Gökkuşağı Alabalıklarından Izole Edilen Chryseobacterium sp. C-204 Suşunun Fenotipik ve Genom Özelliklerinin Belirlenmesi

Yıl 2020, Cilt: 17 Sayı: 3, 303 - 311, 01.12.2020

Izzet Burcin Satıcıoğlu Muhammed Duman Soner Altun

https://doi.org/10.32707/ercivet.828829

Cited By: 2

Öz

Son yıllarda Chryseobacterium cinsinde bulunan türler, birçok ülkede balıklarda ölümlere neden olabilen fırsatçı balık patojenleri olarak ortaya çıkmaktadır. Yalnızca son on yılda C. aahli, C. oncorhynchi, C. chaponense ve C. pis-cicola’nın balıklarda sistemik enfeksiyonlara neden olduğu bildirilmiştir. Bu çalışmada, Chryseobacterium sp. C-204, anormal yüzme, sırt lezyonu, renkte koyulaşma ve iki taraflı egzoftalmi gibi klinik belirtiler gösteren 1 gram ağırlığındaki gökkuşağı alabalığından izole edildi. C-204'ün 106 farklı testi içeren fenotipik özellikleri API 20NE ve BIOLOG GEN III sistemi ile karakterize edildi. C-204'ün antimikrobiyal duyarlılığı, su ürünleri yetiştiriciliğinde yaygın olarak kullanılan beş farklı antimikrobiyal ajana karşı broth mikrodilüsyon yöntemiyle belirlendi. Dizi analizi bazlı tür tanımlama, 27F ve 1387R primerleri kullanılarak yapıldı. C-204'ün tüm genom analizinde, yeni nesil dizilime sistemi kullanıldı ve toplam 24195304 okuma elde edildi. Bu okumalar birleştirilerek 4012452 baz uzunluğunda taslak genom elde edildi. C-204'ün genoma dayalı tür tanımlaması, 100 farklı korunmuş gen bölgesi ve en yakın 50 Chryseobacterium türü ile autoMLST sisteminde (https://automlst.ziemertlab.com) yapıldı. C-204 genomundaki antimikrobiyal direnç genleri (AMR) ve virülans genleri, NCBI referans antimikrobiyal direnç genleri veritabanı ve Virülans Faktör Veritabanı (VFDB) kullanılarak tanımlandı. C-204 izolatının 16S rRNA gen bölgesinin, GenBankta C. aquaticum ile %99.65 ve C. green-landense ile %98.95 oranında benzerliklere sahip olduğu belirlenmiştir. Ortak yapılmış olan 19 biyokimyasal testte, C-204 izolatı nitrat indirgeyebilmesi ve glikozdan asit üretememesi testleri ile diğer tip suşlardan ayrılabildiği belirlenmiştir. Antimikrobiyal duyarlılıkla ilgili olarak, C-204 izolatının yüksek antimikrobiyal konsantrasyonlarında bile üreyebildiği tespit edilmiştir. Genom bazlı tür tanımlamasına göre, C-204 izolatı, Chryseobacterium aquaticum subsp green-landense olarak tanımlandı. Ayrıca, C-204 suşunun genomunda 13 virülans ve sekiz AMR geni tespit edildi. Çalışmamızda sonuç olarak, C-204 izolatının 106 biyokimyasal özellik içeren detaylı fenotipik ve tüm genom dizi ana-lizine dayalı genom yapısı belirlenmiştir.

Anahtar Kelimeler

Antimikrobiyal direnç geni, Chryseobacterium sp., virülens geni

Determination of Phenotypic and Genome Characteristics of Chryseobacterium sp. C-204 Strain Isolated from Rainbow Trout

Öz

In recent years, species in the Chryseobacterium genus have emerged as opportunistic fish pathogens that can cause death in fish in many countries. In the last decade, C. aahli, C. oncorhynchi, C. chaponense, and C. piscico-la have been reported to cause systemic infections in fish. In the present study, Chryseobacterium sp. C-204 was iso-lated from 1g weight rainbow trout showing clinical signs such as abnormal swimming, dorsal skin ulceration, darkening in color, and bilateral exophthalmos. The detailed phenotypic characteristics of the C-204 were characterized by API 20NE, and the BIOLOG GEN III system includes 106 phenotypes. Antimicrobial susceptibility of the C-204 was also determined by the broth microdilution method against five antimicrobial agents commonly used in the Aquaculture. Sequence-based identification was done using 16S rRNA genome sequencing. The genome structure of the C-204 was revealed by using next-generation genome sequencing with reading a total of 24195304 bases and assembled in 4012452 base. Genome-based species delineation of C-204 was done 100 different housekeeping gene regions and 50 the closest Chryseobacterium species with Automated Multi-Locus Species Tree (autoMLST, https://automlst.ziemertlab.com). Antimicrobial resistance genes (AMR) and virulence genes in the C-204 genome were iden-tified using the Virulence Factor Database (VFDB) NCBI-reference antimicrobial resistance genes database. The 16S rRNA sequence of C-204 isolate had similarities with the C. aquaticum (99.65%) and C. greenlandense (98.95%) in GenBank. In parallel 19 biochemical tests, C-204 isolate can be differentiated from the closest type strains by nitrate reduction and inability to produce acid from glucose. With regard to antimicrobial susceptibility, the C-204 isolate can grow even at high antimicrobial concentrations determined for Flavobacteriaceae. According to genome-based species delineation, the C-204 isolate was identified as Chryseobacterium aquaticum subsp greenlandense. 13 virulence and eight AMR genes were detected in the genome of the C-204 isolate. Conclusively, the detailed phenotypic characteris-tic includes 106 biochemical test and genome structure of C-204 isolate by whole genome sequencing were deter-mined.

Anahtar Kelimeler

Antimicrobial resistance genes, Chryseobacterium sp., virulence genes

Kaynakça

• Akhlaghi M, Sharifiyazdi H, Fereidouni MS. Isolation and identification of Chryseobacterium aquaticum from caudal fin rot and peduncle erosion in rain-bow trout (Oncorhynchus mykiss). Iran J Vet Clin Sci 2012; 6: 19-27.
• Alanjary M, Steinke K, Ziemert N. AutoMLST: an au-tomated web server for generating multi-locus species trees highlighting natural product potential. Nucleic Acids Res 2019; 47(W1): W276-82.
• Balta F, Sandalli C, Kayis S., Ozgumus OB. Molecu-lar analysis of antimicrobial resistance in Yersinia ruckeri strains isolated from rainbow trout (Oncorhynchus mykiss) grown in commercial fish farms in Turkey. Bull Eur Ass Fish Pathol 2010; 30(6): 211-9.
• Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV. SPAdes: A new genome assembly algorithm and its applica-tions to single-cell sequencing. J Comput Biol 2012; 19(5): 455-77.
• Bellais S, Naas T, Nordmann P. Genetic and bio-chemical characterization of CGB-1, an Ambler class B carbapenem-hydrolyzing β-lactamase from Chryseobacterium gleum. Antimicrob Agents Chemother 2002; 46(9): 2791-6.
• Bernardet JF, Vancanneyt M, Matte-Tailliez O, Grisez L, Tailliez P, Bizet C, Nowakowski M, Kerouault B, Swings J. Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals. Syst Appl Microbiol 2005; 28(7): 640-60.
• BSGM. Su Ürünleri İstatı̇stı̇klerı̇. https://www.tarimorman.gov.tr/sgb/Belgeler/SagMenuVeriler/BSGM.pdf; Accessed Date: 22.09.2020.
• CLSI. Methods for Broth Dilution Susceptibility Test-ing of Bacteria Isolated from Aquatic Animals, Ap-proved Guideline 2014a; VET04–A2: 1-20.
• CLSI. Performance Standards for Antimicrobial Sus-ceptibility Testing of Bacteria Isolated from Aquatic Animals; Second Informational Supplement 2014b; VET03/04‐S2: 1-25.
• Cooper A, Lambert D, Koziol AG, Seyer K, Carrillo CD. Draft genome sequence of Salmonella enter-ica subsp. enterica serovar mishmarhaemek iso-lated from bovine feces. Genome Announc 2015; 3(5): e01210-5.
• Duman M, Saticioglu IB, Buyukekiz AG, Balta F, Al-tun S. Molecular characterization and antimicrobial resistance profile of atypical Citrobacter gillenii and Citrobacter sp. isolated from diseased rainbow trout (Oncorhynchus mykiss). J Glob Antimicrob Resist 2017; 10: 136-42.
• Durmaz Y, Onuk EE, Ciftci A. Investigation of the presence and antibiotic susceptibilities of Flavo-bacterium psychrophilum in rainbow trout farms (Oncorhynchus mykiss Walbaum, 1792) in The Middle and Eastern Black Sea Regions of Turkey. Ankara Üniv Vet Fak Derg 2012; 59: 141-6.
• Feldgarden M, Brover V, Haft DH, Prasad AB, Slotta DJ, Tolstoy I, Tyson GH, Zhao S, Hsu CH, McDer-mott PF, Tadesse DA. Validating the AMRFinder tool and resistance gene database by using anti-microbial resistance genotype-phenotype correla-tions in a collection of isolates. Antimicrob Agents Chemother 2019; 63(11): e00483-19.
• García-López M, Meier-Kolthoff JP, Tindall B, Gronow S, Woyke T, Kyrpides NC, Hahnke RL, Göker M. Analysis of 1,000 type-strain genomes improves taxonomic classification of Bacteroide-tes. Front Microbiol 2019; 10: 2083.
• Hugo C, Bernardet JF, Nicholson A, Kämpfer P. Chryseobacterium . In Bergey's Manual of System-atics of Archaea and Bacteria (eds ME. Trujillo, S. Dedysh, P. DeVos, B. Hedlund, P. Kämpfer, F.A. Rainey and W.B. Whitman) 2019; p. 1-110.
• Ilardi P, Fernandez J, Avendano-Herrera R. Chryseo-bacterium piscicola sp. nov., isolated from dis-eased salmonid fish. Int J Syst Evol Microbiol 2009; 59(12): 3001-5.
• Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Mar-kowitz S, Duran C, Thierer T. Geneious Basic: an integrated and extendable desktop software plat-form for the organization and analysis of sequence data. Bioinformatics 2012; 28(12): 1647-9.
• Kim KK, Lee KC, Oh HM, Lee JS. Chryseobacterium aquaticum sp. nov., isolated from a water reser-voir. Int J Syst Evol Microbiol 2008; 58(3): 533-7.
• Lee YH, Tung KC, Cheng JF, Wu ZY, Chen SY, Hong YK, Huang YT, Liu PY. Genomic characteri-zation of carbapenem-resistant Shewanella algae isolated from Asian hard clam (Meretrix lusoria). Aquaculture 2019; 500: 300-4.
• Lin YT, Jeng YY, Lin ML, Yu KW, Wang FD, Liu CY. Clinical and microbiological characteristics of Chryseobacterium indologenes bacteremia. J Mi-crobiol Immunol Infect 2010; 43(6): 498-505.
• Liu B, Zheng D, Jin Q, Chen L, Yang J. VFDB 2019: a comparative pathogenomic platform with an in-teractive web interface. Nucleic Acids Res 2019; 47(D1): D687-92.
• Loch TP, Faisal M. Emerging flavobacterial infections in fish: a review. J Adv Res 2015a; 6(3): 283-300.
• Loch TP, Faisal M. Polyphasic characterization re-veals the presence of novel fish-associated Chrys-eobacterium spp. in the Great Lakes of North America. Dis Aquat Org 2015b; 113(2): 113-25.
• Loch TP, Faisal M. Chryseobacterium aahli sp. nov., isolated from lake trout (Salvelinus namaycush) and brown trout (Salmo trutta), and emended de-scriptions of Chryseobacterium ginsenosidimutans and Chryseobacterium gregarium. Int J Syst Evol Microbiol 2014; 64(5): 1573-9.
• Loch TP, Fujimoto M, Woodiga SA, Walker ED, Marsh TL, Faisal M. Diversity of fish-associated flavobacteria of Michigan. J Aquat Anim Health 2013; 25(3): 149-64.
• Loveland-Curtze J, Miteva V, Brenchley J. Novel ul-tramicrobacterial isolates from a deep Greenland ice core represent a proposed new species, Chryseobacterium greenlandense sp. nov. Ex-tremophiles 2010; 14(1): 61-9.
• Michel C, Matte‐Tailliez O, Kerouault B, Bernardet JF. Resistance pattern and assessment of pheni-col agents' minimum inhibitory concentration in multiple drug resistant Chryseobacterium isolates from fish and aquatic habitats. J Appl Microbiol 2005; 99(2): 323-32.
• Mudarris M, Austin B. Systemic disease in turbot Scophthalmus maximus caused by a previously unrecognised Cytophaga-like bacterium. Dis Aquat Org 1989; 6(3): 161-6.
• Onuk EE, Tanriverdi ÇY, Çoban AY, Ciftci A, Balta F, Didinen BI, Altun S. Determination of antimicrobial susceptibility patterns of fish and rearing water originated Aeromonas isolates. Ankara Üniv Vet Fak Derg 2017; 64(1): 69-73.
• Saticioglu IB, Duman M, Smith P, Wiklund T, Altun S. Antimicrobial resistance and resistance genes in Flavobacterium psychrophilum isolates from Tur-key. Aquaculture 2019; 512: 734293.
• Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 2016; 144(14): 6614-24.
• Verner-Jeffreys DW, Brazier T, Perez RY, Ryder D, Card RM, Welch TJ, Hoare R, Ngo T, McLaren N, Ellis R, Bartie KL. Detection of the florfenicol re-sistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule?. FEMS Microbiol Ecol 2017; 93(4): fix015.