GÖKKUŞAĞI ALABALIĞINDAN İZOLE EDİLEN LACTOCOCCUS PETAURİ KSUB4'ÜN TÜM GENOM SEKANS ANALİZİ ve GENOMİK KARAKTERİZASYONU

Öz

Laktokokkozis, dünya çapında balıklarda görülen önemli bakteriyel enfeksiyon hastalıklarından biridir. Bu hastalık, etiyolojik açıdan başlıca Lactococcus garvieae ve yakın zamanda tanımlanan L. petauri türleri ile ilişkilendirilmektedir. Geleneksel olarak, L. garvieae bu hastalığın en önemli ve en iyi karakterize edilmiş patojeni olarak kabul edilmektedir. Bununla birlikte, son yıllarda yapılan çalışmalar, L. petauri'nin hastalığın epidemiyolojisindeki rolünün giderek arttığını ortaya koymuştur. Bu çalışmada, 2022 yılında Karadeniz bölgesindeki bir alabalık işletmesinden izole edilen ve PCR ile Lactococcus sp. olarak doğrulanan bir izolat (KSUB4), Tüm Genom Sekans Analizi (WGS) kullanılarak tanımlanmıştır. İzolatın genom dizilemesi, Illumina HiSeqX platformunda gerçekleştirilmiş ve ham sekans okumaları kalite kontrol işlemlerinden geçirildikten sonra Unicycler v0.4.8 yazılımı kullanılarak birleştirilmiştir. Genomun anotasyonu, NCBI Prokaryotik Genom Otomatik Anotasyon Sistemi (PGAP) aracılığıyla yapılmıştır. İzolatın tür düzeyinde tanımlanması, Type Strain Genome Server (TYGS; https://tygs.dsmz.de/) platformu kullanılarak gerçekleştirilmiştir. Virülans faktörleri ve antimikrobiyal direnç (AMR) genlerinin belirlenmesi için sırasıyla Virulence Factor Database (VFDB) ve Comprehensive Antibiotic Resistance Database (CARD) veri tabanları kullanılmıştır. Analizler sonucunda, KSUB4 izolatının genomunun 2.014.614 baz çifti (bp) uzunluğunda ve %38,13 G+C içeriğine sahip olduğu belirlenmiştir. Tür tanımlama analizleri, KSUB4 izolatının L. garvieae ATCC 49156^T suşu ile %51, L. petauri 159469T suşu ile ise %85,6 dijital DNA-DNA hibridizasyon (dDDH) benzerliği gösterdiğini ortaya koymuştur. KSUB4 genomunda, %35-71 benzerlik oranına sahip 46 virülans geni ve %53-95 benzerlik oranına sahip 12 AMR geni tespit edilmiştir. Bu bulgular, L. petauri'nin balık patojenitesindeki genetik potansiyeline ışık tutmakta ve bu türün antimikrobiyal direnç profili hakkında önemli bilgiler sağlamaktadır.

Anahtar Kelimeler

• Lactococcus petauri
• Tüm Genom Sekans Analizi
• Virülens Genleri
• Antibiyotik Direnci
• Akuakültür

Whole Genome Sequence Analysıs And Genomıc Characterızatıon Of Lactococcus Petaurı Ksub4 Isolated From Raınbow Trout

Öz

Lactococcosis is one of the significant bacterial infectious diseases observed in fish worldwide. Etiologically, this disease is primarily associated with Lactococcus garvieae and, more recently, the newly identified species L. petauri. Traditionally, L. garvieae has been recognized as the most important and best-characterized pathogen of this disease. However, recent studies have revealed that the role of L. petauri in the epidemiology of the disease is increasingly significant. In this study, an isolate (KSUB4) obtained from a trout farm in the Black Sea region in 2022 and confirmed as Lactococcus sp. by Polymerase Chain Reaction (PCR) was identified using Whole Genome Sequencing (WGS) analysis. The genome sequencing of the isolate was performed on the Illumina HiSeqX platform, and the raw sequence reads were assembled using the Unicycler v0.4.8 software after undergoing quality control processes. Annotation of the genome was carried out through the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAP). Species-level identification of the isolate was performed using the Type Strain Genome Server (TYGS; https://tygs.dsmz.de/) platform. Virulence factors and antimicrobial resistance (AMR) genes were identified using the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD), respectively. The analyses revealed that the genome of the KSUB4 isolate was 2,014,614 base pairs (bp) in length with a G+C content of 38.13%. Species identification analyses demonstrated that the KSUB4 isolate exhibited 51% digital DNA-DNA hybridization (dDDH) similarity with L. garvieae ATCC 49156T and 85.6% dDDH similarity with L. petauri 159469T. In the KSUB4 genome, 46 virulence genes with 35-71% similarity and 12 AMR genes with 53-95% similarity were detected. These findings shed light on the genetic potential of L. petauri in fish pathogenicity and provide significant insights into the antimicrobial resistance profile of this species.

Anahtar Kelimeler

• Lactococcus petauri
• Whole Genome Sequencing
• Virulence Genes
• Antibiotic Resistance
• Aquaculture

Kaynakça

1. Abraham, T., Yazdi, Z., Littman, E., Shahin, K., Heckman, T. I., Quijano Cardé, E. M., ... & Soto, E. (2023). Detection and virulence of Lactococcus garvieae and L. petauri from four lakes in southern California. Journal of Aquatic Animal Health, 35(3), 187-198.
2. 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.
3. Altun, S., Duman, M., Ajmi, N., Tasci, G., Ozakin, C., Tuzemen, N. U., ... & Saticioglu, I. B. (2025). Comparative proteomic and genomic analysis of Lactococcus garvieae and Lactococcus petauri isolates from Turkey. Aquaculture, 596, 741887.
4. Brettin, T., Davis, J. J., Disz, T., Edwards, R. A., Gerdes, S., Olsen, G. J., ... & Xia, F. (2015). RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Scientific reports, 5(1), 8365.
5. Chan, Y. X., Cao, H., Jiang, S., Li, X., Fung, K. K., Lee, C. H., ... & Ho, P. L. (2024). Genomic investigation of Lactococcus formosensis, Lactococcus garvieae, and Lactococcus petauri reveals differences in species distribution by human and animal sources. Microbiology Spectrum, 12(6), e00541-24.
6. Collins, M. D., Farrow, J. A. E., Phillips, B. A., & Kandler, O. (1983). Streptococcus garvieae sp. nov. and Streptococcus plantarum sp. nov. Microbiology, 129(11), 3427-3431.
7. Colussi, S., Pastorino, P., Prearo, M., Sciuto, S., Bondavalli, F., Acutis, P. L., ... & Salerno, A. (2023). First report of human urinary tract infection caused by Lactococcus petauri. Microorganisms, 11(10), 2583.
8. Diler, O., Altun, S. O. N. E. R., Adiloglu, A. K., Kubilay, A. Y. Ş. E. G. Ü. L., & Istklt, B. (2002). First occurrence of Streptococcosis affecting farmed rainbow trout (Oncorhynchus mykiss) in Turkey. Bulletin-European Association of Fish Pathologists, 22(1), 21-26.

9. Egger, R. C., Rosa, J. C. C., Resende, L. F. L., de Pádua, S. B., de Oliveira Barbosa, F., Zerbini, M. T., ... & Figueiredo, H. C. P. (2023). Emerging fish pathogens Lactococcus petauri and L. garvieae in Nile tilapia (Oreochromis niloticus) farmed in Brazil. Aquaculture, 565, 739093.
10. Goodman, L. B., Lawton, M. R., Franklin-Guild, R. J., Anderson, R. R., Schaan, L., Thachil, A. J., ... & Kovac, J. (2017). Lactococcus petauri sp. nov., isolated from an abscess of a sugar glider. International journal of systematic and evolutionary microbiology, 67(11), 4397-4404.
11. Heckman, T. I., Yazdi, Z., Older, C. E., Griffin, M. J., Waldbieser, G. C., Chow, A. M., ... & Soto, E. (2024). Redefining piscine lactococcosis. Applied and Environmental Microbiology, 90(5), e02349-23.
12. Kotzamanidis, C., Malousi, A., Bitchava, K., Vafeas, G., Chatzidimitriou, D., Skoura, L., ... & Zdragas, A. (2020). First report of isolation and genome sequence of L. petauri strain from a rainbow trout lactococcosis outbreak. Current Microbiology, 77, 1089-1096.
13. Kusuda, R., Kawai, K., Salati, F., Banner, C. R., & Fryer, J. L. (1991). Enterococcus seriolicida sp. nov., a fish pathogen. International Journal of Systematic and Evolutionary Microbiology, 41(3), 406-409.
14. Liu, B., Zheng, D., Zhou, S., Chen, L., & Yang, J. (2022). VFDB 2022: a general classification scheme for bacterial virulence factors. Nucleic acids research, 50(D1), D912-D917.
15. Mahmoud, M. M., Abdelsalam, M., Kawato, S., Harakawa, S., Kawakami, H., Hirono, I., & Kondo, H. (2023). Comparative genome analyses of three serotypes of Lactococcus bacteria isolated from diseased cultured striped jack (Pseudocaranx dentex). Journal of Fish Diseases, 46(8), 829-839.
16. Martinovic, A., Cabal, A., Nisic, A., Sucher, J., Stöger, A., Allerberger, F., & Ruppitsch, W. (2021). Genome sequences of Lactococcus garvieae and Lactococcus petauri strains isolated from traditional Montenegrin brine cheeses. Microbiology Resource Announcements, 10(31), 10-1128.
17. McArthur, A. G., Waglechner, N., Nizam, F., Yan, A., Azad, M. A., Baylay, A. J., ... & Wright, G. D. (2013). The comprehensive antibiotic resistance database. Antimicrobial agents and chemotherapy, 57(7), 3348-3357.
18. Meier-Kolthoff, J. P., & Göker, M. (2019). TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nature communications, 10(1), 2182.
19. 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.
20. Olson, R. D., Assaf, R., Brettin, T., Conrad, N., Cucinell, C., Davis, J. J., ... & Stevens, R. L. (2023). Introducing the bacterial and viral bioinformatics resource center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic acids research, 51(D1), D678-D689.
21. Öztürk, R. Ç., Ustaoglu, D., Ture, M., Bondavalli, F., Colussi, S., Pastorino, P., ... & Altinok, I. (2024). Epidemiological cutoff values and genetic antimicrobial resistance of Lactococcus garvieae and L. petauri. Aquaculture, 593, 741340.
22. Palacios, M. A., Zamora, M. J., Velazquez, J., Zamora, E., & Duran, A. (1993). Streptococcosis in rainbow trout(Oncorhynchus mykiss) in Spain. Bollettino Societa Italiana di Patologia Ittica, 5(13), 11-16.
23. Ringø, E., & Gatesoupe, F. J. (1998). Lactic acid bacteria in fish: a review. Aquaculture, 160(3-4), 177-203.
24. Rosário, A. E. C. D., Barbanti, A. C. C., Matos, H. C., Maia, C. R. M. D. S., Trindade, J. M., Nogueira, L. F. F., ... & Tavares, G. C. (2024). Antimicrobial Resistance in Lactococcus spp. Isolated from Native Brazilian Fish Species: A Growing Challenge for Aquaculture. Microorganisms, 12(11), 2327.
25. Saticioglu, I. B., Onuk, E. E., Ay, H., Ajmi, N., Demirbas, E., & Altun, S. (2023). Phenotypic and molecular differentiation of Lactococcus garvieae and Lactococcus petauri isolated from trout. Aquaculture, 577, 739933.
26. Sciuto, S., Esposito, G., Pastorino, P., Shahin, K., Varello, K., Trabunella, E., ... & Colussi, S. (2024). First Detection of Lactococcus petauri in Domestic Dogs in Italy. Animals, 14(22), 3279.
27. Stoppani, N., Colussi, S., Pastorino, P., Prearo, M., Sciuto, S., Altinok, I., ... & Fernández-Garayzábal, J. F. (2023). 16S-23S rRNA Internal Transcribed Spacer Region (ITS) sequencing: A potential molecular diagnostic tool for differentiating Lactococcus garvieae and Lactococcus petauri. Microorganisms, 11(5), 1320.
28. Vela, A. I., del Mar Blanco, M., Colussi, S., Kotzamanidis, C., Prearo, M., Altinok, I., ... & Fernández-Garayzábal, J. F. (2024). The association of Lactococcus petauri with lactococcosis is older than expected. Aquaculture, 578, 740057.
29. Vendrell, D., Balcázar, J. L., Ruiz-Zarzuela, I., De Blas, I., Gironés, O., & Múzquiz, J. L. (2006). Lactococcus garvieae in fish: a review. Comparative immunology, microbiology and infectious diseases, 29(4), 177-198.
30. Wattam, A. R., Davis, J. J., Assaf, R., Boisvert, S., Brettin, T., Bun, C., ... & Stevens, R. L. (2017). Improvements to PATRIC, the all-bacterial bioinformatics database and analysis resource center. Nucleic acids research, 45(D1), D535-D542.
31. Wick, R. R., Judd, L. M., Gorrie, C. L., & Holt, K. E. (2017). Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS computational biology, 13(6), e1005595.
32. Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., & Chun, J. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International journal of systematic and evolutionary microbiology, 67(5), 1613-1617.