Determination of the Antibiotic Susceptibility of Pathogenic Bacteria Isolated from Cultivated Rainbow Trout (Oncorhynchus mykiss)

Abstract

The overuse of antibiotics in aquaculture is considered a major concern in terms of resistant pathogens that can lead to infectious diseases. In this study, antibiotic resistance profiles were determined in pathogenic bacteria isolated from fish during outbreak in six different rainbow trout farms located in the Adana region. Vitek 2 (automated systems) was used for advanced bacterial identifications following standard conventional method. After all, bacterial isolates were identified as Aeromonas sobria, Pseudomonas fluorescens, and Lactococcus garvieae with 90–95% similarity rates. In determining the antibiotic resistance profiles of the isolates, their susceptibility to 14 different antibiotics were evaluated with the Kirby Bauer disk diffusion method, and also, the presence of various antibiotic resistance genes of the isolates was investigated by the conventional PCR method using specific primer pairs. When the antibiogram test results were examined, it was determined that each isolate had different levels of antibiotic sensitivity, and all isolates were found to be sensitive only to tetracycline, gentamicin, and levofloxacin. According to the MAR (Multiple Antibiotic Resistance) index values, A. sobria, and L. garvieae (L2) isolates remained below the 0.2 critical value, while the index values of other isolates were calculated above this critical value. The presence of tetA and tetC resistance genes could not be detected among the isolates.

Keywords

• Rainbow Trout
• VITEK 2 (Automated System)
• Antibiotic Susceptibility
• Multidrug Antibiotic Resistance

Kültür Gökkuşağı Alabalıklarında (Oncorhynchus mykiss) Tanımlanan Patojen Bakterilerin Antibiyotik Dirençliliğinin Belirlenmesi

Abstract

Su ürünleri yetiştiriciliğinde aşırı antibiyotik kullanımı, bulaşıcı hastalıklara yol açabilecek dirençli patojenler açısından büyük bir endişe kaynağı olarak değerlendirilmektedir. Bu çalışmada Adana bölgesinde yer alan gökkuşağı alabalığı üretimi yapan 6 farklı özel işletmede hastalık belirtisi gösteren balıklardan izole edilen patojen bakterilerde antibiyotik dirençlilik profilleri belirlenmiştir. Bakteri izolatlarının tanımlanmasında geleneksel kültür çalışmalarının ardından kit bazlı tam otomatize tanımlama sistemi (Vitek 2) kullanılmıştır. Bakteri izolatları Aeromonas sobria, Pseudomonas fluorescens ve Lactococcus garvieae %90-95 benzerlik oranlarıyla tanımlanmıştır. İzolatların on dört antibiyotiğe karşı antibiyotik duyarlığı, Kirby Bauer disk difüzyon yöntemi kullanılarak CLSI önerileri doğrultusunda gerçekleştirilmiş, genotipik olarak ise antibiyotik direnç genlerinin varlığı konvansiyonel PCR yöntemleriyle araştırılmıştır. Elde edilen sonuçlara göre antibiyotik duyarlığının her bir izolat için farklı düzeyde olduğu, sadece tetrasiklin, gentamisin ve levofloksasine karşı tüm izolatların duyarlı olduğu belirlenmiştir. ÇAD (Çoğul Antibiyotik Direnç) indeksi değerlerine göre A.sobria, ve L.garvieae (L2) izolatları 0,2 kritik indeks değerinin altında kalırken, diğer türlere ait indeks değerleri bu kritik değerin üzerinde hesaplanmıştır. İzolatlar arasında tetA, tetC, direnç genlerinin varlığı ise saptanmamıştır

Keywords

• Gökkuşağı Alabalık
• VİTEK 2 otomatize sistemi
• Antibiyotik Duyarlılık
• Çoklu Antibiyotik Dirençliliği

References

1. Altinok, I., Ozturk, R.C., Ture, M. (2022). NGS analysis revealed that Lactococcus garvieae Lg‐Per was Lactococcus petauri in Türkiye. Journal of Fish Diseases, 45(12): 1839-1843.
2. Andersson, D.I., Levin, B.R. (1999). The Biological Cost of Antibiotic Resistance, Current Opinion in Microbiology, 2: 489-493.
3. Aoki, T. (1997). Resistance plasmids and the risk of transfer. In: Bernoth EM, ed. Furunculosis: multidisciplinary fish disease research. London, Academic Press, pp. 433-440.
4. Aksoy, H.M. (2001). Balık Çiftliklerinde Üretilen Tatlı Su Balıklarında P. fluorescens' in Varlığı ve Antibiyotiklere Duyarlılığı. Etlik Veteriner Mikrobiyoloji Dergisi, 12(1-2): 12-22.
5. Balta, F., Balta, Z.D. (2019). The Isolation of Lactococcus garvieae from Eyes of Diseased Rainbow Trout (Oncorhynchus mykiss) with Exopthalmia. Journal of Anatolian Environmental and Animal Sciences, 4(1): 27-33. doi: 10.35229/jaes.527258.
6. Bhat, R., Altinok, I. (2023). Antimicrobial resistance (AMR) and alternative strategies for combating AMR in aquaculture. Turkish Journal of Fisheries and Aquatic Sciences, 23(11): TRJFAS24068. doi: 10.4194/TRJFAS24068.
7. BSGM (2023). Su Ürünleri İstatistikleri. T.C. Tarım ve Orman Bakanlığı, Balıkçılık ve Su Ürünleri Genel Müdürlüğü, Ankara.
8. Capkin, E., Terzi, E., Altinok, I. (2015). Occurrence of antibiotic resistance genes in culturable bacteria isolated from Turkish trout farms and their local aquatic environment. Diseases of Aquatic organisms, 114(2): 127-137.

9. Capkin, E., Ozdemir, S., Ozturk, R. C., 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.
10. Cipriano, R.C. (2001). Aeromonas hydrophila and motile aeromonad septicemias of fish. Fish disease leaflet 68. U.S. Dept. of the Interior, Fish and Wildlife Service, Division of Fishery Research, Washington, DC, pp. 25.
11. CLSI, 2013. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement. Clinical and Laboratory Standards Institute.
12. CLSI M100, (2020). Performance standards for antimicrobial susceptibility testing. 30th ed. CLSI Supplement M100, Clinical and Laboratory Standards Institute, Wayne, PA, USA.
13. CLSI M100, (2021). Performance standards for antimicrobial susceptibility testing. 31th ed. CLSI Supplement M100, Clinical and Laboratory Standards Institute, Wayne, PA, USA.
14. CLSI VET01S, (2020). Performance standards for antimicrobial susceptibility testing. 30th ed. CLSI Supplement M100, Clinical and Laboratory Standards Institute, Wayne, PA, USA.
15. CLSI, (2018). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Clinical and Laboratory Standart Institute. Wayne, PA, USA, pp. 112.
16. Diler, Ö., Altun, S., Adiloğlu, A.K., Kubilay, A., Işıklı, B.I. (2002). First occurence of Streptococcosis affecting farmed rainbow trout (Oncorhynchus mykiss) in Turkey. Bulletin of the European Association of Fish Pathologists, 22(1): 21-26.
17. Dokumacı, A.D. (2024). Antibiyotik Direncinin Aşılması İçin Yeni Stratejiler ve İlaç Geliştirme Çalışmaları: Geleneksel Derleme. Journal of Literature Pharmacy Sciences, 13(1): 57-68.
18. Durmaz, Y., Türk, N. (2009). Alabalık işletmelerinden motil aeromonasların izolasyonu ve antibiyotiklere duyarlılıklarının saptanması. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 15(3): 357-361.
19. Durmaz, Y., Kılıçoğlu, Y. (2015). Detection of naturally infected rainbow trouts (Oncorhynchus mykiss Walbaum, 1792) by Lactococcus garvieae with molecular methods and culture techniques and determination of antibiotic susceptibility profiles of agent in a trout farm. Atatürk Üniversitesi Veteriner Bilimleri Dergisi, 10(2): 109-115.
20. FAO, (2019). Aquaculture development. 8. Recommendations for prudent and responsible use of veterinary medicines in aquaculture. FAO Technical Guidelines for Responsible Fisheries.
21. FAO, (2020). The State of World Fisheries and Aquaculture 2020. Sustainability in action, Rome.
22. FAO, (2022). The state of world fisheries and aquaculture 2022. Towards blue transformation, Rome.
23. FEAP, European Aquaculture Production Report 2015–2021, (2022). Accessed Date: 05/04/2023, https://feap.info/wp-content/uploads/2023/04/2023-04-05-production-report-2023.pdf is retrieved.
24. Filik, N., Önem, E., Kubilay, A. (2021). Aeromonas hydrophila Suşlarının Antibiyotik Direnç Profilleri. Acta Aquatica Turcica, 17(2): 202-213.
25. Gao, P., Mao, D., Luo, Y., Wang, L., Xu, B., Xu, L. (2012). Occurrence of sulfonamide and tetracycline-resistant bacteria and resistance genes in aquaculture environment. Water research, 46(7): 2355-2364.
26. Güngör, N., İpek, Z.Z., Akif, E.R., Kayış, Ş. (2021). Farklı Sucul Sistemlerden İzole Edilen Bakterilerin Antibiyotik Dirençliliklerinin Karşılaştırılması. Journal of Anatolian Environmental and Animal Sciences, 6(1): 25-30.
27. Hancı, İ., Onuk, E.E. (2018). Lactococcus garvieae izolatlarının antimikrobiyal direnç profillerinin fenotipik ve genotipik olarak belirlenmesi. Etlik Vet Mikrobiyoloji Dergisi, 29(2): 94-103.
28. Janssen, K., Chavanne, H., Berentsen, P., Komen, H. (2017). Impact of selective breeding on European aquaculture. Aquaculture, 472(1): 8–16.
29. Kayis, S., Capkin, E., Altinok, I. (2009). Bacteria in rainbow trout (Oncorhynchus mykiss) in the Southern Black Sea Region of Turkey-a survey. The Israeli Journal of Aquaculture – Bamidgeh, 61(4): 339-344.
30. Kav, K., Erganiş, O. (2007). Konya Bölgesinde Bulunan Gökkuşağı Alabalığı (Oncorhynchus mykiss) Çiftliklerinden L. garvieae İzolasyonu, İdentifikasyonu ve Fenotipik Özelliklerinin Belirlenmesi. Veteriner Bilimler Dergisi, 23 (3): 7-17.
31. Krumperman, P.H. (1983). Multiple Antibiotic Resistance Indexing of Escherichia coli to Identify High-Risk Sources of Fecal Contamination of Foods, Applied and Environmental Microbiology, 461: 165-170.
32. Kerrn, M.B., Klemmensen, T., Frimodt-Møller, N., Espersen, F. (2002). Susceptibility of Danish Escherichia coli strains isolated from urinary tract infections and bacteraemia, and distribution of sul genes conferring sulphonamide resistance. Journal of Antimicrobial Chemotherapy, 50: 513−516.
33. Kubilay, A., Altun, S., Uluköy, G., Diler, Ö. (2005). The Determination of Antimicrobial Susceptibilities of Lactococcus garvieae Strains. SDU Eğirdir Su Ürünleri Fakültesi Dergisi, 1(1): 39-48.
34. Meyburgh, C.M., Bragg, R.R., Boucher, C.E. (2017). Lactococcus garvieae: an emerging bacterial pathogen of fish. Diseases of Aquatic Organism, 123 (1): 67–79.
35. Ng, L.K., Martin, I., Alfa, M., Mulvey, M. (2001). Multiplex PCR for the detection of tetracycline resistant genes. Molecular and Cell Probes, 15(4): 209−215.
36. Osundiya, O.O., Oladele, R.O., Oduyebo, O.O. (2013). Multiple antibiotic resistance (MAR) indices of Pseudomonas and Klebsiella species isolates in Lagos University Teaching Hospital. African Journal of Clinical and Experimental Microbiology, 14(3): 164-168.
37. Özer, S., Bulduklu, P., Tezcan, S., Dönmez, E., Aydin, E., Aslan, G., Emekdas, G. (2009). Genetic diversity and antimicrobial susceptibility of motile aeromonads isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) farms. Journal of Applied Ichthyology, 25(2): 195-200.
38. Özkök, S. (2005). Gökkuşağı alabalıklarında (Oncorhynchus mykiss) görülen önemli bakteriyel etkenlerin tespiti ve antibiyotiklere duyarlılıklarının saptanması. Etlik Veteriner Mikrobiyoloji Dergisi, 16(1-2): 1-12.
39. Öztürk, R.Ç., Altınok, İ. (2014). Bacterial and viral fish diseases in Turkey. Turkish Journal of Fisheries and Aquatic Sciences, 14(1): 275-297.
40. Petersen, A., Andersen, J.S., Kaewmak, T., Somsiri, T., Dalsgaard, A. (2002). Impact of integrated fish farming on antimicrobial resistance in a pond environment. Applied and Environmental Microbiology, 68: 6036–6042.
41. Romero, J., Feijoó, C.G., Navarrete, P. (2012). Antibiotics in aquaculture-use, abuse and alternatives. Health and Environment in Aquaculture, 159(1): 159-198. doi:10.5772/28157.
42. Saitanu, A., Chongthaleong, M., Endo, T., Umeda, K., Takami, T., Aoki, T. (1994). Antimicrobial susceptibilities and detection of transferable r-plasmids from Aeromonas hydrophila in Thailand. Asian Fisheries Sciences, (7): 41-46.
43. Schwartz, T., Kohnen, W., Jansen, B., Obst, U. (2003). Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water and drinking water biofilms. FEMS Microbiology Ecology, 43(3): 325−335.
44. Sezgin, S.S., Yilmaz, M., Arslan, T., Kubilay, A. (2023). Current antibiotic sensitivity of Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss) farms from Southwestern Turkey. Journal of Agricultural Sciences, 29(2): 630-642.
45. Toranzo, A.E. (2004). Report about fish bacterial diseases: Mediterranean Aquaculture Laboratories, In: Alvarez-Pellitero, P., Barja, J.L., Basurco, B., Berthe, F., Toranzo, A. E. (Eds.), Mediterranean aquaculture diagnostic laboratories, Santiago de Compostela, Spain. pp. 49-89.
46. Terzi, E. (2013). Alabalık İşletmelerinden İzole Edilen Bakterilerde Antibiyotik Direnç Genlerinin Belirlenmesi. Doktora Tezi, Karadeniz Teknik Üniversitesi Fen Bilimleri Enstitüsü, Trabzon.
47. Türe, M., Alp, H. (2016). Identification of bacterial pathogens and determination of their antibacterial resistance profiles in some cultured fish in Turkey. Journal of Veterinary Research, 60(2): 141-146.
48. Van Dongen, M.B.M., Van Diemen, A.E.A.R., Staarman, I.K., Piera, T. (2008). Antibiotic Resistance in Bacteria from Farmed Fish and Shrimps. InnoTact Consulting BV, Woudenberg, Netherlands.
49. Van, T.T.H., Chin, J., Chapman, T., Tran, L.T, Coloe, P.J. (2008). Safety of raw meat and shellfish in Vietnam: an analysis of Escherichia coli isolations for antibiotic resistance and virulence genes. International Journal of Food Microbiology, 124(3): 217−223.
50. Vendrell, D., Balcazar, J.L., Ruiz-Zarzuela, I., de Blas, I., Girones, O., Múzquiz, J.L. (2006). Lactococcus garvieae in fish: a review. Comparative Immunology, Microbiology and Infectious Diseases, 29(4): 177–198.
51. Vignesh, R., Karthikeyan, B.S., Periyasamy, N., Devanathan, K. (2011). Antibiotics in aquaculture: an overview. South Asian Journal of Experimental Biology, 1(3): 114-120.
52. Yilmaz, M., Arslan, T., Oral, M.A., Kubilay, A. (2024). Antibiotic susceptibility and resistance genes profiles of Vagococcus salmoninarum in a rainbow trout (Oncorhyncus mykiss, Walbaum) farm. PeerJ, 12: e17194.