İzmir, Manisa ve Uşak illerindeki kanatlılardan elde edilen salmonella izolatlarında virülans faktörlerinin, antimikrobiyal ve dezenfektan duyarlılıklarının ve klonal yakınlığın araştırılması
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Dosyalar
Tarih
2017
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Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada kanatlı Salmonella suşlarında, çeşitli antibiyotik ve dezenfektanların etkileri, antibiyotik direnci tespit edilen suşlarda antibiyotik direnç genlerinin tespit edilmesi, çeşitli virülans genlerinin saptanması ve suşlar arasında klonal ilişkinin araştırılması amaçlanmıştır. Türkiye Cumhuriyeti Gıda, Tarım ve Hayvancılık Bakanlığı bünyesinde yer alan Bornova Veteriner Kontrol Enstitüsü'ne bağlı Kanatlı Hastalıkları Teşhis Laboratuvarı'nda 2014 - 2015 yıllarında izole edilen ve serotiplendirme için Etlik Merkez Araştırma Enstitüsü Bakteriyoloji Laboratuvarı (Salmonella Ulusal Referans Laboratuvarı )'na gönderilen 45 kanatlı suşu çalışmaya alındı. İlk olarak 10 değişik antibiyotik kullanılarak Kirky Bauer disk difüzyon yöntemi ile Clinical and Laboratory Standard Institute kriterlerine göre suşların antibiyotik duyarlılık profilleri belirlendi. Daha sonra yaygın kullanılan dezenfektanların suşlar üzerine etkileri sıvı mikrodilüsyon yöntemi ile incelendi. Disk difüzyon testi sonuçlarına göre ß-laktam ve tetrasiklin direnci görülen suşlarda beş direnç geni Polimeraz Zincir Reaksiyonu (PZR) ile araştırıldı. Ayrıca, infeksiyon oluşumunda kritik rolü olan altı virülans geni de PZR ile saptandı. Çalışmanın sonunda suşlar arasındaki klonal ilişki Pulsed Field Jel Elektroforez (PFGE) yöntemi yardımıyla değerlendirildi. ß-Laktam (ampisilin, amoksisilin ve amoksisilin+klavulanik asit) ve tetrasiklin (doksisiklin ve oksitetrasiklin) grubu antibiyotiklere karşı toplam 20 Salmonella suşunda direnç tespit edildi. Diğer test edilen antibiyotiklere (sefepime, levofloksasin, siprofloksasin, sefotaksim, kolistin sülfat) karşı direnç gözlenmedi. ß-Laktam direnci tespit edilen suşlarda Genişlemiş Spektrumlu Beta Laktamaz (GSBL) varlığı blaTEM, blaCTX-M ve blaSHV ile araştırıldı ancak bu genler bulunamadı. Tetrasiklin direnci gösteren suşlarda tetA ve tetB direnç genleri araştırıldı ve 7 suşta sadece tetA bulundu. Sıvı mikrodilüsyon yöntemi ile 45 Salmonella spp. suşunun dört ayrı dezenfektana olan duyarlılıkları çalışıldı. 25 Salmonella suşunda Virkon-S® 'e direnç tespit edildi. Virülans genlerinden invA, sopB, sopE, sifA, pefA, ve pipD araştırıldı. Bu genlerden invA suşların tümünde bulunurken, sopB (%93.3), sopE (%37.7), sifA (%31.1), pefA (%35.5) ve pipD (%95.5) oranlarında belirlendi. Moleküler tiplendirme yöntemi olan PFGE ile 45 suş arasındaki klonal ilişki araştırıldı. Çalışılan suşların dördünde yöntem tekrar edilmesine rağmen patern elde edilemedi. Ortaya çıkan 13 ayrı Salmonella serotipi içeren 41 suşa ait dendogramda 39 ayrı pulsotip tespit edildi. Suşlar arası klonal yakınlık % 30 ile % 100 arasında bulundu. Ayrım / sınır değeri, % 53 olarak alındı ve suşlar arasında sekiz ayrı küme oluşumu gözlendi. Kümelerin sahip olduğu suş sayılarının genetik benzerliklerine dayanarak üç ile 12 arasında değiştiği belirlendi. Çalışmamız iki yıllık bir dönemde İzmir, Manisa ve Uşak illerindeki kanatlılardan izole ve identifiye 45 Salmonella suşunda, ß-Laktam ve tetrasiklin grubu antibiyotiklere karşı direnç olduğunu ve dezenfektan direncinin görüldüğünü ortaya koymaktadır. Antibiyotik dirençli suşlarda GSBL tespit edilmemesine rağmen bazı suşlarda tetrasiklin direnç geni olan tetA saptanmıştır. Farklı virülans genleri değişik serotiplerde belirlenmiş ve çalışılan suşlar arasında yüksek oranlarda klonal yakınlık görülmüştür. Salmonella antibiyotik direnç ve virülans genleri ile ilgili ülkemizde daha fazla araştırmaya ihtiyaç duyulmaktadır.
The aim of this study was to investigate the effects of different antibiotics and disinfectants, antibiotic resistance genes in resistant strains, different virulence genes and clonal relationships in Salmonella isolates of winged animals. The 45 strains of winged animals isolated in 2014-2015 from Poultry Diseases Diagnostic Laboratory of Bornova Veterinary Control Institute related to Turkish Republic Food, Agriculture and Livestock and serotyped in Etlik Central Research Institute Bacteriology Laboratory (Salmonella National Reference Laboratory) were taken into study. At first, Kirky Bauer disc diffusion method with 10 different types of antibiotics was applied in order to determine the antibiotic suspectibility profiles of the strains in accordance with Clinical and Laboratory Standard Institute's criteria. Then, the effects of frequently used disinfectants were examined on strains using broth microdilution method. According to the results of disc diffusion method, five antibiotic resistance genes were investigated using Polimerase Chain Reaction (PCR) in the strains which were resistant to ß-lactams and tetracyclines. Furthermore, six virulence genes which have crucial role in occurence of infection were detected by using PCR. At the end of the study, clonal relationships between the strains were evaluated with the help of Pulsed Field Gel Electrophoresis (PFGE) method. The 20 Salmonella strains were found resistant to ß-lactam (ampicillin, amoxicillin and amoxicillin+clavulanic acid) group and tetracycline (doxycycline and oxytetracycline) group antibiotics. No resistance were observed in strains to other tested antibiotics (cefepime, levofloxacin, ciprofloxacin, cefotaxime and colistin sulphate). The presence of Extended Spectrum Spectrum Beta Lactamase (ESBL) were investigated by using blaTEM, blaCTX-M and blaSHV in strains which were found resistant to ß-lactams but none of the genes were detected. Also tetA and tetB resistance genes were examined in strains which were resistant to tetracyclines and only tetA was found in seven strains. The suspectibilities of 45 Salmonella strains to four different disinfectants were studied by using broth microdilution method. 25 Salmonella strains were detected resistant to Virkon-S® . The virulence genes, invA, sopB, sopE, sifA, pefA, and pipD were investigated. InvA was found in all strains and others were determined at the rates of sopB (93.3%), sopE (37.7%), sifA (31.1%), pefA (35.5%) and pipD (%95.5). The clonal relatedness between 45 strains were researched using PFGE which is a molecular typing method. Although the test was repeated, no patterns were gained in four strains. According to the dendogram which revealed including 41 strains and 13 different serotypes, 39 distinct pulsotypes were detected. The clonal relatedness between strains was in the range of 30% and 100%. The cut-off value was taken as 53% and the formation of eight different clusters were observed. Depending on the genetical relatedness, the number of strains which belonged to clusters was determined as three and 12. Resistance to ß-lactam and tetracycline group antibiotics and a disinfectant was revealed by our study in 45 Salmonella strains which were isolated and identified from winged animals located İzmir, Manisa and Uşak in a two year period. Although ESBL wasn't observed in resistant strains, tetA was detected in some of them. Different virulence genes were found in various serotypes and high clonal relatedness levels were seen between the studied strains. More investigations are needed about Salmonella antibiotic and virulence genes in our country.
The aim of this study was to investigate the effects of different antibiotics and disinfectants, antibiotic resistance genes in resistant strains, different virulence genes and clonal relationships in Salmonella isolates of winged animals. The 45 strains of winged animals isolated in 2014-2015 from Poultry Diseases Diagnostic Laboratory of Bornova Veterinary Control Institute related to Turkish Republic Food, Agriculture and Livestock and serotyped in Etlik Central Research Institute Bacteriology Laboratory (Salmonella National Reference Laboratory) were taken into study. At first, Kirky Bauer disc diffusion method with 10 different types of antibiotics was applied in order to determine the antibiotic suspectibility profiles of the strains in accordance with Clinical and Laboratory Standard Institute's criteria. Then, the effects of frequently used disinfectants were examined on strains using broth microdilution method. According to the results of disc diffusion method, five antibiotic resistance genes were investigated using Polimerase Chain Reaction (PCR) in the strains which were resistant to ß-lactams and tetracyclines. Furthermore, six virulence genes which have crucial role in occurence of infection were detected by using PCR. At the end of the study, clonal relationships between the strains were evaluated with the help of Pulsed Field Gel Electrophoresis (PFGE) method. The 20 Salmonella strains were found resistant to ß-lactam (ampicillin, amoxicillin and amoxicillin+clavulanic acid) group and tetracycline (doxycycline and oxytetracycline) group antibiotics. No resistance were observed in strains to other tested antibiotics (cefepime, levofloxacin, ciprofloxacin, cefotaxime and colistin sulphate). The presence of Extended Spectrum Spectrum Beta Lactamase (ESBL) were investigated by using blaTEM, blaCTX-M and blaSHV in strains which were found resistant to ß-lactams but none of the genes were detected. Also tetA and tetB resistance genes were examined in strains which were resistant to tetracyclines and only tetA was found in seven strains. The suspectibilities of 45 Salmonella strains to four different disinfectants were studied by using broth microdilution method. 25 Salmonella strains were detected resistant to Virkon-S® . The virulence genes, invA, sopB, sopE, sifA, pefA, and pipD were investigated. InvA was found in all strains and others were determined at the rates of sopB (93.3%), sopE (37.7%), sifA (31.1%), pefA (35.5%) and pipD (%95.5). The clonal relatedness between 45 strains were researched using PFGE which is a molecular typing method. Although the test was repeated, no patterns were gained in four strains. According to the dendogram which revealed including 41 strains and 13 different serotypes, 39 distinct pulsotypes were detected. The clonal relatedness between strains was in the range of 30% and 100%. The cut-off value was taken as 53% and the formation of eight different clusters were observed. Depending on the genetical relatedness, the number of strains which belonged to clusters was determined as three and 12. Resistance to ß-lactam and tetracycline group antibiotics and a disinfectant was revealed by our study in 45 Salmonella strains which were isolated and identified from winged animals located İzmir, Manisa and Uşak in a two year period. Although ESBL wasn't observed in resistant strains, tetA was detected in some of them. Different virulence genes were found in various serotypes and high clonal relatedness levels were seen between the studied strains. More investigations are needed about Salmonella antibiotic and virulence genes in our country.
Açıklama
Anahtar Kelimeler
Kanatlı, Salmonella spp., Dezenfektan, Direnç, Virülans, Winged Animals, Salmonella spp., Disinfectant, Resistance, Virulence
