Emine ARSLAN, Kuddisi ERTUĞRUL, Ali İhsan ALBAYRAK

Staphylococcus aureus ve Staphylococcus intermedius Suşlarının REP-PZR Metodu ile Tiplendirilmesi

Mastitis sütçülük işletmelerinde giderlerin artması ve süt veriminin azalması gibi sonuçlara yol açarak önemli ekonomik kayıplara sebep olan yangısal bir bozukluktur. Staphylococcus aureus, mastitise sebep olan etken organizmalar arasında ilk sırada yer almaktadır. Bu çalışmada Konya Bölgesi’ndeki mastitisli ineklerden izole edilen 43 S. aureus 6 S. intermedius suşunun akrabalık derecelerinin REP-PZR yöntemi ile belirlenmesi amaçlanmıştır. REP-PZR sonucunda 500-~3000 bp büyüklüklerinde ve 2-6 arasında değişen sayılarda bantlar gözlemlenmiştir. Bu REP profillerinden elde edilen dendrograma göre S. aureus ve S. intermedius suşları arasındaki en yüksek akrabalık derecesi %82 oranında olduğu belirlenmiştir.

Anahtar Kelimeler:

Mastitis, S. aureus, S. intermedius, REP, PZR

Typing of Staphylococcus aureus and Staphylococcus intermedius Strains with Repetitive Sequence-Based PCR (REP-PCR) Method

Mastitis is an inflammatory anomaly which results in important economic losses causing consequences like increase in prices at small dairies and decrease in milk production. Staphylococcus aureus is the first of the most effective factors which cause mastitis disease. The aim of this study was to determine of the relationship degree by REP-PCR between 43 S. aureus and six S. intermedius strains isolated from bovines mastitis in Konya region. As a result, 2-6 bands sized 500-~3000 bp were observed. According to obtaining a dendrogram from these REP profiles, it was determined that the most high relationship degree between S. aureus and S. intermedius strains was 82% rate.

Keywords:

Mastitis, S. aureus, S. intermedius, REP, PZR,

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[1] Waterman, D.F., Harmon, R.J., Hemken, R.W., Langlois, B.E. Milking Frequency as Related to Udder Health and Milk Production. J Dairy Sci. 66(2):253–258 (1983).
[2] Rajala-Schultz, P.J., Gröhn, Y.T., McCulloch, C.E., Guard, C.L. Effect of Clinical Mastitis on Milk Yield in Dairy Cows. J. Dairy Sci. 82: 1213–1220 (1999).
[3] Shim, E.H., Shanks, R.D., Morin, D.E. Milk Loss and Treatment Costs Associated with Two Treatment Protocols for Clinical Mastitis in Dairy Cows. J Dairy Sci. 87(8):2702–2708 (2004).
[4] Alaçam, E., Tekeli, T., Sezen, Y., Erganiş, O. Sütçü İneklerin Subklinik Mastitislerinde Cefoperazone’un Etkisi Üzerinde Çalışma. S.Ü. Vet. Fak. Derg. 1(2):65–74 (1986).
[5] Bademkıran, S., Yeşilmen, S., Gürbulak, K. Sütçü İneklerde Günlük Sağım Sayısının Klinik Mastitis ve Süt Verimi Üzerine Etkisi. Y.Y.Ü. Vet Fak. Derg. 16 (2):17-21 (2005)
[6] Matthews, K.R., Jayarao, B.M., Oliver, S.P. Plasmid pattern analysis of Staphylococcus species isolated from bovine mammary secretions. J. Dairy Sci. 75(12):3318-23 (1992)
[7] Tenover, F.C., Arbeit, R., Archer, G., Biddle, J., Byrne, S., Goering, R., Hancock, G., Herbert, G.A., Hill, B., Hollis, R., Jarvis, W.R., Kreiswirth B., Eisner, W., Maslow, J., McDougal, L.K., Miller, J.M., Mulligan, M., Pfaller, M.A. Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus. J. Clin. Microbiol. 32:407-415 (1994).
[8] Maslow, J.N., Mulligan, M.E., Arbeit, R.D. Molecular epidemiology: application of contemporary techniques to the typing of microorganisms. Clin. Infect Dis. 17: 153-164 (1993).
[9] van Belkum, A., Kluytamans, van Leeuwen, W., Bax, R., Quint, W., Peters, E., Fluit, A., VandenbrouckeGrauls, C., van den Brule, A., Koelman, H. Multicenter evaluation of arbitrarily primed PCR for typing of Staplycoccus aureus strains. J. Clin. Microbiol. 33:1537-1547 (1995).
[10] Hajek, V., Marsalek, E. The differentiation of pathogenic staphylococci and a suggestion for their taxonomic classification. Zenralbl. Bakteriol. Abt. l. Orig.A217-176-182 (1971).
[11] Devriese, L.A. A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species. J. Appl. Bacteriol. 56,215-220 (1984).
[12] Musser, J., Kapur, V., Clonal analysis of methicillin resistant Staphylococcus aureus strains from intercontinental sources: association of the mec gene with divergent phylogenetic lineages implies dissemination by horizontal transfer and recombination. J. Clin. Microbiol. 30,2058-2063 (1992).
[13] Kapur, V, Sischo, W., Greer, R., Whittam, T, Musser, J. Molecular population genetic analysis of Staphylococcus aureus recovered from cows. J. Clin. Microbiol. 33, 376-380 (1995).
[14] Zadoks, R., van Leeuwen, W., Barkema, H., Sampimon, O., Verbrugh, H., Schukken, Y., van Belkum, A. Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates. J. Clin. Microbiol. 38, 1931-1939 (2000).
[15] Reinoso, E., Bettera, S., Frigerio, C., Di Renzo, M., Calzolari, A., Bogni, C. RAPD-PCR analysis of Staphylococcus aureus strains isolated from bovine and human hosts. Microbiol. Res. 159, 245-255 (2004).
[16] Del Vecchio, V.G., Petroziello, J.M., Gress, M.J., McClesky, F.K., Melcher, G.P., Crouch, H.K. and Lupski, J.R. Molecular genotyping of methicillin-resistant Staphylococcus aureus via fluorophore enhanced repetitive sequence VCR. J. Clin. Microbiol. 33, 2141-2144 (1995).
[17] Versalovic, J., Koeuth, T. and Lupski, J.R. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19, 6823-6831 (1991).
[18] Stern, M.J., Ames, G.F.L., Smith, N. H., Robinson, E. C. and Higgins, C. F. Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell 37:1015-1026 (1984).
[19] Hulton, C.S.J., Pliggins, C.F., Sharp, P.M. ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium, and other enterobacteria. Mol. Microbiol. 5, 825-34 (1991).
[20] Martin, B., Humbert, O., Camara, M., Guenzi, E., Walker, J., Mitchell, T., Andrew, P., Prudhomme, M., Alloing, G., Hackenbeck, R., Morrison, D.A., Boulnois, G.J., Claverys, J.P. A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. Nucleic Acids Res. 20, 3479-83 (1992).
[21] Georghiou, P., Wright, C., Versalovic, J., Koeuth, T., Watson, D., Hamill, R. and Lupski, J. Program Abstr. 32nd lntersci. Conf. Antimicrob. Agents Chemother., abstr. 1415 (1992).
[22] Woods, C.R, Versalovic, J., Koeuth, T. and Lupski, J.R. Analysis of relationships among isolates of Citrobacter diversus by using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reaction. J. Clin. Microbiol. 30:2921-2929 (1992).
[23] Versalovic, J., Kapur, V., Mason, E.O., Shah, U., Koeuth, T., Lupski, J.R. and Musser, J.M. Penicillin resistant Streptococcus pneumoniae strains recovered in Houston, Texas: identification and molecular characterization of multiple clones. J. Infect. Dis. 167:850-856 (1993). [24] Ausubel, F.M., Kingston, R.E., Brent, R., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. Current protocols in molecular biology. Greene Publishing Associates & Wiley Interscience, New York (1991).
[25] Maniatis, R., Fritsh, E.F., Sambrook, J. In Molecular Cloning: A laboratory Manual, Cold Spring Harbos, New York (1982).
[26] van der Zee, A., Verbakel, H., van Zon, J.C., Frenay, I., van Belkum, A., Peeters, M., Butting, A., Bergmans, A. Molecular genotyping of Staphylococcus aureus strains: comparison of repetitive element sequence-based PCR with various typing methods and isolation of a novel epidemicity marker. J. Clin. Microbiol. 37:342-349 (1999).
[27] Reinoso, E.B., El-Sayed, A., Lämmler, C., Bogni, C., Zschöck, M. Genotyping of Staphylococcus aureus isolated from humans, bovine subclinical mastitis and food samples in Argentina. Microbiol. Res. Vol 163:3, 314-322 (2008).
[28] Arslan, E., Açık, L., Uçan, U.S. Mastitisli ineklerden izole edilen S. aureus ve S. intermedius suşlarının RAPD-PCR ile akrabalık derecelerinin belirlenmesi. Vet. Bil. Derg. (2005), 21, 1- 2:65-69 (2005).
[29] Peles, F., Wagner, M., Varga, L., Hein, I., Rieck, P., Gutser, K., Keresztúri, P., Kardos, G., Turcsányi, I., Béri, B., Szabó, A. Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary. Int. J. Food Microbiol. Vol 118:2, 186-193 (2007).
[30] Deplano, A., Vaneechoutte, M., Verschraegen, G. and Struclens, M.J. Typing of Staphylococcus aureus and Staphylococcus epidermidis Strains by PCR Analysis of inter-IS256 spacer length polymorphism. J. Clin. Microbiol. 35:2580-2587 (1997).
[31] Cuny, C, and Witte, W. Typing of Staphylococcus aureus by PCR for DNA sequences flanked by transposon TnP/6 target region and ribosomal binding site. J. Clin. Microbiol. 34:1502-1505 (1996).
[32] Gürtler, V. and Barrie, H.D. Typing of Staphylococcus aureus strains by PCR-amplification of variable-length 16S-23S rDNA spacer regions: characterization of spacer sequences. Microbiol. 141:1255-1265 (1995).
[33] Kumari, D.N.P., Peer, V., Hawkey, P.M., Parnell, P., Joseph, N., Richardson, J.F. and Cookson, B. Comparison and application of ribosome spacer DNA amplicon polymorphisms and pulsed-field gel electrophoresis for differentiation of methicillin-resistant Staphylococcus aureus strains. J. Clin. Microbiol. 35:881-885 (1997).