Gökkuşağı Alabalığı Bağırsaklarından İzole Edilen Laktik Asit Bakterilerinin Lactococcus garvieae’ye Karşı Probiyotik Potansiyelinin İn Vitro Olarak Belirlenmesi

Yıl 2020, , 647 - 654, 31.12.2020

Dilek Pehlivan Ertan Emek Onuk

https://doi.org/10.35229/jaes.821270

Öz

Bu çalışma amacı, gökkuşağı alabalıklarının bağırsak mikroflorasından Lactococcus garvieae’ye karşı antagonostik etkiye sahip Laktik Asit Bakterilerinin (LAB) izole edilmesi ve antagonostik etkiye sahip olduğu belirlenen bakteriyel türlerin olası probiyotik potansiyelinin in vitro olarak ortaya konulmasıdır. Bu amaçla Gökkuşağı alabalıklarının bağırsak florasından LAB türleri konvansiyonel kültür metotları kullanılarak izole edildi. Elde edilen izolatların L. garvieae’ye karşı antagonostik etkinlikleri agar well difüzyon testi ile belirlendi. Sonraki aşamada antagonostik aktiviteye sahip olduğu belirlenen izolatların hidrofobisitesi, pH ve safra tolerans düzeyleri ve antibiyotik duyarlılık profilleri belirlendi. Elde edilen aday probiyotik bakterilerinin genetik identifikasyonu PCR metodu ile gerçekleştirildi. Balıkların bağırsak florasından toplamda 47 izolat elde edildi. Bu izolatların 6’sının L. garvieae’ye karşı antagonostik etkinliğe sahip olduğu belirlendi. İleri testlerde izolatların hidrofobik özellikte oldukları, yüksek safra ve düşük pH şartlarına dirençli oldukları ve izolatların tamamının beş farklı antibiyotiğe karşı dirençli olduğu belirlendi. Genetik identifikasyon sonucu izolatlardan beşi Lactobacillus acidophilus olarak identifiye edildi. Bir izolat ise Lactobacillus spp. olarak tanımlandı. Bu çalışma ile ülkemiz kültür balığı yetiştiriciliğinde yaygın olarak görülen L. garvieae’nın kontrol altına alınmasında kullanılabilecek olası probiyotik bakteriler elde edilmiştir.

Anahtar Kelimeler

Gökkuşağı alabalığı, Laktik asit bakterileri, Lactococcus garvieae, Probiyotik

In Vitro Evaluation of Probiotic Potential of Lactic Acid Bacteria Isolated from Rainbow Trout Gut against Lactococcus garvieae

Öz

The purpose of this study are to isolate Lactic Acid Bacteria (LAB), which have an antagonistic activity against Lactococcus garvieae, in the intestinal microflora of rainbow trouts and to reveal in vitro, the probable probiotic potential of the bacterial species, which were determined to have an antagonistic effect. For this aim LAB species were isolated from the intestinal flora of rainbow trouts using conventional culture methods. The antagonistic efficiencies of the obtained isolates against L. garvieae were determined with the agar well diffusion test. The hydrophobicity, pH and bile tolerance levels and antibiotic susceptibility profiles of the isolates, which were found to have antagonistic activity, were determined in the next stage. Genetic identification of the obtained candidate probiotic bacteria was performed with PCR method. A total of 47 isolates were obtained from the intestinal flora of the rainbow trout. Six of these isolates were determined to have antagonistic activity against L. garvieae. Further tests revealed that the isolates were hydrophobic, resistant to high bile and low pH conditions, and that all isolates were resistant to five different antibiotics. Following genetic identification, five of the isolates were identified as Lactobacillus acidophilus. One of the isolates was defined as Lactobacillus spp. With this study, possible probiotic bacteria were obtained, which can be used to control L. garvieae, widespread in our country's culture fish farming.

Anahtar Kelimeler

Lactic acid bacteria, Lactococcus garvieae, Probiotic, Rainbow trout

Kaynakça

• Altun, S., Diler, Ö. & Adiloğlu A.K. (2004). Genotyping of Lactococcus garvieae strains from Rainbow trout (Oncorhynchus mykiss) by 16S rDNA sequencing. Bulletin of the European Association of Fish Pathologists, 24, 119-125.
• Altun, S., Onuk., E.E., Çiftci, A., Büyükekiz, A.G. & Duman, M. (2013). Phenotypic, genotypic characterisation and antimicrobial susceptibility determination of Lactococcus garvieae strains. Kafkas Universitesi Veteriner Fakultesi Dergisi, 19, 375-381.
• An, Y. & Friedman, R.J. (2000). Handbook of Bacterial Adhesion: Principles, Methods and Applications. Totowa, New Jersey: Humana Press,
• Balcazar, J,L,, Vendrell, D., de Blas, I., Ruiz-Zarzuela, I., Girones, O. & Muzquiz, J.L. (2007). In vitro competitive adhesion and production of antagonistic compounds by lactic acid bacteria against fish pathogens. Veterinary Microbiology, 122, 373-380.
• Balcazar, J.L., Vendrell, D., de Blas, I., Ruiz-Zarzuela, I., Muzquiz, J.L. & Girones, O. (2008). Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish. Aquaculture, 278(1-4), 188-191.
• Cahill, M.M. (1990). Bacterial flora of fishes: a review. Microbial Ecology, 19(1), 21-41.
• Capkin, E. & Altinok, I. (2009). Effects of dietary probiotic supplementations on prevention/treatment of yersiniosis disease. Journal of Applied Microbiology, 106(4), 1147-1153.
• Dhar, A.K., Manna, S.K. & Allnutt, F.T. (2014). Viral vaccines for farmed finfish. Virusdisease, 25(1), 1-17.
• Didinen, B.I., Metin, S., Onuk, E.E., Takmaz, H. & Ersoy, A.T. (2014a). Isolation and characterization of potential probiotic bacteria from rainbow trout Oncorhynchus mykiss,(Walbaum) rearing units against bacterial pathogens. The Israeli Journal of Aquaculture-Bamidgeh, 66, 1006-1014.
• Didinen, B.I., Onuk, E.E., Metin, S. & Cayli, O. (2018). Identification and characterization of lactic acid bacteria isolated from rainbow trout (Oncorhynchus mykiss, Walbaum 1792), with inhibitory activity against Vagococcus salmoninarum and Lactococcus garvieae. Aquaculture Nutrition, 24(1), 400-407.
• Didinen, B.I., Yardimci, B., Onuk, E.E., Metin, S. & Yildirim, P. (2014b). Naturally Lactococcus garvieae infection in rainbow trout (Oncorhyncus mykiss Walbaum, 1792): new histopathological observations, phenotypic and molecular identification. Revue de Medecine Veterinaire, 165(1-2), 12-19.
• Diler, O., Altun, S., Adiloglu, A.K., Kubilay, A. & Isıklı, B. (2002). First occurrence of Streptococcosis affecting farmed rainbow trout (Oncorhynchus mykiss) in Turkey. Bulletin-European Association of Fish Pathologists, 22(1), 21-26.
• Getachew, T. (1989). Stomach pH, feeding rhythm and ingestion rate in Oreochromis niloticus L. (Pisces: Cichlidae) in Lake Awasa, Ethiopia. Hydrobiologia, 174(1), 43-48.
• Gibello, A., Galan-Sanchez, F., Blanco, M.M., Rodríguez-Iglesias, M., Domínguez, L. & Fernández-Garayzábal, J.F. (2016). The zoonotic potential of Lactococcus garvieae: an overview on microbiology, epidemiology, virulence factors and relationship with its presence in foods. Research in Veterinary Science, 109, 59-70.
• Heuer, O.E., Kruse, H., Grave, K., Collignon, P., Karunasagar, I. & Angulo, F.J. (2009). Human health consequences of use of antimicrobial agents in aquaculture. Clinical Infectious Diseases, 49(8), 1248-1253.
• Kim, D.H. & Austin, B. (2008). Characterization of probiotic carnobacteria isolated from rainbow trout (Oncorhynchus mykiss) intestine. Letters in Applied Microbiology, 47(3), 141-147.
• Lavelle, E.C. & Harris, J.E. (1997). The processing of an orally administered protein antigen in the digestive tract of rainbow trout, Oncorhynchus mykiss. Comparative Biochemistry and Physiology Part A: Physiology, 117(2), 263-275.
• Lin, Y.H., Chen, Y.S., Wu, H.C., Pan, S.F., Yu, B., Chiang, C.M., Chiu, C.M. & Yanagida, F. (2013). Screening and characterization of LAB‐produced bacteriocin‐like substances from the intestine of grey mullet (Mugil cephalus L.) as potential biocontrol agents in aquaculture. Journal of Applied Microbiology, 114(2), 299-307.
• Lopez Cazorla, A., Sica, M.G., Brugnoni, L.I., Marucci, P.L. & Cubitto, M.A. (2015). Evaluation of Lactobacillus paracasei subsp. tolerans isolated from Jenyn's sprat (Ramnogaster arcuata) as probiotic for juvenile rainbow trout Oncorhynchus mykiss (Walbaum, 1792). Journal of Applied Ichthyology, 31(1), 88-94.
• Markiewicz, L. & Biedrzycka, E. (2005). Identification of Lactobacillus and Bifidobacterium species with PCR applied to quality control of fermented dairy beverages. Polish Journal of Food and Nutrition Sciences, 14(4), 359-365.
• Marti, E., Variatza, E. & Balcazar, J.L. (2014). The role of aquatic ecosystems as reservoirs of antibiotic resistance. Trends in Microbiology, 22(1), 36-41.
• Meyburgh, C.M., Bragg, R.R. & Boucher, C.E. (2017). Lactococcus garvieae: an emerging bacterial pathogen of fish. Diseases of Aquatic Organisms, 123(1), 67-79.
• Mukherjee, A. & Ghosh, K. (2016). Antagonism against fish pathogens by cellular components and verification of probiotic properties in autochthonous bacteria isolated from the gut of an Indian major carp, Catla catla (Hamilton). Aquaculture Research, 47(7), 2243-2255.
• Nguyen, T.L., Park, C.I. & Kim, D.H. (2017). Improved growth rate and disease resistance in olive flounder, Paralichthys olivaceus, by probiotic Lactococcus lactis WFLU12 isolated from wild marine fish. Aquaculture, 471, 113-120.
• Onuk, E.E. & Fındık, A. (2015). Balıklarda antibiyotik dirençliliği. Türkiye Klinikleri Veteriner Bilimleri-Farmakoloji ve Toksikoloji Özel Dergisi, 1(2), 35-41.
• Perez-Sanchez, T., Balcazar, J.L., Garcia, Y., Halaihel, N., Vendrell, D., De Blas, I., Merrifield, D.L. & Ruiz-Zarzuela, I. (2011). Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae. Journal of Fish Diseases, 34, 499-507.
• Ringo, E., Birkbeck, T.H., Munro, P.O., Vadstein, O. & Hjelmeland, K. (1996). The effect of early exposure to Vibrio pelagius on the aerobic bacterial flora of turbot, Scophthalmus maximus (L.) larvae. Journal of Applied Bacteriology, 81(2), 207-211.
• Ringo, E., Hoseinifar, S.H., Ghosh, K., Doan, H.V., Beck, B.R. & Song, S.K. (2018). Lactic acid bacteria in finfish—An update. Frontiers in Microbiology, 9, 1818.
• Rinkinen, M.L., Koort, J.M., Ouwehand, A.C., Westermarck, E. & Björkroth, K.J. (2004). Streptococcus alactolyticus is the dominating culturable lactic acid bacterium species in canine jejunum and feces of four fistulated dogs. FEMS Microbiology Letters, 230(1), 35-39.
• Safari, R., Adel, M., Lazado, C.C., Caipang, C.M.A. & Dadar, M. (2016). Host-derived probiotics Enterococcus casseliflavus improves resistance against Streptococcus iniae infection in rainbow trout (Oncorhynchus mykiss) via immunomodulation. Fish & Shellfish Immunology, 52, 198-205.
• Sharma, K.K., Soni, S.S. & Meharchandani, S. (2006). Congo red dye agar test as an indicator test for detection of invasive bovine Escherichia coli. Veterinarski Arhiv, 76(4), 363-366.
• Smith, P., Hiney, M.P. & Samuelsen, O.B. (1994). Bacterial resistance to antimicrobial agents used in fish farming: a critical evaluation of method and meaning. Annual Review of Fish Diseases, 4, 273-313.
• Soltani, M., Pakzad, K., Taheri-Mirghaed, A., Mirzargar, S., Shekarabi, S.P.H., Yosefi, P. & Soleymani, N. (2019). Dietary application of the probiotic Lactobacillus plantarum 426951 enhances immune status and growth of rainbow trout (Oncorhynchus mykiss) vaccinated against Yersinia ruckeri. Probiotics and Antimicrobial Proteins, 11(1), 207-219.
• Sorroza, L., Padilla, D., Acosta, F., Roman, L., Grasso, V., Vega, J. & Real, F. (2012). Characterization of the probiotic strain Vagococcus fluvialis in the protection of European sea bass (Dicentrarchus labrax) against vibriosis by Vibrio anguillarum. Veterinary Microbiology, 155(2-4), 369-373.
• Sul, S.Y., Kim, H.J., Kim, T.W. & Kim, H.Y. (2007). Rapid identification of Lactobacillus and Bifidobacterium in probiotic products using multiplex PCR. Journal of Microbiology and Biotechnology, 17(3), 490-495.
• Vlkova, E., Rada, V., Popelarova, P., Trojanova, I. & Killer, J. (2006). Antimicrobial susceptibility of bifidobacteria isolated from gastrointestinal tract of calves. Livestock Science, 105, 253-259.