Genomics of Tenacibaculum Species in British Columbia, Canada

Abstract

Tenacibaculum is a genus of Gram-negative filamentous bacteria with a cosmopolitan distribution. The research describing Tenacibaculum genomes stems primarily from Norway and Chile due to their impacts on salmon aquaculture. Canadian salmon aquaculture also experiences mortality events related to the presence of Tenacibaculum spp., yet no Canadian Tenacibaculum genomes are publicly available. Ribosomal DNA sequencing of 16S and four species-specific 16S quantitative-PCR assays were used to select isolates cultured from Atlantic salmon with mouthrot in British Columbia (BC), Canada. Ten isolates representing four known and two unknown species of Tenacibaculum were selected for shotgun whole genome sequencing using the Oxford Nanopore's MinION platform. The genome assemblies achieved closed circular chromosomes for seven isolates and long contigs for the remaining three isolates. Average nucleotide identity analysis identified T. ovolyticum, T. maritimum, T. dicentrarchi, two genomovars of T. finnmarkense, and two proposed novel species T. pacificus sp. nov. type strain 18-2881-A^T and T. retecalamus sp. nov. type strain 18-3228-7B^T. Annotation in most of the isolates predicted putative virulence and antimicrobial resistance genes, most-notably toxins (i.e., hemolysins), type-IX secretion systems, and oxytetracycline resistance. Comparative analysis with the T. maritimum type-strain predicted additional toxins and numerous C-terminal secretion proteins, including an M12B family metalloprotease in the T. maritimum isolates from BC. The genomic prediction of virulence-associated genes provides important targets for studies of mouthrot disease, and the annotation of the antimicrobial resistance genes provides targets for surveillance and diagnosis in veterinary medicine.

Keywords: Atlantic salmon; Tenacibaculum; antimicrobial resistance; de novo assembly; diversity; mouthrot; phylogenetics; virulence.

Figure 1

Circular Tenacibaculum chromosome plots assembled from Nanopore sequence data. Genomic elements are illustrated by Proksee (https://proksee.ca/, accessed on 28 November 2022). Orientation and origins are based on the rpiB gene. Characters displayed include coding DNA sequences (CDS), transfer-RNA (tRNA), ribosomal-RNA (rRNA), non-coding RNA (ncRNA), transfer-messenger RNA (tmRNA). Tracks outward in include characters of the genome, guanine-cytosine (GC) content, GC skews (+/−), and rRNA.

Figure 2

Multilocus sequence (atpA, dnaK, glyA, gyrB, infB, rlmN, and tgt) maximum-likelihood phylogeny of barcodes 1–10 using a GTR+F+I+G4 model. Branch lengths are proportional to the phylogenetic distance. Bootstrap values (10000 ultrafast bootstraps and 10000 SH-aLRT tests) above 80% are represented by green values at the branch. Non-barcode or outgroup sequences are derived from Tenacibaculum PUBMLST database (https://pubmlst.org/organisms/tenacibaculum-spp, accessed on 28 November 2022), and Korida and Flavobacterium sequences were obtained from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 28 November 2022).

Figure 3

AtpA (A) and fusA (B) maximum-likelihood phylogenies of barcodes 1–10 using GTR+F+G4 and TVM+F+G4 models respectively. Branch lengths are proportional to the phylogenetic distance. Bootstrap values (10,000 ultrafast bootstraps and 10000 SH-aLRT tests) above 80% are represented by green values at the branch. Non-barcode sequences were obtained from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 28 November 2022).

Figure 4

16S rDNA maximum-likelihood phylogeny of barcodes 1–10 using a TVM+F+I+G4 model. Branch lengths are proportional to phylogenetic distance. Bootstrap values (10000 ultrafast bootstraps and 10000 SH-aLRT tests) above 80% are represented by green values at the branch. Non-barcode sequences were obtained from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 28 November 2022).