ALPER KARAGÖZ, Dilek GÜLDEMİR, Rıza DURMAZ

Providencia stuartii moleküler tiplemesi için pulsed-field jel elektroforezinin optimizasyonu

Amaç: Bu çalışmada; Türkiye Halk Sağlığı Kurumuna bağlı Moleküler Mikrobiyoloji Araştırma ve Uygulama Laboratuvarında Providencia stuartiinin tiplemesinde kullanılmak üzere optimize edilmiş bir pulsed-field jel elektroforezi (PFGE) protokolü sunulmuştur. Yöntemler: Gram-negatif bakterilere ait daha önceki PFGE protokolleri ile kıyaslandığında; SpeI enziminin bu bakteri için uygun olduğu ve elektroforez koşullarının; 1. blok için başlangıç vuruş süresi 5 sn, bitiş vuruş süresi 40 sn, vuruş açısı 120°, akım 6 V/cm2 , sıcaklık 14°C, süre 19 saat; 2. blok için başlangıç vuruş süresi 35 sn, bitiş vuruş süresi 45 sn, vuruş açısı 120°, akım 6 V/cm2 , sı- caklık 14°C, süre 5 saat; TBE tamponu pH=8.4 şeklinde olması gerektiği ortaya konmuştur. Filogenetik analizler Bionumerics yazılım sistemi (version 6.01; Applied Maths, Sint-Martens-latem, Belgium) ile değerlendirilmiştir. Bulgular ve sonuç: Optimize edilen yöntem basit, tekrarlanabilir ve P. stuartii için kullanıma uygun bulunmuş- tur. Çalışılan bakterilere bağlı salgınları değerlendirme ve hastane enfeksiyonlarının yaygınlık derecesi hakkında yararlı bilgiler sunma potansiyeli vardır. Optimize edilen bu protokol, farklı merkezlere ait PFGE sonuçlarının birbirleriyle karşılaştırılmasına da olanak sağlayabilir.

Optimization of the pulsed-field gel electrophoresis for molecular typing of Providencia stuartii

Objective: In this study, Pulsed-Field Gel Electrophoresis (PFGE) protocol was optimized for use Providencia stuartiis typing in Molecular Microbiology Research and Application Laboratory, Public Health Agency of Turkey. Methods: This protocol compared with Gram-negative bacteria PFGE protocols, SpeI enzyme is suitable for this bacterium. Electrophoresis conditions should be revealed as 1. block: initial pulse duration 5 sec, ending pulse duration 40 sec, striking angle 120°, the current 6 V/cm2 , temperature 14°C, time 19 hours; 2. block: initial pulse duration 35 sec, ending pulse duration 45 sec, striking angle 120°, the current 6 V/cm2 , temperature 14°C, time 5 hours; TBE buffer pH=8.4. Phylogenetic analyzes were evaluated with Bionumerics software system (version 6.01; Applied Maths, Sint-Martens-Latem, Belgium). Results and conclusion: This procedure is simple, reproducible and suitable for P. stuartii. It has the potential evaluating the outbreaks due to bacteria performed, and providing useful information about the prevalence of nosocomial infections. This optimized protocol is allowed different centers PFGE results to compare with other laboratories results.

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