Comparative genomic analysis of Flavobacterium species causing columnaris disease of freshwater fish in Thailand: insights into virulence and resistance mechanisms

Abstract

Background: Columnaris disease, a prevalent disease among farmed and wild freshwater fish, is caused by the Flavobacterium columnare group, which includes four distinct species: F. columnare, F. oreochromis, F. covae, and F. davisii. Among these, F. oreochromis, F. covae, and F. davisii are particularly prevalent in farmed freshwater fish in Thailand. In this study, a comparative genomic analysis of 22 isolates was conducted to elucidate virulence factors, antibiotic resistance genes (ARGs), genomic islands (GIs), phages, insertion elements (ISs), and clustered regularly interspaced short palindromic repeats (CRISPRs).

Results: A total of 212 putative virulence genes were predicted across three species with F. oreochromis exhibiting the highest number of unique virulence genes, followed by F. davisii, and F. covae. Moreover, 195 genes were predicted as ARGs, with F. oreochromis and F. covae showing an abundance of unique genes associated with resistance to quinolone, fluoroquinolone, and tetracycline antibiotics. Antimicrobial susceptibility testing, assessed with epidemiological cut-off values (ECVs), revealed decreased susceptibility to quinolones, fluoroquinolones and tetracycline in several isolates of F. oreochromis and F. covae. F. oreochromis and F. covae exhibited notable decreased susceptibility to quinolones, with mutations observed in the quinolone resistance-determining regions (QRDRs) of gyrA, including Ser83Phe, Ser83Val, Ser83Ala, and Asp87Tyr, the latter representing a novel mutation among isolates from Thailand. As a result, these findings suggest that gyrA is major target for quinolone in F. oreochromis, F. covae, and F. davisii, while gyrB, parC, parE might be less important to the decreased phenotypic susceptibility to this class of antimicrobials. Moreover, a tetracycline resistance gene (tetA_2) was found in only one F. covae isolate, which exhibited decreased phenotypic susceptibility to this drug, marking the first report of decreased susceptibility in this species.

Conclusions: This study provides insights into the genetic and pathogenic diversity of Flavobacterium species, aiding in the development of strategies to manage columnaris disease in farmed freshwater fish in Thailand.

Keywords: Flavobacterium; Antibiotic resistance; Comparative genome analysis; Freshwater fish; Virulence factor.

Fig. 1

Lesions caused by columnaris disease in red tilapia (Oreochromis sp.), showing necrosis in the gill filaments (red arrow)

Fig. 2

Phylogenetic relationship of F. oreochromis, F. covae, F. columnare, and F. davisii generated from 16S rRNA sequences. Host species are represented by star and circular shapes, Flavobacterium species by triangles, and countries of isolates by rectangles. (Certain color references in this figure legend can be interpreted by the reader through the online version of this article)

Fig. 3

Pan-genome analysis from twenty-two isolates of F. oreochromis, F. covae, and F. davisii. A The number of core genes, accessory genes, and unique genes presented in each isolate. B COG functional categories of core genes, accessory genes, and unique genes among twenty-two isolates. C Core pan-genome curves of power-fit and exponential are indicated by brown lines, and purple lines. The corresponded equations are provided within the figure legend. The blue dots indicated total gene families; the pink dots indicated core gene families. D Phylogenetic analysis based on the core gene sequence of twenty-two isolates of F. oreochromis, F. covae, and F. davisii. (Certain color references in this figure legend can be interpreted by the reader through the online version of this article)

Fig. 4

Phylogenetic analysis of twenty-two isolates of F. oreochromis, F. covae, and F. davisii based on whole genome sequences. Reference isolates, including F. oreochromis (Costa Rica 04–02-TN^T), F. covae (AL-02–36 ^T, 94–081, C#2), F. davisii (90–106 ^T), and F. columnare (ATCC 49512) were used, with F. psychrophilum JIP02/86 as an outgroup

Fig. 5

SEED subsystem categorization of F. oreochromis, F. covae, and F. davisii genomes. Each colored bar indicated the number of genes assigned to specific categories

Fig. 6

Distribution of putative virulence factors among twenty-two Thai isolates of F. oreochromis, F. covae, and F. davisii

Fig. 7

Secretion systems distribution among twenty-two Thai isolates of F. oreochromis, F. covae, and F. davisii

Fig. 8

A Distribution of antimicrobial resistance genes predicted among twenty-two Thai isolates of F. oreochromis, F. covae, and F. davisii. B the percentage amino acid identity of quinolone resistance genes from F. oreochromis, F. covae, and F. davisii isolates compared to the Comprehensive Antibiotic Resistance Database. (Certain color references in this figure legend can be interpreted by the reader through the online version of this article)

Fig. 9

Analysis of genomic islands in twenty-two Thai isolates of F. oreochromis, F. covae, and F. davisii isolates. Representation of prediction methods: Red (integrated methods), Orange (SIGI-HMM), Blue (IslandPath-DIMOB). Identification of incomplete phage components using the PHASTER tool. (Certain color references in this figure legend can be interpreted by the reader through the online version of this article)

Fig. 10

Insertion elements in 22 Thai isolates of F. oreochromis, F. covae, F. davisii