Comparision of Two PCR-Based Methods in Typing of Clinical Staphylococcal Strains

Year 2013, Volume: 41 Issue: 1, 43 - 50, 01.03.2013

Neslihan Idil Nilüfer Aksöz

Abstract

The present study was carried out to investigate usefulness and effectiveness of randomly amplified polymorphic DNA polymerase chain reaction RAPD-PCR and repetitive element sequence-based polymerase chain reaction REP-PCR in differentiating of staphylococcal strains and to compare the results of these methods with those obtained by antibiotyping. Staphylococcal strains, obtained from various clinical samples and collected from different wards, were characterized phenotypically by susceptibility testing and genotypically by using RAPD-PCR and REP-PCR methods. It was found that there was no significant association between genotypes obtained from RAPD and REP-PCR. Strains with a similarity coefficient of 80% and 70% or greater were grouped in a cluster for RAPD-PCR and REP-PCR, respectively. RAPD-PCR was found to be very efficient with the discriminatory index DI of 0.91 whereas discrimination index DI of REP analysis was found to be 0.88 with RW3A primer and combination of REP1R-I, REP2-I primers. The findings of this study indicate that RAPD-PCR reliably distinguish ward and source-related clustering. The RAPD primers provide to discriminate MRSA, MSSA and CNS strains whereas REP analysis could not be as discriminative as RAPD. Therefore, RAPD-PCR, evidenced to be inconsiderably more discriminatory than REP-PCR, is well suited for fast and accurate strain identification.

Keywords

Staphylococcal strains, Antibiotype, RAPD-PCR, REP-PCR

Klinik Stafilokokal Suşların Tiplendirilmesinde İki Farklı PCR Temelli Yöntemin Karşılaştırılması

Abstract

B u çalışmada stafilokokal suşların ayrımında RAPD-PCR Rastgele Çoğaltılmış Polimorfik DNA-Polimeraz Zincir Reaksiyonu ve REP-PCR Tekrarlayan Ekstragenik Elementlerin Polimeraz Zincir Reaksiyonu ile Amplifikasyonu yöntemlerinin etkinliğinin ve kullanılabilirliğinin araştırılmasını ve elde edilen sonuçların antibiyotipleme ile karşılaştırılması amaçlanmaktadır. Çeşitli klinik örneklerden izole edilen ve farklı servislerden toplanan stafilokokal suşlar fenotipik olarak antibiyotik duyarlılık testi, genotipik olarak RAPDPCR ve REP-PCR yöntemleri ile karakterize edilmiştir. RAPD ve REP-PCR ile elde edilen genotipler arasında önemli ölçüde benzerlik bulunamamıştır. RAPD-PCR ve REP-PCR için sırasıyla %80 ve %70 olarak belirlenen benzerlik katsayısı ile suşlar gruplandırılmıştır. RAPD-PCR’ın 0.91 oranında ayrım gücü ile oldukça etkin olduğu tespit edilirken, RW3A primeri, REP1R-I ve REP2-I primerlerinin kombinasyonu ile elde edilen REP analizinin ayrım gücü 0.88 olarak bulunmuştur. Çalışmanın bulguları RAPD-PCR yönteminin klinik örnek ve servisler ile ilişkili kümelemede güvenilir olduğunu göstermektedir. RAPD primerlerinin MRSA, MSSA ve CNS suşların ayrımını sağlayabildiği, REP analizinin RAPD kadar ayırt edici olmadığı bulunmuştur. Böylece RAPD-PCR’ın REP-PCR’dan daha ayırt edici olduğu ve suşların tanımlanmasında hızlı ve doğru bir yöntem olarak uygunluğu kanıtlanmıştır

Keywords

Stafilokokal suşlar, antibiyotip, RAPD-PCR, REP-PCR

References

• M. Nikbakht, M.R. Nahaei, M.T. Akhi, M. Asgharzadeh, S. Nikvash, Molecular fingerprinting of meticillin- resistant Staphylococcus aureus strains isolated from patients and staff of two Iranian hospitals, J. Hosp. Infect., 69 (2008) 46.
• M. Wieser, H.J. Busse Rapid identification of Staphylococcus epidermidis, Int. J. Syst. Evol. Microbiol., 50 (2000) 1087.
• J. Huebner, D.A. Goldmann, Coagulase-negative staphylococci: role as pathogens, Annu. Rev. Med., 50 (1999) 223.
• P. Sharma, K.K. Lahiri, K. Kapila, Conventional and molecular characterization of coagulase-negative staphylococcus in hospital isolates, Indian J. Pathol. Microbiol., 54 (2011) 85.
• A. Shittu, J. Lin, D. Morrison, Antimicrobial susceptibility patterns and characterization of clinical isolates of Staphylococcus aureus in KwaZulu-Natal province, South Africa BMC Infect. Dis., 6 (2006) 125.
• J. Stepán, R. Pantůcek, J. Doskar Molecular diagnostics of clinically important staphylococci, Folia Microbiol., 49 (2004) 353. 7. E. Hyytiä, J. Björkroth, S. Hielm, H. Korkeala, Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR, Int. J. Food. Microbiol., 48 (1999) 179.
• P. Saulnier, C. Bourneix , G. Prévost, A. Andremont, Random amplified polymorphic DNA assay is less discriminant than pulsed-field gel electrophoresis for typing strains of methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol., 31 (1993) 982.
• W.E Kloos, K.H. Schleifer, The genus Staphylococcus. In: PHA Sneath, NS Mair, M.E. Sarpe, J.G. Holt (ed) Bergey’s Manual of Systematic Bacteriology, 2nd edn. Williams and Wilkins, Baltimore, (1986) 1015.
• Clinical and Laboratory Standard Institute, (2005), Performance standards for antimicrobial disk susceptibility tests, CLSI document M100-S15, Clinical and Laboratory Standard Institute (CLSI), Wayne.
• P.R. Hunter, M.A. Gaston, Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity, J. Clin. Microbiol., 26 (1988) 2465.
• A.C. Fluit, C.L.C. Wielders, J. Verhoef, F.J. Schmitz, Epidemiology and Susceptibility of 3,051 Staphylo- coccus aureus isolates from 25 University Hospitals participating in the European SENTRY study, J. Clin. Microbiol., 39 (2001) 3727.
• H.B. Kim, H.C. Jang, H.J. Nam, Y.S. Lee, B.S. Kim, W.B. Park, K.D. Lee, Y.J. Choi, S.W. Park, M.D. Oh, E.C. Kim, K.W. Choe, In-vitro activities of 28 antimicrobial agents against Staphylococcus aureus isolates from tertiary-care hospitals in Korea: a nationwide survey, Antimicrob. Agents Chemother., 48 (2004) 1124.
• F.J. Schmitz, A.C. Fluit, M. Gondolf, , R. Beyrau, E. Lindenlauf, J. Verhoef, H.P. Heinz, M.E. Jones, The prevalence of aminoglycoside resistance and corresponding resistance genes in clinical isolates of staphylococci in 19 European hospitals, J. Antimicrob. Chemother., 43 (1999) 253.