Tenacibaculosis caused by Tenacibaculum dicentrarchi is the third most important bacterial fish infection affecting the Chilean salmon industry. Losses to this disease are most frequently controlled ...by treatments with florfenicol and oxytetracycline. However, recent tenacibaculosis outbreaks were controlled through the extra‐label, oral administration of tiamulin, resulting in high treatment efficiency. In this study, we present an analysis of susceptibility patterns of 32 T. dicentrarchi isolates and the type strain CECT 7612T to tiamulin by determining the minimum inhibitory concentrations (MICs) according to the procedures recommended by the Clinical and Laboratory Standard Institute, but fixing incubation temperature to the more appropriate for the growth of T. dicentrarchi (18 ºC). The MICs of the T. dicentrarchi isolates were unimodally distributed (0.06–1.0 µg/ml range), while the CECT 7612T strain presented an MIC of 0.5 μg/ml. Calculations using Normalized Resistance Interpretation provided epidemiological cut‐off values of ≤1.0 µg/ml, with the 33 T. dicentrarchi classified as wild type. In Chile, tiamulin is authorized for use in other livestock species, but application in salmonids is extra‐label. Our presented in vitro results suggest that tiamulin is a viable alternative to florfenicol, specifically as tiamulin requires comparatively lower concentrations to inhibit T. dicentrarchi. Considering that tiamulin is also exclusively for veterinary use, is classified as “least important” by the World Health Organization and has not resulted in the development of bacterial resistance, pharmaceutical companies should be requested to register tiamulin and provide alternative antimicrobial treatments for the salmonid industry.
Flavobacterium psychrophilum is a pathogenic bacterium affecting Chilean salmonid farms. High antigenic and genetic diversity exists among Chilean F. psychrophilum isolates, but the distribution ...thereof among farms is poorly understood. These epidemiological data are key for developing isolate-specific vaccines. The present study isolated F. psychrophilum in diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) from five freshwater farms between 2018 and 2019. Each farm only raised one salmonid species and was geographically separated from and did not share culturing water with the other farms. Antigenic and genetic analyses were conducted to shed light on the possibility of isolates coexisting within the same farm during outbreaks. A total of 68 Chilean F. psychrophilum isolates were recovered from skin lesions, gills, fins, kidney and spleen of moribund and live fish. Among the 68 Chilean isolates, mPCR-serotyping indicated three major serotypes (i.e. 23.5% type 0; 47.1% type 2; and 26.5% type 4) and, to a lesser degree, serotype 1 (2.9%). Sixteen antigenic groups were detected by slide agglutination. Genetic characterizations by 16S rRNA alleles identified 71% of the isolates as the virulent genogroup CSF259-93 allele. A predominant serotype was associated with each farm, with types 0 and 4 related to Atlantic salmon and types 1 and 2 to rainbow trout. Notwithstanding, several antigenic groups coexisted within some farms. Likewise, the experimental intramuscular challenges (n = 20) demonstrated that the type-2 isolates from rainbow trout were the most pathogenic among isolates recovered from infectious outbreaks in Atlantic salmon, especially as compared to those from types-0 and -4. These results allow us to suggest that prevention measures, specifically vaccines, should be developed according to dominant isolates and with specificity to each farm, that is the use of autogenous or site-specific vaccines.
Chile is the second largest producer of salmon (only behind Norway), and high amounts of antimicrobials are used during the marine stage, mainly florfenicol. Intensive antimicrobials use is a ...relevant concern, prompting the urgent need for use reduction and advancements towards more restricted, efficient application. This review describes the historic and current use of antimicrobials in Chilean salmonid farming. Further covered is the experimental evidence reported to date, thus providing a clearer understanding for the pathogenic nature of Piscirickettsia salmonis—the most important bacterial pathogen and the main target of antimicrobials used in the Chilean industry. This review also assesses research exploring the impacts of antimicrobial treatment on the environment, as well as investigations on the antibiotic susceptibility of salmonid pathogens. This review of the scientific literature contributes to a more unbiased appraisal of the current situation in Chile. Unfortunately, various previous reports dealing with/on the antimicrobial use in the Chilean salmon farming industry have been arrived to insufficiently supported conclusions, thus the present review aims to clarify outdated or poorly substantiated information and assumptions in existing literature. The consequences of antimicrobials in fish farming are covered by this review, which is particularly relevant considering potential impacts on aquatic environments. Focus is given to the selection and spread of drug‐resistant bacteria and associated resistance‐encoding genes. Finally, herein are provided several recommendations from a One Health perspective for decreasing amounts of used antimicrobials, effectively controlling primary bacterial fish pathogens, developing proper prevention strategies and reducing the dissemination of antimicrobial resistance.
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 7612
. 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.
Flavobacterial infection associated with diseased fish is caused by multiple bacterial species within the family Flavobacteriaceae. In the present study, the Chilean isolate FP99, from the gills of a ...diseased, farmed rainbow trout (Oncorhynchus mykiss), was characterized using phenotypic and genomic analyses. Additionally assessed was pathogenic activity. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that isolate FP99 belonged to the genus Epilithonimonas, an average nucleotide identity value of 100% was detected with the Chilean isolate identified as Epilithonimonas sp. FP211‐J200. In silico genome analysis, mechanisms for toxins production, and superantigens, adhesion, or other genes associated with virulence were not detected. However, genes encoding proteins for antibiotic resistance were found, including the chrA gene and the nucleotide sequence that encodes for multiple antibiotic resistance MarC proteins. Furthermore, the blaESP‐1 gene (87.85% aminoacidic sequence identity), encoding an extended‐spectrum subclass B3 metallo‐β‐lactamase and conferring carbapenem‐hydrolysing activity, and the tet(X) gene, which encodes a monooxygenase that catalyses the degradation of tetracycline‐class antimicrobials were carried by this isolate. Phenotyping analyses also supported assignment as E. ginsengisoli. Challenge trials against healthy rainbow trout resulted in no observed pathogenic effect. Our findings identify for the first time the species E. ginsengisoli as associated with fish farming, suggesting that this isolate may be a component of the microbiota of the freshwater system. Notwithstanding, poor environmental conditions and any stressors associated with aquaculture situations or lesions caused by other pathogenic bacteria, such as F. psychrophilum, could favour the entry of E. ginsengisoli into rainbow trout.
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.
The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a ...vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot‐blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross‐reactions, identifying three main serological patterns. When the PCR‐based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4–0; and most isolates, including the rainbow trout isolate, were 3–1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1–0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.
A strategy for vaccine design involves identifying proteins that could be involved in pathogen–host interactions. The aim of this proteomic study was to determine how iron limitation affects the ...protein expression of Tenacibaculum dicentrarchi, with a primary focus on virulence factors and proteins associated with iron uptake. The proteomic analysis was carried out using two strains of T. dicentrarchi grown under normal (control) and iron‐limited conditions, mimicking the host environment. Our findings revealed differences in the proteins expressed by the type strain CECT 7612T and the Chilean strain TdCh05 of T. dicentrarchi. Nonetheless, both share a common response to iron deprivation, with an increased expression of proteins associated with iron oxidation and reduction metabolism (e.g., SufA, YpmQ, SufD), siderophore transport (e.g., ExbD, TonB‐dependent receptor, HbpA), heme compound biosynthesis, and iron transporters under iron limitation. Proteins involved in gliding motility, such as GldL and SprE, were also upregulated in both strains. A negative differential regulation of metabolic proteins, particularly those associated with amino acid biosynthesis, was observed under iron limitation, reflecting the impact of iron availability on bacterial metabolism. Additionally, the TdCh05 strain exhibited unique proteins associated with gliding motility machinery and phage infection control compared to the type strain. These groups of proteins have been identified as virulence factors within the Flavobacteriaceae family, including the genus Tenacibaculum. These results build upon our previous report on iron acquisition mechanisms and could lay the groundwork for future studies aimed at elucidating the role of some of the described proteins in the infectious process of tenacibaculosis, as well as in the development of potential vaccines.
Renibacterium salmoninarum, a Gram‐positive intracellular pathogen, is the causative agent of bacterial kidney disease (BKD), the impacts of which are high mortalities and economic losses for the ...salmon industry. This study provides novel analyses for the whole‐genome sequences of 50 R. salmoninarum isolates and the reference strain ATCC 33209 using a pan‐genomic approach to elucidate phylogenomic relationships and identify unique and shared genes associated with pathogenicity and infection mechanisms. Genome size varied from 3,061,638 to 3,155,332 bp; gene count from 3452 to 3580; and predicted coding sequences from 3402 to 3527. Comparative analyses revealed an open, but approaching closed, pan‐genome. The pan‐genome analysis recovered 4064 genes, with a core genome containing 3306 genes. Phylogenetic analysis of R. salmoninarum showed high genomic homogeneity, apart from one isolate obtained from Salmo trutta in Norway. All genomes presented the 57‐kDa protein (p57). Strain ATCC 33209 and the Chilean isolates H‐2 and DJ2R presented two copies of the msa gene, while the remaining isolates had one copy. The pan‐genome analysis further identified differences in the number of copies and length of the signalling peptide for p57, the principal virulence factor reported for this bacterium. This heterogeneity could be associated with the secretion levels of p57, potentially influencing virulence. Additionally identified were numerous common genes related to iron uptake, the stress response and regulation, and cell signalling—all of which constitute the pathogenic repertoire of R. salmoninarum. This investigation provides information that is applicable in future studies for identifying therapeutic targets and/or for designing new strategies (e.g., vaccines) to prevent BKD infections in salmon farming.
