The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov.

Authors: Lafrentz, Benjamin; KRÁLOVÁ, STANISLAVA - Masaryk University; BURBICK, CLAIRE - Washington State University; ALEXANDER, TREVOR - Washington State University; PHILLIPS, CONNER - Washington State University; GRIFFIN, MATT - Mississippi State University; Waldbieser, Geoffrey - Geoff; Garcia, Julio; DE ALEXANDRE, SEBASTIÃO F - University Of California; SOTO, ESTEBAN - University Of California; LOCH, THOMAS - Michigan State University; LILES, MARK - Auburn University; SNEKVIK, KEVIN - Washington State University

Submitted to: Systematic and Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2021
Publication Date: 1/10/2022
Publication URL: https://handle.nal.usda.gov/10113/7611579
Citation: Lafrentz, B.R., Králová, S., Burbick, C.R., Alexander, T.L., Phillips, C.W., Griffin, M.J., Waldbieser, G.C., Garcia, J.C., De Alexandre, S., Soto, E., Loch, T.P., Liles, M.R., Snekvik, K.R. 2022. The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov. Systematic and Applied Microbiology. 45:126293. https://doi.org/10.1016/j.syapm.2021.126293.
DOI: https://doi.org/10.1016/j.syapm.2021.126293

Interpretive Summary: Flavobacterium columnare is a devastating fish pathogen and the causative agent of columnaris disease in wild and cultured fish populations worldwide. In aquaculture, high mortality rates are common and result in significant economic losses due to fish mortality, reduced feeding activity during epizootics, and increased treatment expenditures. Research in our laboratory previously demonstrated four phylogenetically distinct genetic groups within F. columnare, suggesting that the species designation requires revision. In the present research, the taxonomic status of the four genetic groups was determined. A broad research approach was undertaken to characterize the four genetic groups including phylogenetic analyses, whole genome sequencing, biochemical and physiological characterization, antimicrobial susceptibility, chemotaxonomic analyses, and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The results demonstrate the genetic groups of F. columnare represent four different species. Quantitative differences in fatty acid profiles and MALDI-TOF were useful for discriminating between the four genetic groups. In silico DNA-DNA hybridization and ANI value calculations fully support these groups as unique species with values well below the thresholds used to distinguish between members of closely related species. Based on these findings, it is proposed that genetic group 1 isolates remain recognized as F. columnare while isolates of genetic groups 2, 3, and 4 be recognized as novel Flavobacterium species for which the names Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov., and Flavobacterium oreochromis sp. nov., respectively are proposed. Since these pathogens are globally distributed and have significant impacts on wild and cultured fish species, recognition of the four species will advance and improve research to define host-pathogen-environment relationships, epidemiology, and develop effective control and prevention measures in aquaculture. Such research needs to target the correct bacterial species and research findings can be properly interpreted by correct and consistent taxonomic assignment.

Technical Abstract: Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish and four discrete genetic groups exist within the species, suggesting that the species designation requires revision. The present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches and included five representative isolates from each genetic group (including type strain ATCC 23463(T); genetic group 1). 16S rRNA gene sequence analysis revealed genetic group 2 isolate AL-02-36(T), genetic group 3 isolate 90-106(T), and genetic group 4 isolate Costa Rica 04-02-TN(T) shared less than <98.8 % sequence identity to F. columnare ATCC 23463(T). Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463(T), genetic group 2 isolate AL-02-36(T), genetic group 3 isolate 90-106(T), and genetic group 4 isolate Costa Rica 04-02-TN(T) were less than 90.84% and 42.7%, respectively. Biochemical and physiological characteristics were similar among the four genetic groups; however, quantitative differences in fatty acid profiles were detected and MALDI-TOF analyses demonstrated numerous distinguishing peaks unique to each genetic group. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. Herein, the names F. covae sp. nov. [AL-02-36(T)], F. davisii sp. nov. [90-106(T)], and F. oreochromis sp. nov. [Costa Rica 04-02-TN(T)] are proposed to represent genetic groups 2, 3, and 4, respectively.