Yersinia ruckeri’ye Karşı Bakteriyofaj İzolasyonu ve Konak Aralığının Belirlenmesi

Abstract

Yersinia ruckeri'nin neden olduğu enterik kızıl ağız hastalığı, balıklarda önemli ekonomik kayıplara neden olan bakteriyel hastalıklardan biridir. Bakteriyel balık hastalıklarının tedavisinde antibiyotikler kullanılmaktadır. Ancak son yıllarda antibiyotik direncinin artması, alternatif tedavilerin geliştirilmesini zorunlu kılmıştır. Bakteriyofaj tedavisi, antibiyotiklere dirençli bakteriyel enfeksiyonların tedavisinde, antibiyotiklere karşı umut vadeden bir alternatif olarak giderek daha fazla önem kazanmaktadır. Bu çalışmanın amacı, Türkiye ve dünyanın birçok bölgesinde yaygın olarak görülen bir hastalık olan yersiniosise karşı faj izole etmek ve izole edilen fajın konak aralığını belirlemektir. Bu çalışmada, belediye atık sularından bakteriyofaj izolasyonu için zenginleştirme ve çift katmanlı agar yöntemleri kullanılmıştır. Belediye atık sularından bir bakteriyofaj izole edilmiştir. İzole edilen faja, konak genusu ve suşuna göre YP4M adı verilmiştir. Bu fajın konak aralığı, spot test kullanılarak belirlenmiştir. Fajların konakçı aralığını belirlemek için 18 Y. ruckeri izolatı, Lactococcus garvieae (ATCC), Aeromonas hydrophila (ATCC) ve Vibrio anguillarum (ATCC) suşları ile birlikte kullanıldı. YP4M, 18 Y. ruckeri izolatından 3, 4, 8, 9, 13, 14, 15 ve 17 numaralı bakterilere karşı litik aktivite gösterirken, Lactococcus garvieae (ATCC), Aeromonas hydrophila (ATCC) ve Vibrio anguillarum (ATCC) suşlarına karşı litik aktivite göstermedi. Fajların karakterizasyonunun ve faj kokteyllerinin hazırlanmasının, su ürünleri yetiştiriciliğinde önemli bir hastalık olan yersiniosisin kontrolüne önemli katkı sağlayacağı düşünülmektedir.

Keywords

• Bakteriyofaj
• Yersinia ruckeri
• Yersiniosis
• Antibiyotik Direnci
• Faj Terapisi

Isolation of Bacteriophage Against Yersinia ruckeri and Determination of Host Range

Abstract

Enteric red mouth disease caused by Yersinia ruckeri is one of the bacterial diseases that cause significant economic losses in fish. Antibiotics are used in the treatment of bacterial fish diseases. However, the rise in antibiotic resistance in recent years has necessitated the development of alternative therapies. Bacteriophage therapy is gaining importance as a promising alternative to antibiotics in the treatment of antibioticresistant bacterial infections. The aim of this study was to isolate and characterize bacteriophages against local Yersinia ruckeri isolates obtained from rainbow trout farms in Türkiye, and to determine the host range of the isolated phage using the spot assay method. In this study, enrichment and double-layer agar methods were used to isolate bacteriophage from municipal wastewater. One bacteriophage was isolated from municipal wastewater. The isolated phage was named YP4M based on the host species and strain. The lytic activity of phage YP4M was determined by spot assay against 18 Y. ruckeri isolates, as well as Lactococcus garvieae ATCC 49156, Aeromonas hydrophila ATCC 7966 and Vibrio anguillarum ATCC 43305. YP4M exhibited lytic activity against bacteria numbered 3, 4, 8, 9, 13, 14, 15, and 17 from the 18 Y. ruckeri isolates, but did not exhibit lytic activity against Lactococcus garvieae ATCC 49156, Aeromonas hydrophila ATCC 7966 and Vibrio anguillarum ATCC 43305 strains. It is believed that the characterization of phages and the preparation of phage cocktails will significantly contribute to the control of yersiniosis, an important disease in aquaculture.

Keywords

• Antibiotic Resistance
• Bacteriophage
• Phage Therapy
• Yersinia ruckeri
• Yersiniosis.

References

1. Adriaenssens, E. M., Brister, J. R. (2017). How to name and classify your phage: An informal guide. Viruses, 9(4), 70. https://doi.org/10.3390/v9040070 Ahmadpour, S., Mardani, K., Tukmechi, A. (2016). Culture and molecular characterization of phages isolated from rainbow trout farms and sewage treatment plants and investigation of their effects on Yersinia ruckeri. Iranian Journal of Fisheries Sciences, 15(1), 267–280.
2. Altinok, I., Ture, M., Ustaoglu, D., Cebeci, A., Öztürk, R. Ç., Aygür, E., & Kaygusuz, Ö. (2025). Isolation and characterization of novel Yersinia ruckeri bacteriophages for potential use in aquaculture. Aquaculture, 612(2), 743219. https://doi.org/10.1016/j.aquaculture.2025.743219
3. Austin, B., Austin, D. A. (2016). Bacterial fish pathogens: Disease of farmed and wild fish (6th ed.). Springer International Publishing. https://doi.org/10.1007/978-3-319-32674-0
4. Aydın, İ., Öztürk, R. Ç., Eroldoğan, O. T., Arslan, M., Terzi, Y., Yılmaz, S., & Sevgili, H. (2025). An in-depth analysis of the finfish aquaculture in Türkiye: Current status, challenges, and future prospects. Reviews in Aquaculture, 17(2), e70010. https://doi.org/10.1111/raq.70010
5. Aydoğan, D. Y., Hadımlı, H. H. (2016). Bakteriyofaj tedavisi [Bacteriophage therapy]. Journal of Etlik Veterinary Microbiology, 27(1), 38–47. https://doi.org/10.35864/evmd.514172
6. Balta, F., Dengiz Balta, Z., Özgümüş, O. B., Çağırgan, H. (2016). Doğu Karadeniz Bölgesi’ndeki gökkuşağı alabalığı (Oncorhynchus mykiss) çiftliklerinde Yersinia ruckeri’nin portörlük yönünden tetkiki ve antimikrobiyal direncin tespiti [Investigation of Yersinia ruckeri in terms of carrier state and determination of antimicrobial resistance in rainbow trout (Oncorhynchus mykiss) farms in the Eastern Black Sea Region]. Journal of Anatolian Environmental and Animal Sciences, 1(3), 72–76. https://doi.org/10.35229/jaes.280741
7. Balta, F., Sandalli, C., Kayis, S., Ozgumus, O. B. (2010). Molecular analysis of antimicrobial resistance in Yersinia ruckeri strains isolated from rainbow trout (Oncorhynchus mykiss) grown in commercial fish farms in Turkey. Bulletin of the European Association of Fish Pathologists, 30(6), 211–219.
8. Bullock, G. L. (1984). Enteric redmouth disease of salmonids (Fish Disease Leaflet 67). U.S. Department of the Interior, Fish and Wildlife Service, Division of Fishery Research. Capkin, E., Ozdemir, S., Öztürk, R. Ç., Altinok, I. (2017). Determination and transferability of plasmid-mediated antibiotic resistance genes of the bacteria isolated from rainbow trout. Aquaculture Research, 48(11), 5561–5575. https://doi.org/10.1111/are.13378

9. Chanishvili, N. (2012). Phage therapy—History from Twort and d'Herelle through Soviet experience to current approaches. In M. Łobocka & W. Szybalski (Eds.), Advances in virus research (Vol. 83, pp. 3–40). Academic Press. https://doi.org/10.1016/B978-0-12-394438-2.00001-3
10. Dinçtürk, E., Tanrıkul, T. T. (2021). Yersinia ruckeri and Pseudomonas fluorescens co-infection in rainbow trout (Oncorhynchus mykiss Walbaum, 1792). Aquaculture Research, 52(10), 4858–4866.https://doi.org/10.1111/are.15320
11. Dkhili, S., Ribeiro, M., Slama, K. B. (2025). A century of bacteriophages: Insights, applications, and current utilization. Antibiotics, 14(11), 1080. https://doi.org/10.3390/antibiotics14111080
12. Donati, V. L., Dalsgaard, I., Sundell, K., Castillo, D., Er-Rafik, M., Clark, J., & Middelboe, M. (2021). Phage-mediated control of Flavobacterium psychrophilum in aquaculture: In vivo experiments to compare delivery methods. Frontiers in Microbiology, 12, Article 628309. https://doi.org/10.3389/fmicb.2021.628309
13. Duman, M., Altun, S., Cengiz, M., Saticioglu, I. B., Buyukekiz, A. G., & Sahintur, P. (2017). Genotyping and antimicrobial resistance genes of Yersinia ruckeri isolates from rainbow trout farms. Diseases of Aquatic Organisms, 125(1), 31–44. https://doi.org/10.3354/dao03132
14. Ghasemi, S. M., Bouzari, M., Emtiazi, G. (2014). Preliminary characterization of Lactococcus garvieae bacteriophage isolated from wastewater as a potential agent for biological control of lactococcosis in aquaculture. Aquaculture International, 22(4), 1469–1480. https://doi.org/10.1007/s10499-014-9760-z
15. Gündoğdu, A., Ulu-Kılıç, A. (2018). Bakteriyofaj terapisi: Unutulmuş bir şifa kaynağı [Bacteriophage therapy: A forgotten source of healing]. Klimik Journal, 31(2), 78–87.
16. Hunter, V., Knittel, M. D., Fryer, J. L. (1980). Stress-induced transmission of Yersinia ruckeri infection from carriers to recipient steelhead trout Salmo gairdneri Richardson. Journal of Fish Diseases, 3(6), 467–472. https://doi.org/10.1111/j.1365-2761.1980.tb00433.x
17. Kumar, G., Menanteau-Ledouble, S., Saleh, M., El-Matbouli, M. (2015). Yersinia ruckeri, the causative agent of enteric redmouth disease in fish. Veterinary Research, 46(1), 103. https://doi.org/10.1186/s13567-015-0238-4
18. Kunttu, H. M., Runtuvuori-Salmela, A., Middelboe, M., Clark, J., Sundberg, L. R. (2021). Comparison of delivery methods in phage therapy against Flavobacterium columnare infections in rainbow trout. Antibiotics, 10(8), 914. https://doi.org/10.3390/antibiotics10080914
19. Kutateladze, M., Adamia, R. (2010). Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends in Biotechnology, 28(12), 591–595. https://doi.org/10.1016/j.tibtech.2010.08.001
20. Lin, D. M., Koskella, B., Lin, H. C. (2017). Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World Journal of Gastrointestinal Pharmacology and Therapeutics, 8(3), 162–173. https://doi.org/10.4292/wjgpt.v8.i3.162
21. Liu, R., Han, G., Li, Z., Cun, S., Hao, B., Zhang, J., & Liu, X. (2022). Bacteriophage therapy in aquaculture: Current status and future challenges. Folia Microbiologica, 67(4), 573–590. https://doi.org/10.1007/s12223-022-00965-6
22. Lu, Z., Breidt, F., Jr., Fleming, H. P., Altermann, E., &Klaenhammer, T. R. (2003). Isolation and characterization of a Lactobacillus plantarum bacteriophage, ΦJL-1, from a cucumber fermentation. International Journal of Food Microbiology, 84(2), 225–235. https://doi.org/10.1016/S0168-1605(03)00111-9
23. Nikolich, M. P., Filippov, A. A. (2020). Bacteriophage therapy: Developments and directions. Antibiotics, 9(3), 135. https://doi.org/10.3390/antibiotics9030135
24. Ohtani, M., Villumsen, K. R., Strøm, H. K., Lauritsen, A. H., Aalbæk, B., Dalsgaard, I., Nowak, B., & Bojesen, A. M. (2019). Effects of fish size and route of infection on virulence of a Danish Yersinia ruckeri O1 biotype 2 strain in rainbow trout (Oncorhynchus mykiss). Aquaculture, 503, 519–526. https://doi.org/10.1016/j.aquaculture.2019.01.041
25. Onuk, E., Didinen, B., Çiftci, A., Yardımcı, B., Pekmezci, G. (2019). Molecular characterisation of antibiotic resistance in Yersinia ruckeri isolates from Turkey. Bulletin of the European Association of Fish Pathologists, 39(4), 145–155.
26. Pallavi, B., Puneeth, T. G., Shekar, M., Girisha, S. K. (2021). Isolation, characterization and genomic analysis of vB-AhyM-AP1, a lytic bacteriophage infecting Aeromonas hydrophila. Journal of Applied Microbiology, 131(2), 695–705. https://doi.org/10.1111/jam.14997
27. Reza, M. S., Alam, M. M., Khan, M. F. R., Rahman, M. (2024). Phage therapy to combat antibiotic resistance in aquaculture. Journal of Aquaculture & Marine Biology, 13(3), 104–106. https://doi.org/10.15406/jamb.2024.13.00403
28. Rodgers, C. J. (2001). Resistance of Yersinia ruckeri to antimicrobial agents in vitro. Aquaculture, 196(3–4), 325–345. https://doi.org/10.1016/S0044-8486(01)00546-4
29. Sambrook, J., Russell, D. W. (2001). Molecular cloning: A laboratory manual (3rd ed.). Cold Spring Harbor Laboratory Press.
30. Schar, D., Klein, E. Y., Laxminarayan, R., Gilbert, M., Van Boeckel, T. P. (2020). Global trends in antimicrobial use in aquaculture. Scientific Reports, 10(1), 21878. https://doi.org/10.1038/s41598-020-78849-3
31. Silva, Y. J., Costa, L., Pereira, C., Mateus, C., Cunha, A., Calado, R., & Almeida, A. (2014). Phage therapy as an approach to prevent Vibrio anguillarum infections in fish larvae production. PLOS ONE, 9(12), e114197. https://doi.org/10.1371/journal.pone.0114197
32. Sulakvelidze, A., Alavidze, Z., Morris, J. G., Jr. (2001). Bacteriophage therapy. Antimicrobial Agents and Chemotherapy, 45(3), 649–659. https://doi.org/10.1128/AAC.45.3.649-659.2001
33. Tobback, E., Decostere, A., Hermans, K., Haesebrouck, F., Chiers, K. (2007). Yersinia ruckeri infections in salmonid fish. Journal of Fish Diseases, 30(5), 257–268. https://doi.org/10.1111/j.1365-2761.2007.00816.x
34. Ture, M., Altinok, I., Cebeci, A., Caliskan, N. (2022a). Characterization of novel bacteriophage HP-T19 that targets Hafnia alvei. Aquaculture Research, 53(2), 694–699. https://doi.org/10.1111/are.15592
35. Ture, M., Aygur, E., Caliskan, N. K., Cebeci, A., Polat, E. K., & Altinok, I. (2025). Effects of oral application of bacteriophage cocktails to treat aeromoniasis caused by Aeromonas hydrophila. Turkish Journal of Fisheries and Aquatic Sciences, 25(3), TRJFAS26235. https://doi.org/10.4194/TRJFAS26235
36. Ture, M., Cebeci, A., Altınok, İ., Aygür, E., Çalışkan, N. (2022b). Isolation and characterization of Aeromonas hydrophila-specific lytic bacteriophages. Aquaculture, 558, 738371. https://doi.org/10.1016/j.aquaculture.2022.738371
37. Wintachai, P., Naknaen, A., Pomwised, R., Voravuthikunchai, S. P., Smith, D. R. (2019). Isolation and characterization of Siphoviridae phage infecting extensively drug-resistant Acinetobacter baumannii and evaluation of therapeutic efficacy in vitro and in vivo. Journal of Medical Microbiology, 68(7), 1096–1108. https://doi.org/10.1099/jmm.0.001002
38. Wrobel, A., Leo, J. C., Linke, D. (2019). Overcoming fish defences: The virulence factors of Yersinia ruckeri. Genes, 10(9), 700. https://doi.org/10.3390/genes10090700
39. Yu, H., Zhang, L., Feng, C., Chi, T., Qi, Y., Raza, S. H. A., & Zhang, L. (2022). A phage cocktail in controlling phage resistance development in multidrug resistant Aeromonas hydrophila with great therapeutic potential. Microbial Pathogenesis, 162, 105374. https://doi.org/10.1016/j.micpath.2021.105374