In this study, we isolated, identified and characterized isolates of Tenacibaculum dicentrarchi in Atlantic salmon (Salmo salar) farmed in Chile for the first time. In 2010 and 2014, mortalities were observed in Atlantic salmon (average weight 25-30 and 480-520 g, respectively) at an aquaculture centre in Puerto Montt, Chile. Severe tail rots, frayed fins and, in some cases, damaged gills were detected. Wet smear analyses of these lesions revealed a high occurrence of Gram-negative, filamentous bacteria. Microbiological analysis of infected gill and tail tissues yielded six bacterial isolates. All were identified as T. dicentrarchi through polyphasic taxonomy, which included phenotypic characterization, 16S rRNA sequencing and multilocus sequence typing. The latter method revealed a close relationship of the Chilean genotype with the T. dicentrarchi type strain and two Norwegian Atlantic cod (Gadus morhua) isolates. The pathogenic potential of the TdChD05 isolate was assessed by challenging Atlantic salmon and rainbow trout (Oncorhynchus mykiss) for one hour, which resulted in mean cumulative mortality rates of 65% and 93%, respectively, as well as clinical signs 14 days post-challenge. However, challenged Coho salmon (Oncorhynchus kisutch) presented no mortalities or clinical signs of infection. These findings indicate that the geographical and host distribution of T. dicentrarchi is wider than previously established and that this bacterium may have negative impacts on salmonid cultures.

Results of previous multilocus sequence and whole-genome-based analyses have suggested that a homogeneous group of isolates belonging to the genus Tenacibaculum , represented by strain TNO020T and associated with skin ulcer development in sea-farmed fish, represents an as-yet-undescribed species. Comparative whole-genome analysis performed in the present study clustered five isolates, including TNO020T, in a distinct lineage within the genus Tenacibaculum . Phenotypic differences, high intra-cluster average nucleotide identity (ANI) values and low ANI values with other Tenacibaculum species support the proposal of a novel species, for which we propose the name Tenacibaculum piscium sp. nov. with strain TNO020T (=CCUG 73833T=NCIMB 15240T) as the type strain. Further, large-scale genome analyses confirmed the existence of two different phylogenetic lineages within ‘ T. finnmarkense ’, a species effectively but not validly published previously. ANI values just above the species delineation threshold of 95–96 % confirmed that both lineages belong to the same species. This result was also supported by DNA–DNA hybridization values. Phenotypically, the two conspecific lineages are distinguishable by differences in growth temperature range and ability to degrade l-proline. For the group of isolates already commonly known as ‘ T. finnmarkense ’, we propose the name Tenacibaculum finnmarkense sp. nov., with strain TNO006T (=CCUG 73831T=NCIMB 15238T) as the type strain. We further propose the subdivision of T. finnmarkense sp. nov. into two genomovars, T. finnmarkense genomovar finnmarkense with strain TNO006T (=CCUG 73831T=NCIMB 15238T) as the type strain and T. finnmarkense genomovar ulcerans with strain TNO010T (=CCUG 73832T=NCIMB 15239T) as the type strain.

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Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socioeconomic impacts for Chilean salmonid aquaculture. The identification of major environmental reservoirs for P. salmonis has long been ignored. Most microbial life occurs in biofilms, with possible implications in disease outbreaks as pathogen seed banks. Herein, we report on an in vitro analysis of biofilm formation by P. salmonis Psal-103 (LF-89-like genotype) and Psal-104 (EM-90-like genotype), the aim of which was to gain new insights into the ecological role of biofilms using multiple approaches. The cytotoxic response of the salmon head kidney cell line to P. salmonis showed interisolate differences, depending on the source of the bacterial inoculum (biofilm or planktonic). Biofilm formation showed a variable-length lag-phase, which was associated with wider fluctuations in biofilm viability. Interisolate differences in the lag phase emerged regardless of the nutritional content of the medium, but both isolates formed mature biofilms from 288 h onwards. Psal-103 biofilms were sensitive to Atlantic salmon skin mucus during early formation, whereas Psal-104 biofilms were more tolerant. The ability of P. salmonis to form viable and mucus-tolerant biofilms on plastic surfaces in seawater represents a potentially important environmental risk for the persistence and dissemination of piscirickettsiosis. Piscirickettsiosis, or salmon rickettsial septicemia, is a fish disease caused by the facultative intracellular bacterium Piscirickettsia salmonis. First described in relation to the Chilean salmon farming industry 1 , this pathogen has since been reported in North America and Europe 2. A decade ago, P. salmonis was considered an obligate intracellular pathogen 3 , but later research achieved growth on artificial cell-free media 4,5. P. salmonis can survive outside of hosts for a long time as free-living cells, having been detected around salmon farms by qPCR up to 30 days after cage emptying 6 or as a biofilm mode-of-growth in a marine broth medium for 15-30 days 7. Biofilm formation is a multi-step process that involves bacterial attachment to surfaces, microcolony formation, growthdependent maturation, and cell detachment from mature biofilms to colonize new habitats 8. One persisting question is if the survival behavior of P. salmonis as a free-living bacterium is linked with a biofilm lifestyle in marine habitats. Bacterial biofilms are nearly ubiquitous in all major habitats 9 , including some synthetic habitats, such as the "plastisphere" of marine environments 10-12 .

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty-two Chilean isolates and the DSM20767 type strain grew in the presence of 2,2'-Dipyridyl at varying concentrations (250-330 μm), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200 μm of 2,2'-Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron-limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100 μm), it did facilitate growth at lower iron concentrations (10 μm and 1 μm). An exception was the H-2 isolate, which only grew with 100 μm of iron sulphide. In turn, 100 μm of haemin was toxic when isolates were grown in normal KDM-2. In silico R. salmoninarumATCC 33209 genome analysis detected various genes coding iron uptake-related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.

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This is the first study to isolate, identify and characterize Streptococcus iniae as the causative disease agent in two tilapia (Oreochromis aureus) populations. The populations were geographically isolated, of distinct origins, and did not share water sources. Affected fish showed various external (e.g., exophthalmia and cachexia, among others) and internal (e.g., granulomatous septicaemia and interstitial nephritis, among others) signs. All internal organ samples produced pure cultures, two of which (one from each farm, termed S-1 and S-2) were subjected to biochemical, PCR and 16S rRNA sequencing (99.5% similarity) analyses, confirming S. iniae identification. The two isolates presented genetic homogeneity regardless of technique (i.e., RAPD, REP-PCR and ERIC-PCR analyses). Pathogenic potentials were assessed through intraperitoneal injection challenges in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). Rainbow trout mortalities were respectively 40% and 70% at 10 and 10 CFU per fish with the S-1 isolate, while 100% mortality rates were recorded in zebrafish at 10 and 10 CFU per fish with the S-2 isolate. The obtained data clearly indicate a relationship between intensified aquaculture activities in Mexico and new disease appearances. Future studies should establish clinical significances for the tilapia industry.