Soner ALTUN, ERTAN EMEK ONUK, ALPER ÇİFTCİ, Ayşe Gül BÜYÜKEKİZ, MUHAMMED DUMAN

Phenotypic, genotypic characterisation and antimicrobial susceptibility determination of lactococcus garvieae strains

Araştırmada, gökkuşağı alabalığı işletmelerinden izole edilmiş olan 10 adet Lactococcus garvieae suşunun 3 adet referans suşla (İspanya, İngiltere ve ATCC 43921) karşılaştırmalı olarak fenotipik ve genotipik özellikleri incelenmiştir. L. garvieae suşlarının fenotipik özelliklerinin belirlenmesinde konvansiyonel mikrobiyolojik ve Rapid 32 STREP testleri kullanılmıştır. Rapid 32 STREP sistemde suşlar arasında β-Glucuronidase, D-ribose, sorbitol, lactose, raffinose, alanyl-phenylalanyl-proline arylamidase, pyrrolidonyl arylamidase, hippurate hydrolysis, urease testleri yönüyle farklıklar olmasına rağmen API Webde tüm suşlar L. garvieae olarak doğrulanmıştır. ERIC2 primerinin kullanıldığı RAPD PCR analizinde L. garveae izolatları %70 benzerlik katsayısına göre 3 farklı genotipe ayrılmış ve Türkiye kökenli izolatların büyük bir bölümü (8 izolat) predominant tip olan LG1 genotipine dahil olduğu belirlenmiştir. Ayrıca bu çalışmada L. garvieae suşlarının antibiyotik duyarlılık testlerinde ülkemizde balıklarda sıklıkla kullanılan gentamisin, neomycin, lincomycin, sulphamethoxazoletrimetoprim, oksitetrasiklin, eritromycin, amoxycillin, florfenikol ve doksisikline karşı direnç geliştirmiş oldukları belirlenmiştir.

Lactococcus garvieae suşlarının fenotipik, genotipik karakterizasyonu ve antimikrobiyal duyarlılıklarının belirlenmesi

In this study, phenotypic and genotypic features of 10 L. garvieae strains isolated from rainbow trout farms were examined with 3 reference strains (Spain, England and ATCC 43921) comperativly. Rapid 32 STREP and conventional microbiologic tests were used for determining phenotipic features of L. garvieae strains. Altough there are diff erences, in Rapid 32 STREP system, between strains in terms of β-Glucuronidase, D-ribose, sorbitol, lactose, raffinose, alanyl-phenylalanyl-proline arylamidase, pyrrolidonyl arylamidase, hippurate hydrolysis, urease tests, all strains have been confirmed as L. garvieae by API web. In RAPD PCR analysis, in which ERIC 2 primer is used, L. garvieae isolates were genotyped within 3 separate clusters according to similarity coefficient index of 70%, and it was detected that a vast majority of isolates with Turkey-origin (8 isolates) belongs to predominant type LG1 genotype. In addition to this, antimicrobial tests of L. garvieae strains shows that there are resistance against gentamycin, neomycin, lincomycin, sulfamethoxazole-trimethoprim, oxytetracycline, erythromycin, amoxicillin, florfenicol and doxycycline, which are frequently used on fish in our country.

PDF

___

1. Vendrell D, JL Balcazar, Ruiz-Zarzuela I, de Blas I, Girones O, Muzquiz JL: Lactococcus garvieae in fish: A review. Comp Immun Microbiol Infect Dis, 29, 177-198, 2006.
2. Kusuda K, Kawai K, Salati F, Banner CR, Fryer JL: Enterococcus seriolicida sp. nov., a fish pathogen. Int J Syst Bacteriol, 41, 406-409, 1991.
3. Algöet M, Bayley AE, Roberts EG, Feist SW, Wheeler RW, Verner- Jeffreys DW: Susceptibility of selected freshwater fish species to a UK Lactococcus garvieae isolate. J Fish Dis, 32, 825-834, 2009.
4. Evans JJ, Klesius PH, Shoemaker CA: First isolation and characterization of Lactococcus garvieae from Brazilian Nile tilapia, Oreochromis niloticus (L.), and pintado, Pseudoplathystoma corruscans (Spix & Agassiz). J Fish Dis, 32, 943-951, 2009.
5. Eyngor M, Zlotkin A, Ghittino C, Prearo M, Douet DG, Chilmonczyk S, Eldar A: Clonality and diversity of the fish pathogen Lactococcus garvieae in Mediterranean countries. Appl Environ Microbiol, 70 (9): 5132-5137, 2004.
6. Foschino R, Nucera D, Volponi G, Picozzi C, Ortoffi M, Bottero MT: Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing. J Applied Microbiol, 105 (3): 652-662, 2008.
7. Tejedor JL, Vela AI, Gibello A, Casamayor A, Domınguez L, Fernandez- Garayzabal JF: A genetic comparison of pig, cow and trout isolates of Lactococcus garvieae by PFGE analysis. Lett Appl Microbiol, 53, 614-619, 2011.
8. Eldar A, Ghittino C: Lactococcus garvieae and Streptococcus iniae infections in rainbow trout (Onchorhynchus mykiss): Similar, but diff erent diseases. Dis. Aquat Org, 36, 227-231, 1999.
9. Eldar A, Ghittino C, Asanta L, Bozzetta E, Goria M, Prearo M, Bercovier H: Enterococcus seriolicida is a junior synonym of Lactococcus garvieae, a causative agent of septicemia and meningoencephalitis in fish. Curr Microbiol, 32, 85-88, 1996.
10. Ravelo C, Magarinos B, Lopez-Ramade S, Toranzo AE, Romalde JL: Molecular fingerprinting of fish-pathogenic Lactococcus garvieae strains by random amplified polymorphic DNA analysis. J Clin Microbiol, 41 (2): 751-756, 2003.
11. Altun S, A Kubilay A, Ekici S, Didinen BI, Diler O: Oral Vaccination against Lactococcosis in Rainbow trout (Oncorhynchus mykiss) using sodium alginate and poly(lactide-co-glycolide) carrier. Kafkas Univ Vet Fak Derg, 16 (Suppl-B): S211-S217, 2010.
12. Diler Ö, Altun S, Adiloğlu AK, Kubilay A, Işıklı B: First Occurance of Streptococcosis aff ecting farmed rainbow trout (Oncorhynchus mykiss) in Turkey. Bull Eur Ass Fish Pathol, 22 (1): 21-26, 2002.
13. Zlotkin A, Hershko H, Eldar A: Possible transmission of Streptococcus iniae from wild fish to cultured marine fish. Appl Environ Microbiol, 64, 4065- 4067, 1998.
14. Ravelo C, Magarinos B, Romalde JL, Toranzo AE: Conventional versus miniaturized systems for the phenotypic characterization of Lactococcus garvieae strains. Bull Eur Ass Fish Pathol, 21, 136-144, 2001.
15. Eldar A, Goria M, Ghittino C, Zlotkin A, Bercovier H: Biodiversity of Lactococcus garvieae strains isolated from fish in Europe, Asia and Australia. Appl Environ Microbiol, 65 (3): 1005-1008, 1999.
16. Vela AI, Vazquez J, Gibello A, Blanco MM, Moreno MA, Liebana P, Albendea C, Alcala, A. Mendez B, Domınguez L, Fernandez-Garayzabal JF: Phenotypic and genetic characterization of Lactococcus garvieae isolated in Spain from lactococcosis outbreaks in comparison with isolates of other countries and sources. J Clin Microbiol, 38, 3791-3795, 2000.
17. Whitman KA: Finfish and Shellfish Bacteriology Manual: Techniques and Procedures. pp. 107-155, Iowa State Press. Ames, Iowa, 2004.
18. Austin B, Austin DA: Bacterial Fish Pathogens Disease in Farmed and Wild Fish. 4th (Revised) ed., p. 552, Praxis, Publishing, Chichester, UK, 2008.
19. Versalovic J, Koeuth T, Lupski R: Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res, 19 (24): 6823-6831, 1991.
20. Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement: M100-S20 & M100-S-20-U, 2010.
21. Altun S, Diler Ö, Adiloğlu AK: Genotyping of Lactococcus garvieae strains from Rainbow trout (Oncorhynchus mykiss) by 16S rDNA sequencing. Bull Eur Ass Fish Pathol, 24, 119-125, 2004.
22. Teixeira LM, Merquior VLC, Vianni MCE, Carvalho MGS, Fracalanzza SEL, Steigerwalt AG, Brenner DJ, Facklam RR: Phenotypic and genotypic characterization of atypical Lactococcus garvieae strains isolated from water buff alos with subclinical mastitis and confirmation of L. garvieae as a senior subjective synonym of Enterococcus seriolicida. Int J Syst Bacteriol, 46, 664-668, 1996.
23. Valdebenito S, Avendano-Herrera R: Phenotypic, serological and genetic characterization of Flavobacterium psychrophilum strains isolated from salmonids in Chile. J Fish Dis, 32 (4): 321-333, 2009.
24. Onuk EE, Çiftçi A, Fındık A, Çiftçi G, Altun S, Balta F, Özer S, Çoban AY: Phenotypic and molecular characterization of Yersinia ruckeri isolates from Rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) in Turkey. Berl Munch Tierarztl Wochenschr, 124 (7-8): 320-328, 2011.
25. Castro N, Toranzo AE, Bastardo A, Barja JL, Magarinos B: Intraspecific genetic variability of Edwardsiella tarda strains from cultured turbot. Dis Aquat Org, 95 (3): 253-258, 2011.
26. Labella A, Manchado M, Alonso MC, Castro D, Romalde JL, Borrego JJ: Molecular intraspecific characterization of Photobacterium damselae ssp. damselae strains affecting cultured marine fish. J Appl Microbiol, 108 (6): 2122-2132, 2010.
27. Balboa S, Ferguson HW, Romalde JL: Phenotypic, serological and genetic characterization of Pseudomonas anguilliseptica strains isolated from cod, Gadus morhua L., in northern Europe. J Fish Dis, 30 (11): 657- 664, 2007.
28. Magarinos B, Toranzo AE, Barja JL, Romalde JL: Existence of two geographically-linked clonal lineages in the bacterial fish pathogen Photobacterium damselae subsp. piscicida evidenced by random amplified polymorphic DNA analysis. Epidemiol Infect, 125, 213-219, 2000.
29. Fındık A, İça T, Onuk EE, Perçin D, Kevenk TO, Çiftçi A: Molecular typing and cdt gene prevalence of Campylobacter jejuni isolates from various sources. Trop Anim Health Prod, 43, 711-719, 2011.
30. Welsh J, McClelland M: Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res, 18, 7213-7218, 1990.
31. Colorni A, Ravelo C, Romalde JL, Toranzo AE, Diamant A: Lactococcus garvieae in wild Red Sea wrasse Coris aygula (Labridae). Dis Aquat Org, 56, 275-278, 2003.
32. Kawanishi M, Kojima A, Ishihara K, Esaki H, Kijima M, Takahashi T, Suzuki S, Tamura Y: Drug resistance and pulsed-field gel electrophoresis patterns of Lactococcus garvieae isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan. Lett Appl Microbiol, 40, 322-328, 2005.
33. Kubilay A, Altun S, Uluköy G, Diler Ö: Lactococus garvieae suşlarının antimikrobiyal duyarlılıklarının belirlenmesi. S.D.Ü. Eğirdir Su Ürün Fak Derg, 1 (1): 39-48, 2005.