Molecular detection of exfoliative toxin in Staphylococcus intermedius isolates from dogs with pyoderma

Abstract

Staphylococcus aureus and S. intermedius are considered as the most significant species causing skin infections in dogs. The aim of this study was to develop a Polymerase Chain Reaction technique for the detection of siet (S. intermedius exfoliative toxin) gene encoding exfoliative toxin in S. intermedius and to investigate its presence in S. intermedius isolates from dogs with pyoderma. A total of 41 isolates (35 S. intermedius, 4 S. aureus, one S. capitis subsp. ureolytica and one S. chromogenes) from dogs with pyoderma were included in the study. Original primers specifically amplifying 145 bp of siet gene and 182 bp of agr (accessory gene regulator) gene locus of S. intermedius were designed in the study. agr gene was detected in all S. intermedius isolates, but not in other isolates. siet gene was detected in all S. intermedius isolates. siet Polymerase Chain Reaction assay was found to be specific since no amplifications were observed with siet negative S. intermedius and other bacterial control strains. Rapid and reliable detection of staphylococci causing skin lesions in dogs and their virulence markers like siet gene will provide important data for clinical practice to manage the disease more effectively by means of treatment and prevention

Keywords

• Canine pyoderma
• exfoliative toxin
• siet gene
• Staphylococcus intermedius

Piyodermalı köpeklerden izole edilen Staphylococcus intermedius’larda eksfoliatif toksin varlığınınmoleküler olarak belirlenmesi

Abstract

Staphylococcus aureus ve S. intermedius’un köpeklerde deri enfeksiyonlarına neden olan en önemli türler olduğu kabul edilmektedir. Bu çalışmada, S. intermedius’un eksfoliatif toksinini kodlayan siet (S. intermedius eksfoliatif toksin) genini belirlemek için bir Polimeraz Zincir Reaksiyonu (PCR) tekniği geliştirilmesi ve piyodermalı köpeklerden izole edilen S. intermedius izolatlarında bu genin araştırılması amaçlandı. Piyodermalı köpeklerden izole edilen toplam 41 izolat (35 S. intermedius, 4 S. aureus, bir S. capitis subsp. ureolytica ve bir S. chromogenes) çalışmaya dahil edildi. Bu çalışmada, siet geninin 145 bp’lik ve S. intermedius’un agr (accessory gene regulator) geninin 182 bp’lik bölümünü spesifik olarak çoğaltan özgün primerler tasarlandı. agr geni diğer stafilokok türlerinde değil sadece S. intermedius izolatlarında tespit edildi. Bütün S. intermedius’larda siet geni saptandı. Diğer kontrol suşlarında ve siet negatif S. intermedius suşunda amplifikasyon görülmediği için siet PCR yöntemi spesifik bulundu. Köpeklerde deri lezyonu oluşturan stafilokokların ve bu stafilokokların virulens özelliklerinden siet geninin hızlı ve güvenilir olarak belirlenmesi, tedavi ve korunma anlamında hastalıkla daha etkili mücadele için klinik olarak önemli bilgiler sunacaktır

Keywords

• Eksfoliatif toksin
• köpek
• piyoderma
• siet geni
• Staphylococcus intermedius

References

1. Ahrens P, Andresen LO (2004): Cloning and sequence analysis of genes encoding Staphylococcus hyicus exfoliative toxin types A, B, C, and D. J Bacteriol, 186, 1833-37.
2. Andresen LO (2005): Production of exfoliative toxin by isolates of Staphylococcus hyicus from different countries. Vet Rec, 157, 376-378.
3. Andresen LO, Ahrens P, Daugaard L, Bille-Hansen V (2005): Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes. Vet Rec, 105, 291-300.
4. Ardic N, Ozyurt M, Sareyyupoglu B, Haznedaroglu T (2005): Investigation of erythromycin and tetracycline resistance genes in methicillin-resistant staphylococci. Int J Antimicrob Agents, 26, 213-218.
5. Baron F, Cochet M, Pellerini JL, Zakour NB, Lebon A, Navarro A, Proudy I, Le Loir Y, Gaultier M (2004): Development of a PCR test to differentiate between Staphylococcus aureus and Staphylococcus intermedius. J Food Prot, 67, 2302-05.
6. Becker K, Roth R, Peters G (1998): Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene. J Clin Microbiol, 36, 2548-53.
7. Bes M, Slim LS, Becharnia F, Meugnier H, Vandenesch F, Etienne J, Freney J (2002): Population diversity of Staphylococcus intermedius isolates from various host species: typing by 16S-23S intergenic ribosomal DNA spacer polymorphism analysis. J Clin Microbiol, 40, 2275- 77.
8. Biberstein EL, Jang SS, Hirsh DC (1984): Species distribution of coagulase-positive staphylococci in animals. J Clin Microbiol, 19, 610-615.

9. Devriese LA, Vancanneyt M, Baele M, Vaneechoutte M, De Graef E, Snauwaert C, Cleenwerck I, Dawyndt P, Swings J, Decostere A, Haesebrouck F (2005): Staphylococcus pseudintermedius sp. nov., a coagulase- positive species from animals. Int J Syst Evol Microbiol, 55, 1569-73.
10. Futagawa-Saito K, Sugiyama T, Karube S, Sakurai N, Ba-Thein W, Fukuyasu T (2004a): Prevalence and characterization of leukotoxin-producing Staphylococcus intermedius isolates from dogs and pigeons. J ClinMicrobiol, 42, 5324-26.
11. Futagawa-Saito K, Suzuki M, Ohsawa M, Ohshima S, Sakurai N, Ba-Thein W, Fukuyasu T (2004b): Identification and prevalence of an enterotoxin-related gene, se-int, in Staphylococcus intermedius isolates from dogs and pigeons. J Appl Microbiol, 96, 1361-66.
12. Hajek V (1976): Staphylococcus intermedius, a new species isolated from animals. Int J Syst Bacteriol, 26, 401-408.
13. Hajek V, Balusek J, Horak V, Koukalova D (1991): Characterization of coagulase-positive staphylococci isolated from free living birds. J Hyg Epidemiol Microbiol Immunol, 35, 407-418.
14. Hartmann FA, White DG, West SEH, Walker RD, De Boer DJ (2005): Molecular characterization of Staphylococcus intermedius carriage by healthy dogs and comparison of antimicrobial susceptibility patterns to isolates from dogs with pyoderma. Vet Microbiol, 108, 119-131.
15. Hauschild T, Wojcik A (2007): Species distribution and properties of staphylococci from canine dermatitis. Res Vet Sci, 82, 1-6.
16. Ihrke PJ (1987): An overview of bacterial skin disease in the dog. Br Vet J, 143, 112-118.
17. Kanbar T, Voytenko AV, Alber J, Lammler C, Weiss R, Zschöck M, Shilov IA, Dmitrenko OA, Gintsburg AL (2006): Prevalence of genes encoding exfoliative toxins among Staphylococcus hyicus isolated in Russia and Germany. J Vet Med B Infect Dis Vet Public Health, 53, 429-433.
18. Kloos WE (1980): Natural population of the genus Staphylococcus. Annu Rev Microbiol, 34, 559-592.
19. Lautz S, Kanbar T, Alber J, Lammler C, Weiss R, Prenger-Berninghoff E, Zschöck M (2006): Dissemination of the gene encoding exfoliative toxin of Staphylococcus intermedius among strains isolated from dogs during routine microbiological diagnostics. J Vet Med B Infect Dis Vet Public Health, 53, 434-438.
20. Medleau L, Long RE, Brown J, Miller WH (1986): Frequency and antimicrobial susceptibility of Staphylococcus species isolated from canine pyodermas. Am J Vet Res, 47, 229-231.
21. Prevost G, Couppie P, Monteil H (2003): Staphylococcal epidermolysins. Curr Opin Infect Dis, 16, 71-76.
22. Raus J, Love DN (1983): Characterization of coagulase- positive Staphylococcus intermedius and Staphylococcus aureus isolated from veterinary clinical specimens. J Clin Microbiol, 18, 789-792.
23. Robertson J, Fox L, Hancock D, Gay J, Besser T (1996): Prevalence of coagulase-positive staphylococci, other than Staphylococcus aureus, in bovine mastitis. Am J Vet Res, 57, 54-58.
24. Sambrook J, Fritsch EF, Maniatis T (1989): Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, New York.
25. Sato H, Matsumori Y, Tanabe T, Saito H, Shimizu A, Kawano J (1994): A new type of staphylococcal exfoliative toxin from a Staphylococcus aureus strain isolated from a horse with plegmon. Infect Immun, 62, 3780-85.
26. Sato H, Watanabe K, Higuchi K, Teruya K, Ohtake A, Murata Y, Saito H, Aizawa C, Danbara H, Maehara N (2000): Chromosomal and extrachromosomal synthesis of exfoliative toxin from Staphylococcus hyicus. J Bacteriol, 182, 4069-4100.
27. Strommenger B, Kettlitz C, Werner G, Witte W (2003): Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol, 41, 4089-94.
28. Talan DA, Staatz D, Staatz A, Goldstein E, Singer K, Overturf G (1989): Stapyhlococcus intermedius in canine gingiva and canine-inflicted wound infections: a newly recognized zoonotic pathogen. J Clin Microbiol, 27, 78-81.
29. Terauchi R, Sato H, Hasegawa T, Yamaguchi T, Aizawa C, Maehara N (2003a): Isolation of exfoliative toxin from Staphylococcus intermedius and its local toxicity in dogs. J Vet Microbiol, 94: 19-29.
30. Terauchi R, Sato H, Endo Y, Aizawa C, Maehara N (2003b): Cloning of the gene coding for Staphylococcus intermedius exfoliative toxin and its expression in Escherichia coli. Vet Microbiol, 94, 31-38.
31. Vandenesch F, Celard M, Arpin D, Bes M, Greenland T, Etienne J (1995): Catheter-related bacteremia associated with coagulase-positive Staphylococcus intermedius. J Clin Microbiol, 33, 2508-10.
32. Vannuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauters G, Gala JL (1995): Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. J Clin Microbiol, 33, 2864-67.
33. White SD, Brown AE, Chapman PL, Jang SS, Ihrke PJ (2005): Evaluation of aerobic bacteriologic culture of epidermal collarette specimens in dogs with superficial pyoderma. J Am Vet Med Assoc, 226, 904-908.
34. Yamaguchi T, Nishifuji K, Sasaki M, Fudaba Y, Aepfelbacher M, Takata T, Ohara M, Komatsuzawa H, Amagai M, Sugai M (2002): Identification of the Staphylococcus aureus ETD pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B. Infect Immun, 70, 5835-45.
35. Yamashita K, Shimizu S, Kawano J, Uchida E, Haruna A, Igimi S (2005): Isolation and characterization of Staphylococci from external auditory meatus of dogs with or without otitis externa with special reference to Staphylococcus schleiferi subsp. coagulans isolates. J Vet Med Sci, 67, 263-268.
36. Yoo JH, Yoon JW, Lee SY, Park HM (2010): High prevalence of fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius isolates from canine pyoderma and otitis externa in veterinary teaching hospital. J Microbiol Biotechnol, 20, 798-802.
37. Youn J, Yoon JW, Koo HC, Lim S, Park YH (2011): Prevalence and antimicrogram of Staphylococcus intermedius group isolates from veterinary staff, companion animals, and the environment veterinary hospitals in Korea. J Vet Diagn Invest, 23, 268-274.