Sinop ilindeki hamsi ve zargana balıklarından Vibrio spp. izolasyonu ve karakterizasyonu

Öz

Amaç: Bu çalışmada, Sinop İlindeki balıkçılardan temin edilen hamsi ve zargana balık örneklerinden izole edilen Vibrio türlerinin karekterizasyonu ile bu izolatların antibiyotik dirençlilikleri, plazmid DNA ve SDS-PAGE sodyum dodesil sülfat poliakrilamid jel elektroforezis toplam hücre protein profillerinin belirlenmesi amaçlanmıştır.Yöntem: Balık örnekleri taze olarak alınmış, buz üzerinde korunarak laboratuvara getirilmiş ve hemen analize alınmıştır. Balık örnekleri 25 g tartılarak zenginleştirme amacıyla %3 NaCl içeren steril alkali peptonlu su 225 mL besiyerine ilave edilmiş ve 37 OC’de 18-24 saat inkübasyona bırakılmıştır. İnkübasyon sonrası Tiyosülfat Sitrat Safra Sükroz TCBS agar besiyerinde gelişen sarı, yeşil veya mavi-yeşil koloniler Tripton Soya Agar TSA besiyerine ekilmiş ve izolatların daha sonraki tanımlaması için bir gece süreyle inkübe edilmiştir. İzolatlar morfolojik, fizyolojik ve biyokimyasal testlere göre tanımlanmıştır. Bunlara ek olarak izolatların yedi farklı antibiyotiğe karşı dirençliliği disk-difüzyon yöntemiyle belirlenmiştir. Ayrıca, izolatların SDSPAGE hücresel protein ve plazmid DNA profilleri de çıkartılmıştır. Bulgular: Toplam 44 adet balık örneği Vibrio türlerinin belirlenmesine yönelik çalışılmış ve morfolojik, biyokimyasal İnceleme sonucunda; 12 adet Vibrio spp. belirlenmiştir. özellikleri yönünden incelenmiştir. properties. At the end of the analysis; 12 Vibrio species were determined. The isolates were resistant against to ceftazidime 66.6%, gentamisine 25%, tetracycline 16.6%, ceftriaxone 91.6%, amikacine 16.6%, ofloxacine 25% and penicillin G 58.3%. In addition, The MARI Multiple Antibiotic Resistance Index values of tested strains were found at the rate of 0.41. The numerical analysis of SDS-PAGE whole-cell protein profiles of Vibrio spp. isolates revealed two major clusters at similarity levels of 80% and above. The 1-4 plasmide range 1590 - 27000 bp in all of the isolates were determined.Conclusion: As a result, it is possible to say that microbial infections can be prevented by adequate freezing and cooking, proper storage and processing after harvesting and avoidance of cross-contamination during fish handling. In addition, the coastal contamination due to bacteria leads to decline in the quality of seawater resources and so leads to risks to human health and economic losses. Furthermore, our study indicated that the presence of antibiotic resistant Vibrio spp. strains isolated from fishes might be potential risk for health

Anahtar Kelimeler

• Vibrio, balık, SDS-PAGE, plazmid, antibiyogram

Isolation and characterization of Vibrio spp. from anchovy and garfish in the Sinop province

Abstract

Objective: In this study it was aimed to characterize the Vibrio species isolated from anchovy and garfish samples obtained from fishermen and to determine the antibiotic resistances, plasmide DNA and SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis whole cell protein profiles of these isolates.Method: The fish samples were taken as fresh, kept in ice during transport to the laboratory and immediately taken for analysis. The fish samples weighed as 25 grams were added into 225 mL of sterile alkaline peptone water supplemented with 3% NaCl broth for enrichment purpose and incubated at 37 OC for 18 to 24 hours. After incubation, yellow, green or blue-green colonies growing on Thiosulphate Citrate Bile Salt Sucrose TCBS agar were subcultured on Triptone Soya Agar TSA and incubated overnight for further identification of the isolates. The isolates were identified according to morphological, physiological and biochemical tests. Moreover, the resistances against seven different antibiotics of isolates were determined by disk-diffusion method. In addition, the SDS-PAGE cell protein and plazmid DNA profiles of isolates were determined.Results: Totallly 44 fish samples were tested for determining Vibrio species and were analyzed according to their morphological, biochemical properties. At the end of the analysis; 12 Vibrio species were determined. The isolates were resistant against to ceftazidime 66.6%, gentamisine 25%, tetracycline 16.6%, ceftriaxone 91.6%, amikacine 16.6%, ofloxacine 25% and penicillin G 58.3%. In addition, The MARI Multiple Antibiotic Resistance Index values of tested strains were found at the rate of 0.41. The numerical analysis of SDS-PAGE whole-cell protein profiles of Vibrio spp. isolates revealed two major clusters at similarity levels of 80% and above. The 1-4 plasmide range 1590 - 27000 bp in all of the isolates were determined.Conclusion: As a result, it is possible to say that microbial infections can be prevented by adequate freezing and cooking, proper storage and processing after harvesting and avoidance of cross-contamination during fish handling. In addition, the coastal contamination due to bacteria leads to decline in the quality of seawater resources and so leads to risks to human health and economic losses. Furthermore, our study indicated that the presence of antibiotic resistant Vibrio spp. strains isolated from fishes might be potential risk for health

Keywords

• Vibrio, fish, SDS-PAGE, plasmide, antibiogram

References

1. Ustaçelebi Ş. Temel ve Klinik Mikrobiyoloji. Ankara: Güneş Kitabevi, 1999.
2. Uchiyama H. Distribution of Vibrio species isolated from aquatic environments with TCBS agar. Environ Health Preven Med, 2000; 4 (4): 199-204.
3. Boe B, Gjerde J. Fatty acid patterns in the classification of some representatives of the families Enterobacteriaceae and Vibrionaceae. J Gen Microbiol, 1980; 116 (1): 41-9.
4. Han F, Walker RD, Janes ME, Prinyawiwatkul W, Ge B. Antimicrobial susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus isolates from Louisiana Gulf and retail raw oysters. Appl Environ Microbiol, 2007; 73 (21): 7096-8.
5. Alavandi SV, Manoranjita V, Vijayan KK, Kalaimani N, Santiago TC. Phenotypic and molecular typing of V. harveyi isolates and their pathogenicity to tiger shrimp larvae. Let Appl Microbiol, 2006; 43 (5): 566–70.
6. Bej AK, Harold N, Vickery MCL, Brasher C, Jeffreys A, Jones DD, et al. Use of PCR to determine genomic diversity and distribution of siderophore- mediated iron acquisition genes in clinical and environmental isolates of V. vulnificus, abstr. 97th General Meeting of the American Society for Microbiology. American Society for Microbiology- Washington D.C., 1997.
7. Amorim A, Vasconcelos V. Dynamics of microcystins in the mussel Mytilus galloprovincialis. Toxicon, 1999; 37 (7): 1041-52.
8. Farmer III JJ, Hickman Brener FW. The Genera Vibrio and Photobacterium. In: Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E, eds. The Prokaryotes: A Handbook on the Biology of Bacteria. 3rd edn. New York: Springer, 2006: 508-64.

9. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disc method. Am J Clin Pathol, 1966; 45 (4): 493–6.
10. Anonymous. National Committee for Clinical Laboratory Standards. Approved Standard M100-S20 performance standards for antimicrobial susceptibility testing; twentieth informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.
11. Sarter S, Nguyen HNK, Hung LT, Lazard J, Montet D. Antibiotic resistance in Gram-negative bacteria isolated from farmed catfish. Food Control, 2007; 18 (11): 1391-6.
12. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970; 227: 680-5.
13. Sambrook J, Fritsch EF, Maniatis T. Molecular Clonning: a Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989.
14. Panicker G, Call DR, Krug MJ, Bej AK. Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays. Appl Environ Microbiol, 2004; 70 (12): 7436–44.
15. Amos K. Procedures for the detection and identification of certain fish pathogens. 3rd. edn. Bethesda, Maryland: American Fisheries Society, 1985.
16. Håstein T, Hjeltnes B, Lillehaug A, Skare JV, Berntssen M, Lundebye AK. Food safety hazards that occur during the production stage: challenges for fish farming and the fish industry. Rev Sci Tech off Int Epiz, 2006; 25: 607-25.
17. Morris JG. Cholera and other types of Vibriosis: a story of human pandemics and oysters on the half shell. Clin Infect Dis, 2003; 37 (2): 272-80.
18. Anonymous. Vibrio cholerae izolasyonu ve identifikasyonu standart laboratuvar prosedürleri. Ankara: Laboratuvarları Daire Başkanlığı, Ulusal Enterik Patojenler Referans Laboratuvarı, 2012. Referans
19. Korun J. Kültürü yapılan çipuralarda (Sparus aurata L.) görülen Listonella anguillarum enfeksiyonu üzerine bir çalışma. Ege JFAS, 2006; 23 (Ek 1/2): 259-63.
20. Aydın A, Soyutemiz E. Bazı balık türlerinden ve kum midyelerinden (Venus gallina) Vibrio parahaemolyticus izolasyonu ve identifikasyonu. Turk J Vet Anim Sci, 2002; 26: 1249-53.
21. İnal T, Yurtyeri A, Ambarcı I. Untersuchungen über das Vorkommen von Vibrio parahaemolyticus im Jahre 1971 in der Türkei. Fleischwirtschaft, 1973; 53(9): 1299.
22. İnal T, Yurteri A, Ambarcı I, Tolgay Z, Tezcan I. Untersuchungen über das vorkommen von Vibrio parahaemolyticus an der Schawarzmeerküste der Türkei. Alimenta, 1977; 16: 129-33.
23. Manjusha S, Sarita GB, Elyas KK, Chandrasekaran M. Multiple antibiotic resistances of Vibrio isolates from coastal and brackish water areas. Am J Biochem Biotech, 2005; 1(4): 201-6.
24. Rojas MVR, Matté MH, Dropa M, Silva ML, Matté GR. Characterization of Vibrio parahaemolyticus isolated from oysters and nussels in São Paulo, Brazil. Rev Inst Med Trop Sao Paulo, 2011; 53 (4): 201-5.
25. Marhual NP, Das BK, Samal SK. Characterization of Vibrio alginolyticus and Vibrio parahaemolyticus isolated from Penaeus monodon: antimicrobial resistance, amplification of polymorphic DNA analysis. Afr J Microbiol Res, 2012; 6 (20): 4270-6. and random
26. Yoon LC, Ryu J, Oh SS, Hwang IG. Detection and identification of Vibrio species using whole-cell protein pattern analysis. J Microbiol Biotech, 2012; 22 (8): 1107-12.
27. Benediktsdóttir E, Verdonck L, Spröer C, Helgason S, Swings J. Characterization of Vibrio viscosus and Vibrio wodanis isolated at different geographical locations: a proposal for reclassification of Vibrio viscosus as Moritella viscosa comb. nov. Int J Syst Evol Microbiol, 2000; 50 (2): 479-88.