S.aureus isolates, identified according tothe results of disk diffusion and automated system, was calculated as19%. Vancomycin and linezolid resistance were not observed inisolates while gentamicin 2%, penicillin 100%, methicillin 19%,tetracycline 18% resistance were identified using the automatedsystem. According to the results of molecular analysis aac(6’)/aph(2’’),blaZ, mecA, femA, tetK and tetM genes frequencies were determinedas 2%, 100%, 19%, 100%, 17% and 3% respectively, but vanA, vanBand cfr genes were not amplified by PCR. In order to determine therelationship between antibiotic resistance and plasmid presence,plasmids were isolated from identified bacterial isolates. It is foundthat most of bacterial isolates (79%) contain different numbersplasmids. Rapid and reliable method for antibiotic susceptibility isimportant to determine the appropriate therapy decision. PCR can beused for confirmation of the results obtained by automated system orcould be used as an alternative diagnostic method in the routinediagnosis for rapid, sensitive, and specific detection of MRSAassociated antibiotic resistance genes.

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