The Emerging Fish Pathogen Flavobacterium spartansii Isolated from Chinook Salmon: Comparative Genome Analysis and Molecular Manipulation

Abstract

Flavobacterium spartansii strain T16^T was isolated from a disease outbreak in hatchery-reared Chinook salmon (Oncorhynchus tshawytscha) fingerlings. To gain insight into its genomic content, structure and virulence pathogenesis factors, comparative genome analyses were performed using genomes from environmental and virulent Flavobacterium strains. F. spartansii shared low average nucleotide identity (ANI) to well-known fish-pathogenic flavobacteria (e.g., F. columnare, F. psychrophilum, and F. branchiophilum), indicating that it is a new and emerging fish pathogen. The genome in T16^T had a length of 5,359,952 bp, a GC-content 35.7%, and 4,422 predicted protein-coding sequences. Flavobacterium core genome analysis showed that the number of shared genes decreased with the addition of input genomes and converged at 1182 genes. At least 8 genomic islands and 5 prophages were predicted in T16^T. At least 133 virulence factors associated with virulence in pathogenic bacteria were highly conserved in F. spartansii T16^T. Furthermore, genes linked to virulence in other bacterial species (e.g., those encoding for a type IX secretion system, collagenase and hemolysin) were found in the genome of F. spartansii T16^T and were conserved in most of the analyzed pathogenic Flavobacterium. F. spartansii was resistant to ampicillin and penicillin, consistent with the presence of multiple genes encoding diverse lactamases and the penicillin-binding protein in the genome. To allow for future investigations into F. spartansii virulence in vivo, a transposon-based random mutagenesis strategy was attempted in F. spartansii T16^T using pHimarEm1. Four putative gliding motility deficient mutants were obtained and the insertion sites of pHimarEm1 in the genome of these mutants were characterized. In total, study results clarify some of the mechanisms by which emerging flavobacterial fish pathogens may cause disease and also provide direly needed tools to investigate their pathogenesis.

Keywords: Flavobacterium spartansii; genome analysis; mutation; virulence factors.

Figure 1

Pan and core genome evolution according to the number of sequenced Flavobacterium genomes. (A) Total number of genes (pan-genome) for a given number of genomes sequentially added. (B) Number of shared genes (core genome) as a function of the number of genomes sequentially added.

Figure 2

Venn diagram of shared and unique genes in the selected Flavobacterium. The unique and shared genome among the compared genomes were determined using the BLAST score ratio approach of EDGAR 2.0 with a cutoff of 30% (Blom et al., 2016).

Figure 3

Genomic islands in F. spartansii T16^T. (A) The 8 genomic islands were predicted by Islandviewer. (B) The schematic of GI-associated features. The relative locations of the 8 GIs were shown in the predicted genome. The conjugation protein genes belonging to “tra” and “GG” in CTnFs were centered in the GI-7 and GI-8 regions, respectively.

Figure 4

Demonstration of the hemolytic activity in F. spartansii. The hemolytic activity on sheep blood agar plate was observed after 48-h incubation. (A) F. spartansii, (B) S. aureus MSU001, the β-hymolysin control, and (C) E. meningoseptica ATCC 13253, α-hemolysin control.

Figure 5

Comparation of the cell growth in PY2 broth and gliding motility on the PY2 agar between the WT and mutants. Transposon pHimarEm1 was introduced into F. spartansii T16^T and erythromycin-resistant conjugants were sub-cultured in PY2 broth. The cell growth was compared by determining the OD600nm after overnight culture (up panel). The motility was evaluated by culture the cells on PY2 agar after 48 h (low panel).