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

Abstract

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.

Keywords

• Antimikrobiyal direnç geni
• Chryseobacterium sp.
• virülens geni

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

Abstract

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.

Keywords

• Antimicrobial resistance genes
• Chryseobacterium sp.
• virulence genes

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