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.