Süheyla TÜRKYILMAZ, Emel İNEGÖL

Determination of SCCmec types in methicillin resistant staphylococci isolated from cows and farm workers

Özet: Bu çalışma sığır ve çiftlik çalışanlarından izole edilen metisilin dirençli stafilokokların (MRS) stafilokokal kaset kromozom mec (SCCmec) tiplerini belirlemek amacıyla gerçekleştirildi. Toplam 145 subklinik mastitisli inek sütü ve 91 burun sıvap örneği (56 inekten ve 35 çiftlik çalışanından) MRS varlığı yönünden incelendi. Metisilin direnci sefoksitin disk difüzyon yöntemi ile belirlendi. Bakteriyel identifikasyon ve metisilin direnci sırasıyla 16S rRNA sekansı ve mecA PCR ile doğrulandı. Bu izolatların SCCmec tipleri mec ve ccr, IS elementi ve plazmidler için multipleks PCR ile incelendi. Bu 236 örnekten 181 stafilokok izole edilirken bunların 59’u metisilin dirençli olarak belirlendi. Elli dokuz izolatın 37 (%62.7)’si sığır ve 22 (%37.3)’si insan orjinliydi. 16S rRNA sekans analizi ile incelenen 59 MRS içinde 10 tür [S. haemolyticus (18), S. epidermidis (17), S. aureus (12), S. xylosus (4), S. cohnii (3), S. equorum (1), S. sciuri (1), S. succinus (1), S. pseudointermedius (1), S. saprophyticus (1)] identifiye edildi. SCCmec tip II, III, IV ve V sırasıyla 2, 38, 18 ve 1 MRS izolatında belirlendi. SCCmec Tip II ve III’ü taşıyan 40 izolat hastane kökenli, SCCmec Tip IV ve V’i içeren 19 izolat toplum kökenli olarak tespit edildi. Hemen tüm S. haemolyticus ve S. aureus izolatlarının hastane kökenli SCCmec tiplerini taşıdığı belirlendi. Sığır ve insan stafilokok izolatlarının taşıdığı SCCmec tipleri arasındaki benzerlik hayvanlardan insanlara ya da tam tersi bir bulaşma olabileceğini düşündürmektedir. Sığır ve çiftlik çalışanlarından izole edilen MRS’lar arasındaki klonal ilişkinin belirlenebilmesi için ileri moleküler teknikler kullanılarak gerçekleştirilecek olan çalışmaların yapılmasına ihtiyaç duyulmaktadır.

Sığırlardan ve sektör çalışanlarından izole edilen metisilin dirençli stafilokoklarda SCCmec tiplerinin belirlenmesi

Summary: This study was conducted to determine staphylococcal cassette chromosome mec (SCCmec) types of methicillin resistant staphylococci (MRS) isolated from cattle and farm workers. A total of 145 subclinic mastitic bovine milk and 91 nasal swab samples (56 from cows and 35 from farm workers) were studied for presence of MRS. Methicillin resistance was detected by cefoxitin disc diffusion test. Bacterial identification and methicillin resistance was confirmed by 16S rRNA sequencing and mecA PCR, respectively. SCCmec types of these isolates were determined by multiplex PCR targeting for mec and ccr genes, associated IS element and plasmids. Among 236 total samples, 181 staphylococci, 59 of which were found as methicillin resistant were isolated. Fifty nine methicillin resistant isolates originated from 37 (62.7%) were bovine and 22 (37.3%) were human. Ten Staphylococcus species [S. haemolyticus (18), S. epidermidis (17), S. aureus (12), S. xylosus (4), S. cohnii (3), S. equorum (1), S. sciuri (1), S. succinus (1), S. pseudointermedius (1) and S. saprophyticus (1)] among 59 MRS were identified by 16S rRNA sequence analysis. SCCmec types of 2, 38, 18, and 1 MRS isolates were II, III, IV, and V, respectively. A total of 40 isolates including type II and III were hospital acquired whereas 19 isolates including type IV and V were community acquired. Almost all S. haemolyticus and S. aureus isolates carried hospital acquired type SCCmec. The similarity of SCCmec types between human and cattle isolates suggest evidence of transmission from animals to humans, or vice versa. Further studies are needed to establish clonal relationship of MRS isolated from bovine and farm workers with advanced molecular techniques.

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1. Baştan A (2002): İneklerde Meme Hastalıkları. Hatipoğlu Basım ve Yayım San Tic Ltd Şti, Ankara.
2. Clinical and Laboratory Standards Institute (2008): Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. 3th. Edition: Approved Standard M31-A3. Wayne, PA, USA.
3. Febler TA, Billerbeck C, Kadlec K, Schwarz S (2010): Identification and characterization of methicillin-resistant coagulase-negative staphylococci from bovine mastitis. J Antimicrob Chemother, 65, 1576–1582.
4. Hadimli HH, Ateş M, Güler L, Kav K, Öncel T (2001): Mastitisli süt ineklerinden izole edilen stafilokokların β- laktamaz aktiviteleri ve antibiyotiklere duyarlılıkları, Vet Bil Derg, 17, 21–25.
5. International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWGSCC) (2009): Classification of Staphylococcal Cassette Chromosome mec (SCCmec) Elements. Antimicrob Agents Chemother, 53, 4961-4967.
6. Ito T, Katayama Y, Hiramatsu K (1999): Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrob. Agents Chemother, 43, 1449–1458.
7. Ito Y, Ma XX, Takeuchi F, Okuma K, Yuzawa H, Hiramatsu K (2004): Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC. Antimicrob Agents Chemother, 48, 2637–2651.
8. Jevons MP (1961): Celbenin-resistant staphylococci. Br Med J, 124, 124–125.
9. Juhász-Kaszanyitzky É, Jánosi S, Somogyi P, Dán Á, Bloois LG, Duijkeren E, Wagenaar JA (2007): MRSA transmission between cows and humans. Emerg Infect Dis, 13, 630–632.
10. Kaynarca S, Türkyılmaz S (2010): Methicillin resistance and slime positivity of staphylococci isolated from bovine mastitis. Kafkas Univ Vet Fak Derg, 16, 567-572.
11. Kırkan Ş, Göksoy EÖ, Kaya O (2005): Identification and antimicrobial susceptibility of Staphylococcus aureus and coagulase negative staphylococci from bovine mastitis in the Aydın region of Turkey. Turk J Vet Anim Sci, 29, 791– 796.
12. Kim CH, Khan M, Morin DE, Hurley WE, Tripathy DN, Jr Kerhli M, Oluoch AO, Kakoma I (2001): Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk. J Dairy Sci, 84, 74–83.
13. Kireçci E, Çolak A (2002): Kuru dönem başlangıcında subklinik mastitisli ineklerden izole edilen stafilokok suşlarında metisilin direnci. Kafkas Üniv Vet Fak Derg, 8, 98-100.
14. Murakami K, Minamide W, Wada K, Nakamura E, Teraoka H, Watanabe S (1991): Identification of methicillin resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol, 29, 2240-44.
15. Murray PR (2003): Manuel of Clinical Microbiology, 8th ed. ASM Press, Washington D.C., USA.
16. Oliveira DC, Tomasz A, de Lencastre H (2001): The evolution of pandemic clones of methicillin resistant Staphylococcus aureus: identification of two ancestral genetic backgrounds and the associated mec elements. Microb Drug Resist, 7, 349–361.
17. Oliveira DC, de Lencastre H (2002): Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrobial Chemother, 46, 2155-61.
18. Oztop AY, Pinarbasi H, Kocagoz S, Bakici MZ, Bakir M (2004): Molecular genotyping of methicillin-resistant Staphylococcus aureus strains in a teaching hospital in Turkey. Microb Drug Resist, 10, 154–159.
19. Rohrer S, Bischoff M, Rossi J, Berger-Bachi B (2003): Mechanisms of methicillin resistance. In: Fluit, A.C., Schmitz, F.-J. (Eds.), MRSA. Current Perspectives. Caister Academic Press, Wymondham.
20. Ruppe´ E, Barbier F, Mesli Y et al. (2009): Diversity of staphylococcal cassette chromosome mec structures in methicillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus strains among outpatients from four countries. Antimicrob Agents Chemother, 53, 442–449.
21. Sghir A, Antonopoulos D, Mackie RI (1998): Design and evaluation of a lactobacillus group-specific ribosomal RNA-targeted hybridization probe and its application to the study of intestinal microecology in pigs. Syst Appl Microbiol, 21, 291-296.
22. Suau A, Bonnet R, Sutren M, Godon JJ, Gibson G, Collins MD, Dore´J (1999): Direct rDNA community analysis reveals a myriad of novel bacterial lineages within the human gut. Appl Environ Microbiol, 65, 4799-4807.
23. Türkyılmaz S, Tekbıyık S, Oryasin E, Bozdogan B (2010): Molecular epidemiology and antimicrobial resistance mechanisms of methicillin-resistant Staphylococcus aureus isolated from bovine milk. Zoonoses Public Health, 57, 197–203.
24. Türütoğlu H, Erçelik S, Öztürk D (2006): Antibiotic resistance of Staphylococcus aureus and coagulase negative staphylococci isolated from bovine mastitis. Bull Vet Inst Pulawy, 50, 41-45.
25. Ünal S (2006): Toplumda kazanılmış metisilin dirençli Staphylococcus aureus. Genetik özellikler. Ankem Derg, 20, 100–1.
26. Van Hoovels L, Vankeerberghen A, Boel A et al. (2006): First case of Staphylococcus pseudintermedius infection in a human. J Clin Microbiol, 44, 4609–4612.
27. Vangerhaeghan W, Cerpentier T, Adriaesen C, Vicca J, Hermans K, Butaye P (2010): Methicillin resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows. Vet Microbiol, doi: 10.1016/j.vet-mic.200912.044
28. Zhang K, McClure JA, Elsayed S (2005): Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette choromosome mec types I to V in methicillin resistant Staphylococcus aureus. J Clin Microbiol, 43, 5026-33.
29. Zhang Y, Agidi S, LeJeune JT (2009): Diversity of staphylococcal cassette chromosome in coagulasenegative staphylococci from animal sources. J Appl Microbiol, 107, 1375–1383.