Molecular Typing with BOX-PCR of Staphylococcus aureus Isolated from Milk Samples

Year 2021, , 42 - 48, 21.03.2021

Elif Gülbahçe Mutlu , Emine Arslan

https://doi.org/10.17798/bitlisfen.819731

Abstract

The aim of this study is to characterize 98 Staphylococcus aureus isolated from bovine subclinical mastitis in Konya region with BOX primer. A total of 28 band profiles ranging from ~ 950-300 bp were generated by the BOX primer. This band profile was evaluated according to Nei homology and a dendogram (UPGMA) was obtained. According to the data obtained, 95 isolates with genetic similarity varying between 50-100% were in the same group. This study demonstrates that the BOX-PCR technique provides a fast and reliable tool to investigate epidemiology in S. aureus.

Keywords

Molecular typing, bovine mastitis, repetitive DNA sequences, Staphylococcus aureus

Supporting Institution

TUBITAK

Project Number

108T290

Thanks

This research was supported by TUBITAK (Project No: 108T290). We would like to thank Prof. Dr. Uçkun Sait UÇAN for his help in bacterial identification and for the milk collected. In addition, this study was presented as an oral presentation at the 24th National Biochemistry Congress.

Molecular Typing with BOX-PCR of Staphylococcus aureus Isolated from Milk Samples

Abstract

Bu çalışmanın amacı Konya bölgesindeki sığır subklinik mastitinden izole edilen 98 Staphylococcus aureus'u REP ve BOX primerleri ile karakterize etmektir. İlk olarak REP primeri tarafından oluşturulan bant profili ve BOX primeri tarafından oluşturulan bant profili ayrı ayrı değerlendirildi. Daha sonra her iki primerin oluşturduğu bant profili birlikte değerlendirildi. Bant profillerinin değerlendirilmesi Nei homolojisine göre yapıldı ve bir dendogram (UPGMA) elde edildi. ~ 4000-300 bp arasında değişen toplam 44 bant profili değerlendirildi. Elde edilen verilere göre, hemen hemen aynı izolatlar aynı gruptaydı. Her iki primerin kombinasyonuna dayanan dendrogram, S. aureus suşlarının çoğunun % 62'den fazla benzer olduğunu gösterdi. Bu çalışma, kombine REP ve BOX-PCR tekniğinin S. aureus'ta epidemiyolojiyi araştırmak için hızlı ve güvenilir bir araç sağladığını göstermektedir.

Keywords

Moleküler tiplendirme, sığır mastitis, tekrar eden DNA dizileri, Staphylococcus aureus

Project Number

108T290

References

• Han H.R., Pak S.II., Kang S.W., Jong W.S., Youn C.J. 2000. Capsular polysaccharide typing of domestic mastitis causing Staphylococcus aureus strains and its potential exploration of bovine mastitis vaccine development, I. Capsular polysaccharide typing, isolation and purification of the strains. Journal of Veterinary Science, 1: 53-60.
• Reinoso E., Bettera S., Odierno L., Bogni C. 2007. Rep-PCR of Staphylococcus aureus strains isolated from bovine mastitis in Argentina. Brazilian Journal of Veterinary Research and Animal Science, 44: 115-121.
• Pellegrino M.S., Frola I.D., Odierno L.M., Bogni C.I. 2011. Mastitis bovina: resistencia a antibióticos de cepas de Staphylococcus aureus asiladas de leche. Revista electrónica de Veterinaria., 12: 1-14.
• Silva E.R., Pereira A.M.G., Moraes W.S., Santoro K.R., Silva T.R.M. 2012. Perfil de sensibilidade antimicrobiana in vitro de Staphylococcus aureus isolado de mastite subclínica bovina. Revista Brasileira de Saúde e Produção Animal, 13: 701-711.
• Teixeira J.P., Silva N., Fonseca L.M., Costa G.M. 2014. Uso de PCR Duplex para detecção dos genes femA e mecA e determinação da concentração inibitória mínima (CIM) em Staphylococcus aureus isolados de leite cru. Revista do Instituto Adolfo Lutz, 73: 272-279.
• Xavier A.R.E.O., Almeida A.C, Souza C.N, Silva L.M.V., Ruas A.X.A., Sanglard D.A., Júnior A.F.M, Oliveira A.M.E., Xavier M.A.S. 2017. Phenotypic and genotypic characterization of Staphylococcus aureus isolates in milk from flocks diagnosed with subclinical mastitis. Genetics and molecular research, 29: 16 (2).
• Ganda E.K., Bisinotto R.S., Decter D.H., Bicalho R.C. 2016. Evaluation of an on-farm culture system (accumast) for fast identification of milk pathogens associated with clinical mastitis in dairy cows. PLoS One, 13, 11 (5): e0155314.
• 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. 1994. Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus. Journal of Clinical Microbiology, 32: 407-415.
• Del Vecchıo V.G., Petroziello J.M., Gress M.J., Mcclesky F.K., Melcher G.P., Crouch H.K., Lupski J.R. 1995. Molecular genotyping of methicillin-resistant Staphylococcus aureus via flu-orophore enhanced repetitive sequence VCR. Journal of Clinical Microbiology, 33: 2141-2144.
• van Belkum A., Sluijuter M., de Groot R., Verbrugh H., Hermans P.W. 1996. Novel BOX repeats PCR assay for high-resolution typing of Streptococcus pneumoniae strains. Journal of Clinical Microbiology, 34: 1176-1179.
• Wieser M., Busse H.J. 2000. Rapid identification of Staphylococcus epidermidis. International Journal of Systematic and Evolutionary Microbiology, 50: 1087-1093.
• Versalovic J., Koeuth T., Lupski J.R. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research, 19: 6823-6831.
• Versalovic J., Schneider M., de Bruijn F.J., Lupski J.R. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based PCR (rep-PCR). Methods in Molecular Biology, 5: 25-40.
• Holt J.G., Krieg N.R., Sneath P.H.A., Staley J.T. Willams S.T. 1994. Bergey’s manual of Determinative Bacteriology (Ninth edition), Baltimore: Williams & Wilkins.
• Ausubel F.M., Kingston R.E., Brent R., Moore D.D. Seidman J.G., Smith J.A., Struhl K. 1991. Current protocols in molecular biology. New York: Greene Publishing Associates & Wiley Interscience.
• Louws F.J., Fulbright D.W., Stephens C.T., De Bruijn F.J. 1994. Specific genomic fingerprints of phytopathogenic Xantho-monas and Pseudomonas pathovars and strains, generated with repetitive sequences and PCR. Applied and Environmental Microbiology, 60: 2286-2295.
• El-Sayed A., Awad W., Abdou N.E., Castañeda Vázquez H. 2017. Molecular biological tools applied for identification of mastitis causing pathogens. International Journal of Veterinary Science, 5 (2): 89-97.
• Owen R.J. 1989. Chromosomal DNA fingerprinting-a new method of species and strain identification applicable to microbial pathogens. Journal of Medical Microbiology, 30: 89-99.
• Murray B.E., Singh K.V., Heath J.D., Sharma B.R., Weinstock G.M. 1990. Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. Journal of Clinical Microbiology, 28: 2059-2063.
• Szaluś-Jordanow O., Chrobak D., Pyrgiel M., Lutyńska A., Kaba J., Czopowicz M., Witkowski L., Kizerwetter-Świda M., Binek M., Frymus T. 2013. PFGE and AFLP genotyping of Staphylococcus aureus subsp. anaerobius isolated from goats with Morel’s disease. Archives of Microbiology, 195 (1): 37-41.
• Litwin C.M., Storm A.L., Chipowsky S., Ryan K.J. 1991. Molecular epidemiology of Shigella infections: plasmid profiles, serotype correlation, and restriction endonuclease analysis. Journal of Clinical Microbiology, 29: 104-108.
• Uçan U.S., Açık L., Çelebi A., Erganiş O., Arslan E. 2005. Plasmids and Protein Patterns of Ecsherichia coli Isolated from Bovine Mastitis in Konya, Turkey. Turkish Journal of Veterinary and Animal Sciences, 29: 475-480.
• Arslan E., Çelebi A., Açık L., Uçan U.S. 2009. Characterization of Coagulase Positive Staphylococcus Species Isolated from Bovine Mastitis by Protein and Plasmid Patterns. Turkish Journal of Veterinary and Animal Sciences, 33 (6): 493-500.
• Arslan E., Leyla A., Uçan U.S. 2005. Determination by RAPD-PCR of Relationship Degrees of S. aureus and S. intermedius Strains Isolated from Bovine Mastitis. S. Ü. Veteriner Bilimleri Dergisi, 21 (1-2): 65-69.
• Arslan E., Ertuğrul K., Albayrak A. 2009. Staphylococcus aureus ve Staphylococcus intermedius Suşlarının REP-PZR Metodu ile Tiplendirilmesi. Selçuk Universitesi Fen Fakültesi Fen Dergisi, 2 (34): 85-92.
• Arslan E., Gülbahçe Mutlu E. 2016. Genotyping of Staphylococcus aureus Strains Isolated from Bovine Mastitis in Turkey by using ERIC-PCR Method. Pakistan Journal of Zoology, 48 (6): 1747-1752.
• Cuny C., Witte W. 1996. Typing of Staphylococcus aureus by PCR for DNA sequences flanked by transposon Tn916 target region and ribosomal binding site. Journal of Clinical Microbiology, 34: 1502-1505.
• Gürtler V., Barrie H.D. 1995. Typing of Staphylococcus aureus strains by PCR-amplification of variable-length 16S-23S rDNA spacer regions: characterization of spacer sequences. Microbiology, 141: 1255-1265.
• Deplano A., Schuermans A., Van Eldere J., Witte W., Meugnier H., Etienne J., Grundmann H., Jonas D., Noordhoek G.T., Dijkstra J., van Belkum A., van Leeuwen W., Tassios P.T., Legakis N.J., van der Zee A., Bergmans A., Blanc D.S. Tenover F.C., Cookson B.C., O'Neil G., Struelens M.J. 2000. Multicenter evaluation of epidemiological typing of methicillin resistant Staphylococcus aureus strains by repetitive element PCR analysis. The European Study Group on Epidemiological Markers of the ESCMID. Journal of Clinical Microbiology, 38: 3527-3533.
• van Belkum A., Kluytamans J., van Leeuwen W., Bax R., Quint W., Peters E., Fluit A., Vandenbroucke-Grauls C., van den Brule A., Koelman H. 1995. Multicenter evaluation of arbitrarily primed PCR for typing of Staplycoccus aureus strains. Journal of Clinical Microbiology, 33: 1537-1547.
• Ross T.L., Merz W.G., Farkosh M., Carroll C. 2005. Comparison of an automated repetitive sequence-based PCR microbial typing system to pulsed-field gel electrophoresis for analysis of outbreaks of methicillin-resistant Staphylococcus aureus. Journal of Clinical Microbiology, 43: 5642-5647.
• Kerouanton A., Brisabois A., Grout J., Picard B. 1996. Molecular epidemiological tools for Salmonella dublin typing FEMS Immunology and Medical Microbiology, 14: 25-29.
• Appuhamy S., Parton R., Coote J.G., Gibbs H.A. 1997. Genomic fingerprinting of Haemophilus somnus by a combination of PCR methods. Journal of Clinical Microbiology, 35: 288-291.
• Rajashekara G., Koeuth T. Nevile S., Back A., Nagaraja K.V., Lupski J.R., Kapur V. 1998. SERE, a widely dispersed bacterial repetitive DNA element. Journal of Medical Microbiology, 47: 489-498.
• Lessing M.P., Jordens J.Z., Bowler I.C. 1995. Molecular epidemiology of a multiple strain outbreak of methicillin-resistant Staphylococcus aureus amongst patients and staff. Journal of Hospital Infection, 31: 253-260.
• van Belkum A., Bax R., Peerbooms P., Goessens W.H.F., Van Leeuwen N., Quint W.G.V. 1992. Comparision of phage typing and DNA fingerprinting by polymerase chain reaction for discrimination of methicillin-resistant Staphylococcus aureus strains. Journal of Clinical Microbiology, 31: 798-803.
• Woods C.R., Versalovic J., Koeuth T., Lupski J.R. 1993. Whole-cell repetitive element sequence-based polymerase chain reaction allows rapid assessment of clonal relationship of bacterial isolates. Journal of Clinical Microbiology, 31: 1927-1931.
• van der Zee A., Verbakel H., van Zon J.C., Frenay I., van Belkum A., Peeters M., Butting A., Bergmans A. 1999. Molecular genotyping of Staphylococcus aureus strains: comparison of repetitive element sequence-based PCR with various typing methods and isolation of a novel epidemicity marker. Journal of Clinical Microbiology, 37: 342-349.
• Shutt C.K., Pounder J., Page S.R., Schaecher B.J., 2005. Clinical evaluation of the DiversiLab microbial typing system using repetitive-sequence-based PCR for Characterization of Staphylococcus aureus strains. Journal of Clinical Microbiology, 43: 1187-1192.
• Reinoso E.B., El-Sayed A., Lammler C., Bogni C. Zschock M. 2006. Genotyping of Staphylococcus aureus isolated from humans, bovine subclinical mastitis and food samples in Argentina. Microbiological Research, 163: 314-322.
• Alam S., Brailsford S.R., Whiley R.A., Beighton D. 1999. PCR-Based methods for genotyping viridans group streptococci. Journal of Clinical Microbiology, 37: 2772-2776.
• Currie B.J., Gal D., Mayo M., Ward L., Godoy D., Spratt B.G., Lipuma J.J. 2007. Using BOX-PCR to exclude a clonal outbreak of melioidosis. BMC Infectious Diseases, 7: 68.