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-AT and T. retecalamus sp. nov. type strain 18-3228-7BT. 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.

Tenacibaculosis in wild-caught, captive Chinook salmon (Oncorhynchus tshawytscha) in British Columbia, Canada

“…T. maritimum and T. dicentrarchi may be especially abundant in and around farms sites off the coast of BC in warm temperature summer months (Nowlan et al, 2021a) while T. finnmarkense outbreaks have been primarily associated with colder water temperatures on Norwegian farms (Spilsberg et al, 2022). Overall, diversity of the Tenacibaculum genus is greater than previously understood (Nowlan et al, 2023), and further genetic sequencing will be needed to understand the range of species that are involved in disease in BC and globally. Regardless of the source, or the possible impacts of environmental conditions, these findings demonstrate that T. dicentrarchi can result in infection, development of tenacibaculosis, and mortality in wild Chinook salmon.…”

“…These have both been detected on salmon farms, can co-occur with T. maritimum , and may be responsible for some cases of tenacibaculosis (Alvarez and Santos, 2018; Avendaño-Herrera et al 2016; Smage et al, 2016, 2018; Olsen et al, 2017; Mimeault et al, 2020). On Atlantic salmon in BC, at least six species of Tenacibaculum have been confirmed, including T. maritimum, T. dicentrarchi and T. finnmarkense (Smage et al, 2018; Nowlan et al 2021a, b; Nowlan et al,2023). The latter three species have been established through international challenge studies as causal agents of tenacibaculosis (Smage et al, 2018; Frisch et al, 2018; Klakegg et al, 2019; Nowlan et al, 2021b; Valdes et al, 2021).…”

Tenacibaculum ovolyticum 16S rDNA Quantitative-PCR Assay Development and Field Testing

“…Isolates and DNA Used A T.ovo isolate (20-4135-2 [in-house name]) was obtained by swabbing a mouth lesion on an Atlantic salmon that exhibited mouthrot at a BC marine net-pen site in the Broughton Archipelago, and was cultured on Flexibacter maritimus media supplemented with kanamycin (50 µg•mL −1 ) at 12 • C. T.ovo 20-4135-2 (Genbank Accession #: OP629685) was most similar to T.ovo da5A-8 based on 16S rDNA sequencing using universal 27F (5 -AGAGTTTGATCATGGCTCAG -3 ) and 1492R (5 -GGTTACCTTGTTACGACTT -3 ) primers (Table 1). T.ovo 20-4135-2 was also sequenced using MinION nanopore long-read technologies [19]. A FastANI [20] comparison against the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov [accessed on 1 August 2022]) sequences of T.ovo da5A-8 (GCF_001641405.1_ASM164140v1), T.ovo EKD 002 T (DSM 18103) (GCF_000430545.1_ASM43054v1), and T.ovo To-7Br (GCF_021852385.1_ASM2185238v1) were over 95% similar, passing the threshold for Tenacibaculum species-level identification [19,21,22].…”

“…T.ovo 20-4135-2 was also sequenced using MinION nanopore long-read technologies [19]. A FastANI [20] comparison against the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov [accessed on 1 August 2022]) sequences of T.ovo da5A-8 (GCF_001641405.1_ASM164140v1), T.ovo EKD 002 T (DSM 18103) (GCF_000430545.1_ASM43054v1), and T.ovo To-7Br (GCF_021852385.1_ASM2185238v1) were over 95% similar, passing the threshold for Tenacibaculum species-level identification [19,21,22]. Recent work on the complete genome of T.ovo 20-4135-2 identified six copies of 16S rDNA and a single nucleotide polymorphism [19].…”

“…Rough estimates for the limit of detection (LOD), were based on the complete genome length (~4.1-4.2 Mb) of T.ovo EKD 002 T (DSM 18103) (NCBI: GCF_000430545.1_ASM43054v1), da5A-8 (NCBI: GCF_001641405.1_ASM164140v1), and 20-4135-2 (19), and the DNA Copy Number, and Dilution Calculator (https://www.thermofisher.com/ca/en/home/brands/ thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biologyresource-library/thermo-scientific-web-tools/dna-copy-number-calculator.html [accessed on 10 September 2021]) using a 'Custom DNA Fragment' and 'Genome Length' of 4.15 Mb, indicating ~223,345 copies of the genome per•ng −1 .…”