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Tenacibaculum dicentrarchi is the second most important pathogen in Chilean salmon farming. This microorganism causes severe skin lesions on the body surface of farmed fish. The bacterium can also adhere to surfaces and form biofilm, survive in fish skin mucus, and possess different systems for iron acquisition. However, the virulence mechanisms are still not fully elucidated. Outer membrane vesicles (OMV) are nanostructures released by pathogenic Gram-negative bacteria during growth, but none has been described yet for T. dicentrarchi. In this study, we provide the first reported evidence of the fish pathogen T. dicentrarchi producing and releasing OMV from 24 h after incubation, increasing thereafter until 120 h. Analyses were conducted with T. dicentrarchi TdCh05, QCR29, and the type strain CECT 7612T . The OMV sizes, determined via scanning electron microscopy, ranged from 82.25 nm to 396.88 nm as per the strain and incubation time point (i.e., 24 to 120 h). SDS-PAGE revealed that the number of protein bands evidenced a drastically downward trend among the T. dicentrarchi strains. In turn, the OMV shared five proteins (i.e., 22.2, 31.9, 47.7, 56.3, and 107.1 kDa), but no protein pattern was identical. A heterogeneous amount of protein, RNA, and DNA were obtained, depending on the time at which OMV were extracted. Purified OMV were biologically active and induced a cytotoxic effect in macrophage-enriched cell cultures from rainbow trout (Oncorhynchus mykiss) head kidneys. This is the first step towards understanding the role that OMV could play in the pathogenesis of T. dicentrarchi.

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RESUMO

Iron uptake during infection is an essential pathogenicity factor of several bacteria, including Tenacibaculum dicentrarchi, an emerging pathogen for salmonid and red conger eel (Genypterus chilensis) farms in Chile. Iron-related protein families were recently found in eight T. dicentrarchi genomes, but biological studies have not yet confirmed functions. The investigation reported herein clearly demonstrated for the first time that T. dicentrarchi possesses different systems for iron acquisition-one involving the synthesis of siderophores and another allowing for the utilization of heme groups. Using 38 isolates of T. dicentrarchi and the type strain CECT 7612T , all strains grew in the presence of the chelating agent 2.2'-dipyridyl (from 50 to 150 µM) and produced siderophores on chrome azurol S plates. Furthermore, 37 of the 38 T. dicentrarchi isolates used at least four of the five iron sources (i.e. ammonium iron citrate, ferrous sulfate, iron chloride hexahydrate, haemoglobin and/or hemin) when added to iron-deficient media, although the cell yield was less when using hemin. Twelve isolates grew in the presence of hemin, and 10 of them used only 100 µM. Under iron-supplemented or iron-restricted conditions, whole cells of three isolates and the type strain showed at least one membrane protein induced in iron-limiting conditions (c.a. 37.9 kDa), regardless of the isolation host. All phenotypic results were confirmed by in-silico genomic T. dicentrarchi analysis. Future studies will aim to establish a relationship between iron uptake ability and virulence in T. dicentrarchi through in vivo assays.

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RESUMO

Tenacibaculum dicentrarchi is an emerging pathogen for salmonid cultures and red conger eel (Genypterus chilensis) in Chile, causing high economic losses not only in Chile but also to the global salmon industry. Infected fish show severe gross skin lesions that are sometimes accompanied by bone exposure. Despite pathogenicity demonstrated by Koch's postulates, no knowledge is currently available regarding the virulence machinery of T. dicentrarchi strains. Comparisons between the genome sequences of the eight T. dicentrarchi strains obtained from G. chilensis and Atlantic salmon (Salmo salar) provide insights on the existence of genomic diversity within this bacterium. The T. dicentrarchi type strain 3509T was used as a reference genome. Depending on the T. dicentrarchi strain, the discovered diversity included genes associated with iron acquisition mechanisms, copper homeostasis encoding, resistance to tetracycline and fluoroquinolones, pathogenic genomic islands and phages. Interestingly, genes encoding the T9SS membrane protein PorP/SprF were retrieved in all of the analysed T. dicentrarchi strains, regardless of the host fish (i.e. red conger eel or Atlantic salmon). However, the T6SS core component protein VgrG was identified in only one Atlantic salmon strain. Three types of peptidase genes and proteins associated with quorum sensing were detected in all of the T. dicentrarchi strains. In turn, all eight strains presented a total of 17 proteins associated with biofilm formation, which was previously confirmed through physiological studies. This comparative analysis will help elucidate and describe the genes and pathways that are likely involved in the virulence process of T. dicentrarchi. All or part of these predicted genes could aid the pathogen during the infective process in fish, making further physiological research necessary for clarification.

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RESUMO

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.

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