O‐Antigen Gene Cluster Reveals Genomic Variation in Chilean <i>Tenacibaculum dicentrarchi</i> Through Multiplex <scp>PCR</scp>‐Based Genotyping Scheme

Tenacibaculum dicentrarchi emerged as a major pathogen in Chilean salmon farming in early 2023, causing 32.9% of Atlantic salmon (Salmo salar) mortalities. Although recent studies have provided valuable insights into T. dicentrarchi through virulence mechanisms and genome analysis, genetic diversity based on the O-antigen gene cluster remains largely unexplored. In this study, we conducted a comparative genomic analysis of O-antigen biosynthesis genes in 30 whole-genome sequenced strains, including the T. dicentrarchi type strain. The analysis identified a single O-antigen gene cluster (O-AGC) consisting of 20 genes involved in the biosynthesis of this component of the lipopolysaccharide layer. Variations in the O-AGC revealed antigenic diversity within the species, allowing classification into four distinct groups, designated Types 1 to 4. Based on these findings, we developed a multiplex PCR-based genotyping scheme, which was successfully applied to 25 bacterial isolates from Chilean fish farms. Most isolates were identified as Type 1 and 3, while Types 2 and 4 were less common, with the same number of isolates. We also investigated whether core genome phylogeny correlated with O-AGC Types by including publicly available genomes from Chile, Norway and Canada. Notably, T. dicentrarchi isolates clustered into two groups: one comprising isolates from Norway and Canada, all belonging to Type 1. Another group consisted of Chilean isolates with diverse O-AGC Types (i.e., 1, 2, 3 and 4), including the type strain. This multiplex PCR approach provided a valuable tool for rapid and reliable typing of T. dicentrarchi, facilitating epidemiological studies and aiding in the selection of appropriate isolates for the vaccine development against tenacibaculosis in fish farms.