Determination of genetic relationship between methicillin-sensitive and methicillin- resistant Staphylococcus aureus clinical isolates by RAPD-PCR Method

Abstract

The increase in the frequency of methicillin-resistant Staphylococcus aureus MRSA demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. The objective of this study is to establish variations of randomly amplified polymorphic DNA RAPD patterns in S. aureus isolates which are collected from clinical isolates and a systematic relationship between them. Identification of isolates and antimicrobial susceptibility tests were performed by automated system and conventional phe- notypic methods. A total of 30 out of 47 isolates 64% expressed methicillin-resistance. When methicillin-sus- ceptible and resistance isolates were compared, the least active agents were rifampin, all aminoglycosides and penicillines, tetracycline and amoxicillin-clavulonic acid for methicillin-resistance isolates. RAPD-PCR method with 25 different primers was analyzed on 47 clinical isolates of S.aureus. All the data were employed to cons- truct dendograms using the unweighted pair group method using arithmetic UPGMA . In this study, it is clear that, RAPD-PCR assayed with combination of five primers can be successfully applied to assess the genetic relationship between MRSA and MSSA isolates from clinical samples.

Keywords

• Antibiotyping,Staphylococcus aureus,methicillin resistance,RAPD-PCR

RAPD-PCR yöntemi ile metisilin duyarlı ve dirençli Staphylococcus aureus klinik izolatları arasındaki genetik ilişkinin saptanması

Abstract

M etisilin dirençli Staphylococcus aureus MRSA görülme sıklığındaki artış bu suşların karakterizasyonunun hızlı ve güvenilir bir şekilde yapılmasını ve hastanelerde klonal yayılımın tanımlanmasını gerektirmektedir. Bu çalışmanın amacı, klinik örneklerden elde edilen Staphylococcus aureus suşlarına ait rastgele çoğaltılmış polimorfik DNA RAPD bant modellerindeki varyasyonu ve bunlar arasındaki sistematik ilişkiyi ortaya çıkarmaktır. Suşların tanımlanması ve antibiyotik duyarlılıkları otomatize sistem ve geleneksel yöntemler ile belirlendi. Toplam 47 izolatın 30’u %64’ü metisiline dirençli bulundu. Metisilin duyarlı ve dirençli izolatlar karşılaştırıldığında metisilin dirençli izolatlarda en düşük etki gösteren antibiyotikler rifampin, tüm aminoglikozidler ve penisilinler, tetrasiklin ve amoksilin-klavulonik asittir. 47 S.aureus klinik izolat, RAPDPCR tekniği kullanılarak 25 farklı primer ile analiz edildi. Tüm veriler ile ağırlıksız çift grup yöntemi UPGMA kullanılarak dendogram çizildi. Bu çalışmada, beş primerin kombinasyonu kullanılan RAPD-PCR yöntemi klinik örneklerden izole edilen MRSA ve MSSA izolatları arasındaki genetik ilişkinin belirlenmesinde başarılı bir şekilde uygulanabildiği açıkça görülmektedir

Keywords

• Antibiyotipleme,Staphylococcus aureus,Metisilin direnci,RAPD-PCR

References

1. 1. J. Kluytmans, W. Van Leeuwen, W. Goessens, R. Hollis, S. Messer, L. Herwaldt, H. Bruining, M. Heck, J. Rost, N. Van leeuwen, Food-initiated outbreak of Methicillin-resistant Staphylococcus aureus analyzed by pheno- and genotyping, J. Clin. Microbiol., 33 (1995) 1121.
2. 2. F.D. Lowy, Staphylococcus aureus Infections., N. Engl. J. Med. 339 (1998) 520.
3. 3. A. Onasanya, H.D. Mignouna, G. Thottappilly, Genetic fingerprinting and phylogenetic diversity of Staphylococcus aureus isolates from Nigeria, African J. Biotech. ,2 (2003) 246.
4. 4. V. Neela, N.S. Mariana, S. Radu, S. Zamberi, A.R. Raha, R. Rosli, Use of RAPD to investigate the epidemiology of Staphylococcus aureus infection in Malaysian hospitals, World J. Microbiol. & Biotechnol., 21 (2005) 245.
5. 5. A. Van Belkum, R. Bax, P. Peerbooms, W.H. Goessens, N. van Leeuwen, W.G. Quint, Comparison of phage typing and DNA fingerprinting by polymerase chain reaction for discrimination of methicillin-resistant Staphylococcus aureus strains, J. Clin. Microbiol., 31 (1993) 798.
6. 6. A. Deplano, A. Schuermans, J. van Eldere, W. Witte, H. Meugnier, J. Etienne, H. Grundmann, D. Jonas, G. T. Noordhoek, J. Dijkstra, A. Van Belkum, W. Van Leeuwen, P. T. Tassios, N. J. Legakis, A. Van der Zee, A. Bergmans, D. S. Blanc, F. C. Tenover, B. C. Cookson, G. O’neil, M. J. Struelens, and the European Study Group on Epidemiological Markers of the ESCMID, Multicenter evaluation of epidemiological typing of methicillin-resistant Staphylococcus aureus strains by repetitive-element PCR analysis, J. Clin. Microbiol., 38 (2000) 3527.
7. 7. C. Schlichting, C. Branger, J.M. Fournier, W. Witte, A. Boutonnier, C. Wolz, P. Goullet, G. Doring, Typing of Staphylococcus aureus by pulsed-field gel electrophoresis, zymotyping, capsular typing and phage typing: resolution of clonal relationships, J. Clinic. Microbiol., 31 (1993) 227.
8. 8. W. van Leeuwen, C. Libregts, M. Schalk, J. Veuskens, H. Verbrugh, A. van Belkum, Binary typing of Staphylococcus aureus strains through reverse hybridization using digoxigenin-universal linkage system-labeled bacterial genomic DNA, J. Clin. Microbiol., 39 (2001) 328.

9. 9. J. Welsh, M. Mcclelland, Fingerprinting genomes using PCR with arbitrary primers, Nucleic Acids Res., 18 (1990) 7213.
10. 10. J.G.K. Williams, A.R. Kubelik, K.J. Livak, J.A. Rafalski, DNA polymorphisms amplified by arbitrary primers are useful as genetic markers, Nucleic Acids Res., 18 (1990) 6531.
11. 11. K.E. Cheeseman, G.J. Williams, J.Y. Maillard, S.P. Denyer, E. Mahenthiralingam, Typing of Staphylococcus aureus clinical isolates using random amplification of polymorphic DNA method and comparison with antibiotic susceptibility typing, J. Hosp. Infect., 67 (2007) 388.
12. 12. M. Nikbakht, M.R. Nahaei, M.T. Akhi, M. Asgharzadeh, S. Nikvash, Molecular fingerprinting of methicillinresistant Staphylococcus aureus strains isolated from patients and staff of two Iranian hospitals, J. Hosp. Infect., 69 (2008) 46.
13. 13. A. Tambic, E.G.M Power, H. Talsania, R.M. Anthony, G.L. French, Analysis of outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay, J. Clin. Microbiol., 35 (1997) 3092.
14. 14. 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.
15. 15. Y. Duman, E. Serindağ, M.S. Tekerekoğlu, Klinik Örneklerden İzole Edilen Staphylococcus aureus’ların Antimikrobiyallere Direnç Durumu, İnönü Üniversitesi Tıp Fakültesi Dergisi, 16 (2009) 145.
16. 16. Centers for Disease Control and Prevention Update: Staphylococcus aureus with reduced susceptibility to vancomycin—United States MMWR Morb. Mortal. Wkly. Rep., 46 (1997) 813.
17. 17. K. Hiramatsu, H. Hanaki, T. Ino, K. Yabuta, T. Oguri, F.C. Tenover, Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility, J. Antimicrob. Chemother., 40 (1997) 135.
18. 18. S. Assadullah, D.K. Kakru, M.A. Thoker, F.A. Bhat, N. Hussain, A. Shah, Emergence of low level vancomycin resistance in MRSA, Indian J. Med. Microbiol., 21 (2003) 196.
19. 19. S. Artur, N. Malachowa, J. Miedzobrodzki, W. Hryniewichz, Comparison of PCR-based methods for typing Staphylococcus aureus isolates, J. Clin. Microbiol., 44 (2006) 3804.
20. 20. A. Van der Zee, H. Verbakel, J.C. van Zon, I. Frenay, A. van Belkum, M. Peeters, A. Buiting, A. Bergmans, Molecular genotyping of Staphylococcus aureus strains: comparison of repetitive element sequencebased PCR with various typing methods and isolation of novel epidemicity marker, J. Clin. Microbiol., 37 (1999) 342.
21. 21. T.L.T. Fabıano, B.H.P. Avıla, C.C. Dias, R.P. Maluta, F.A. Avila, Genetic similarity between Staphylococcus sp isolated from human and hospital settings, and susceptibility to different antimicrobials, Braz. J. Microbiol., 39 (2008) 652.
22. 22. C.B. Louki, G. Dimitracopoulos, I. Spiliopoulou, Polymerase chain reaction fingerprints of methicillinresistant Staphylococcus aureus clinical isolates in Greece are related to certain antibiotypes, J. Microbiol. Methods., 53 (2003) 417.
23. 23. M.S.V. Pereira, N.C. Leal, T.C.A. Leal, M. Sobreira, A.M.P. De Almeida, J.P. Siqueira-Junior, G.M. CamposTakaki, Typing of human and bovine Staphylococcus aureus by RAPD-PCR and ribotyping-PCR, Lett. Appl. Microbiol., 35 (2002) 32.