Mehmet Reşat CEYLAN, Alper KARAGÖZ, Rıza DURMAZ

Proteus mirabilis'in moleküler tiplemesi için pulsed-field jel elektroforezinin optimizasyonu

Amaç: Proteus mirabilis ile oluşan salgınların saptanması için suşlar arasında klonal ilişkinin belirlenmesine yönelik "pulsed-field jel elektroforezi (PFGE)" gibi yöntemlere ihtiyaç vardır. Bu çalışmanın amacı; Proteus mirabilis'in moleküler tiplemesi için pulsed-field jel elektroforezinin optimizasyonudur.Yöntemler: Bu çalışmada; Proteus mirabilis tiplemesinde kullanılmak üzere optimize edilmiş bir pulsed-field jel elektroforezi protokolü sunulmuştur. Suşların filogenetik analizleri Bionumerics yazılım sistemi (version 6.01; Applied Maths, Sint-Martens-latem, Belgium) ile değerlendirilmiştir.Bulgular: Gram-negatif bakteriler için kullanılan PFGE protokollerinden farklı olarak NotI enziminin bu bakteri için uygun olduğu belirlendi ve elektroforez koşulları: blok 1 için başlangıç vuruş süresi 1 sn, bitiş vuruş süresi 30 sn, vuruş açısı 120°, akım 6 V/cm2, sıcaklık 14°C, süre 8 saat; - blok 2 için başlangıç vuruş süresi 30 sn, bitiş vuruş süresi 70 sn, vuruş açısı 120°, akım 6 V/cm2, sıcaklık 14°C, süre 16 saat; - TBE pH=8.4 olarak saptandı.Sonuç: Bu PFGE protokolünün; P. mirabilis suşlarının tiplendirilmesi ve Bionumerics yazılım sistemi ile klonal ilişkilerinin belirlenmesi için uygun olduğu anlaşıldı. Optimize edilen yöntem basit, tekrarlanabilir ve bu bakteri için kullanıma uygundur. Ayrıca süreyi kısaltması, kullanılan çözelti ve enzimlerin düşük hacimleriyle çalışabilmesi nedeniyle, ekonomik olarak değerlendirilmiştir. 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 sağlayacaktır

Optimization of a pulsed-field gel electrophoresis for molecular typing of Proteus mirabilis

Objective: For the detection of outbreaks caused by Proteus mirabilis, strains clonal relations are determined methods as "pulsed-field gel electrophoresis (PFGE)". The aim of this study was optimization of a pulsed-field gel electrophoresis for molecular typing of P. mirabilis.Methods: In this study, PFGE' protocol is optimized for use in molecular typing of P. mirabilis. Phylogenetic analyzes of strains were evaluated with Bionumerics software system (version 6.01; Applied Maths, Sint-MartensLatem, Belgium).Results: This protocol compared with Gram-negative bacteria PFGE protocols, NotI enzyme is suitable for this bacterium. Electrophoresis conditions should be revealed as; - block 1: initial pulse duration 1 sec, ending pulse duration 30 sec, striking angle 120°, the current 6 V/cm2, temperature 14°C, time 8 hours; - block 2: initial pulse duration 30 sec, ending pulse duration 70 sec, striking angle 120°, the current 6 V/cm2, temperature 14°C, time 16 hours; - TBE, pH=8.4.Conclusion: P. mirabilis strains were typed by PFGE and Bionumerics analysis program were determined clonal relationships. The procedure was simple, reproducible and suitable for these bacteria. Also it was evaluated, because of reducing time, the solution volumes and enzymes can be economically. Outbreaks of nosocomial infections due to bacteria studied assessment and the potential to provide useful information about the degree of prevalence. This optimized protocol is allowed different centers' PFGE results to compare with other laboratories results. J Clin Exp Invest 2013; 4 (3): 306-312

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Kunin CM. Urinary tract infections: detection, preven- tion and management, 5th ed., Williams and Wilkins, Baltimore MD 1997;226-278.
Johnson DE, Russell RG, Lockatell CV, et al. Contribu- tion of Proteus mirabilis urease to persistence, uro- lithiasis, and acute pyelonephritis in a mouse model of ascending urinary tract infection. Infect Immun 1993;61:2748-2754.
Bagattini M, Crivaro V, Di Popolo A, et al. Molecular epidemiology of extended-spectrum beta lactamase- producing Klebsiella pneumoniae in a neonatal inten- sive care unit. J Antimicrob Chemother 2006;57:979- 982.
Centers for Disease Control and Prevention: Standard- ized laboratory protocol for molecular subtyping of Escherichia coli 0157:H7, non-typhoidal Salmonella serotypes, and Shigella sonnei by pulsed- field gel electrophoresis (PFGE). http://www.cdc.gov/pulsenet/ protocols/ecoli_salmonella_shigella_protocols.pdf.
Guducuoglu H, Durmaz R, Yaman G, et al. Spread of a single clone Acinetobacter baumannii strain in an intensive care unit of a teaching hospital in Turkey. New Microbiol 2005;28:337-343.
Mamlouk K, Boutiba-Ben Boubaker I, Gautier V, et al. Emergence and outbreaks of CTX-M beta-lacta- mase-producing Escherichia coli and Klebsiella pneu- moniae strains in a Tunisian hospital. J Clin Microbiol 2006;44:4049-4056.
Maslow JN, Glaze T, Adams P, et al. Concurrent out- break of multidrug-resistant and susceptible sub- clones of Acinetobacter baumannii affecting different wards of a single hospital. Infect Control Hosp Epide- miol 2005;26:69-75.
Maslow JN, Slutsky AM, Arbeit RD. Application of pulsed-field gel electrophoresis to molecular epide- miology. Persing HD, Smith TF, Tenover FC, White TJ (eds): Diagnostic Molecular Microbiology: Prin- ciples and Applications. Washington DC, ASM Press. 1993:563-572.
Naseer U, Natas OB, Haldorsen BC, et al. Nosocomial outbreak of CTX-M-15-producing E. coli in Norway. APMIS 2007;115:120-126.
Ribot EM, Fitzgerald C, Kubota K, et al. Rapid pulsed- field gel electrophoresis protocol for subtyping of Campylobacter jejuni. J Clin Microbiol 2001;39:1889- 1894.
Rodriguez-Bano J, Navarro MD, Romero L et al. Clini- cal and molecular epidemiology of extended- spec- trum beta-lactamase-producing Escherichia coli as a cause of nosocomial infection or colonization: implica- tions for control. Clin Infect Dis 2006;42:37-45.
Seifert H, Dolzani L, Bressan R, et al. Standardiza- tion and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated finger- prints of Acinetobacter baumannii. J Clin Microbiol 2005;43:4328-4335.
Seifert H, Gerner-Smidt P. Comparison of ribotyp- ing and pulsed-field gel electrophoresis for molecu- lar typing of Acinetobacter isolates. J Clin Microbiol 1995;33:1402-1407.
Stephens C, Francis SJ, Abell V, et al. Emergence of resistant Acinetobacter baumannii in critically ill patients within an acute care teaching hospital and a long-term acute care hospital. Am J Infect Control 2007;35:212-215.
Tenover FC, Arbeit RD, Goering RV. How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: a re- view for healthcare epidemiologists. Molecular Typing Working Group of the Society for Healthcare Epide- miology of America. Infect Control Hosp Epidemiol 1997;18:426-439.
Turabelidze D, Kotetishvili M, Kreger A et al. Im- proved pulsed-field gel electrophoresis for typing vancomycin-resistant enterococci. J Clin Microbiol 2000;38:4242-4245.
Zarrilli R, Casillo R, Di Popolo A et al. Molecular epi- demiology of a clonal outbreak of multidrug-resistant Acinetobacter baumannii in a university hospital in Italy. Clin Microbiol Infect 2007;13:481-489.
Pazhani GP, Niyogi SK, Singh AK, et al. Molecular characterization of multidrug-resistant Shigella spe- cies isolated from epidemic and endemic cases of shigellosis in India. J Med Microbiol 2008;57:856-863.
Gaynor K, Park SY, Kanenaka R, et al. International foodborne outbreak of Shigella sonnei infection in air- line passengers. Epidemiol Infect 2009;137:335-341.
Saran B, Erdem B, Tekeli FA, Sahin F, Aysev AD. An- kara'da izole edilen Shigella kökenlerinin antibiyotik direnç modelleri, plazmid profil analizi ve değişken alanlı jel elektroforezi ile incelenmesi. Mikrobiyol Bul 2013;47:35-48.
Akçalı A, Levent B, Akbaş E, Esen B. Türkiye'nin bazı illerinde izole edilen Shigella sonnei suşlarının antimikrobiyal direnç ve "Pulsed Field" jel elektro- forezi yöntemleri ile tiplendirilmesi. Mikrobiyol Bul 2008;42:563-572.
Pfaller M. Molecular approaches to diagnosing and managing infectious diseases: practicality and costs. Emerg Infect Dis 2001;7:312-318.