Draft genome for Flavobacterium psychrophilum isolates from diseased coho salmon (Oncorhynchus kisutch) in Chile.

We present the draft genome sequences of six Flavobacterium psychrophilum isolates recovered from diseased coho salmon (Oncorhynchus kisutch) cultured by two farms in Chile. This study provides the first detailed insights into the genomic characteristics of this fish pathogen recovered from a host with limited information and cultured in Chile.

Genomic features of Pseudomonas sp. RGM2144 correlates with increased survival of rainbow trout infected by Flavobacterium psychrophilum.

This study evaluated the probiotic potential of the biofilm formed by the strain Pseudomonas sp. RGM2144 on rainbow trout survival. When challenged with the fish pathogen Flavobacterium psychrophilum, Pseudomonas sp. RGM2144 increased rainbow trout survival to 92.7 ± 1.2% (control: 35.3 ± 9.5%, p < .0001). The draft genome of Pseudomonas sp. RGM2144 is 6.8 Mbp long, with a completeness 100% and a contamination of 0.4%. The genome contains 6122 protein-coding genes of which 3564 (~60%) have known functions. The genome and phylogeny indicate that Pseudomonas sp. RGM2144 is a new species in the Pseudomonas genus, with few virulence factors, plasmids, and genes associated with antimicrobial resistance, suggesting a non-pathogenic bacterium with protective potential. In addition, the genome encodes for 11 secondary metabolite biosynthetic gene clusters that could be involved in the inhibition of F. psychrophilum. We suggest that Pseudomonas sp. RGM2144 may be applied as a probiotic in salmonid fish farming.

Protective Effect of [Cu(NN1)2](ClO4) Complex in Rainbow Trout Challenged against Flavobacterium psychrophilum.

Previously, we reported an in vitro evaluation regarding antibacterial effects against F. psychrophilum by a new Cu (I) complex, [Cu(NN1)2](ClO4). This study presents the results of an in vivo evaluation of [Cu(NN1)2](ClO4) added as a dietary supplement against F. psychrophilum in rainbow trout. The results showed that the administration of [Cu(NN1)2](ClO4) at 29 and 58 µg/g of fish for 15 days does not affect the growth of rainbow trout. On the other hand, the amount of copper present in the liver, intestine, and muscle of rainbow trout was determined. The results showed that the amount of copper in the liver, when compared between treated fish and control fish, does not change. While, in the intestine, an increase in the fish fed at 58 µg/g of fish was observed. In muscle, a slight decrease at 29 µg/g was obtained. Additionally, copper concentrations in the pond water after 15 days of feeding with the [Cu(NN1)2](ClO4) complex showed the highest levels of copper. Finally, the effect of the administration of [Cu(NN1)2](ClO4) for 15 days at 58 µg/g of fish was evaluated against F. psychrophilum, where a 75% survival was obtained during 20 days of challenge.

Oral Administration of Lactococcus lactis Producing Interferon Type II, Enhances the Immune Response Against Bacterial Pathogens in Rainbow Trout.

Lactic acid bacteria are a powerful vehicle for releasing of cytokines and immunostimulant peptides at the gastrointestinal level after oral administration. However, its therapeutic application against pathogens that affect rainbow trout and Atlantic salmon has been little explored. Type II interferon in Atlantic salmon activates the antiviral response, protecting against viral infection, but its role against bacterial infection has not been tested in vivo. In this work, through the design of a recombinant lactic acid bacterium capable of producing Interferon gamma from Atlantic salmon, we explore its role against bacterial infection and the ability to stimulate systemic immune response after oral administration of the recombinant probiotic. Recombinant interferon was active in vitro, mainly stimulating IL-6 expression in SHK-1 cells. In vivo, oral administration of the recombinant probiotic produced an increase in IL-6, IFNγ and IL-12 in the spleen and kidney, in addition to stimulating the activity of lysozyme in serum. The challenge trials indicated that the administration of the IFNγ-producing probiotic doubled the survival in fish infected with F. psychrophilum. In conclusion, our results showed that the oral administration of lactic acid bacteria producing IFNγ managed to stimulate the immune response at a systemic level, conferring protection against pathogens, showing a biotechnological potential for its application in aquaculture.

Serological diversity in Flavobacterium psychrophilum: A critical update using isolates retrieved from Chilean salmon farms.

Chile is currently the second largest producer of farmed salmon worldwide, but Flavobacterium psychrophilum, as one of the most detrimental pathogens, is responsible for major losses during the freshwater culturing step in salmonid fish farms. An antigenic study conducted 10 years ago reported four serological groups using 20 F. psychrophilum Chilean strains. To reduce disease outbreaks and to develop vaccine candidates, antigenic knowledge needs to be regularly updated using a significant number of additional recent F. psychrophilum isolates. The present study aimed at investigating the serological diversity of 118 F. psychrophilum isolates collected between 2006 and 2018 from farmed Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch). The current study supports an expansion of the known antigenic groups in Chile from 4 to 14. However, the use of the slide-agglutination technique for serotyping is costly, is labour-intensive and requires significant technical expertise. Addressing these points, the mPCR-based procedure was a very useful tool for serotyping the collected Chilean F. psychrophilum isolates. This technique revealed the presence of diverse mPCR serotypes (i.e. types 0, 1, 2 and 4). Therefore, mPCR should be employed to select the bacterial strain(s) for vaccine development and to conduct follow-up, selective breeding or epidemiological surveillance in Chilean fish farms. Given the presented findings, changes to Chilean fish-farming practices are vital for ensuring the continued productivity and well-being of farmed salmonids.

Transcriptomic response of rainbow trout (Oncorhynchus mykiss) skeletal muscle to Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the etiologic agent of rainbow trout fry syndrome (RTFS). This pathogen infects a wide variety of salmonid species during freshwater stages, causing significant losses in the aquaculture industry. Rainbow trout (Oncorhynchus mykiss) infected with F. psychrophilum, presents as the main external clinical sign ulcerative lesions and necrotic myositis in skeletal muscle. We previously reported the in vitro cytotoxic activity of F. psychrophilum on rainbow trout myoblast, however little is known about the molecular mechanisms underlying the in vivo pathogenesis in skeletal muscle. In this study, we examined the transcriptomic profiles of skeletal muscle tissue of rainbow trout intraperitoneally challenged with low infection dose of F. psychrophilum. Using high-throughput RNA-seq, we found that 233 transcripts were up-regulated, mostly associated to ubiquitin mediated proteolysis and apoptosis. Conversely, 189 transcripts were down-regulated, associated to skeletal muscle contraction. This molecular signature was consistent with creatine kinase activity in plasma and oxidative damage in skeletal muscle. Moreover, the increased caspase activity suggests as a whole skeletal muscle atrophy induced by F. psychrophilum. This study offers an integrative analysis of the skeletal muscle response to F. psychrophilum infection and reveals unknown aspects of its pathogenesis in rainbow trout.

Stress Tolerance-Related Genetic Traits of Fish Pathogen Flavobacterium psychrophilum in a Mature Biofilm.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome, and hence this bacterium is placed among the most important salmonid pathogens in the freshwater aquaculture industry. Since bacteria in biofilms differ substantially from free-living counterparts, this study sought to find the main differences in gene expression between sessile and planktonic states of F. psychrophilum LM-02-Fp and NCMB1947T, with focus on stress-related changes in gene expression occurring during biofilm formation. To this end, biofilm and planktonic samples were analyzed by RNA sequencing to detect differentially expressed candidate genes (DECGs) between the two growth states, and decreasing the effects of interstrain variation by considering only genes with log2-fold changes ≤ -2 and ≥ 2 at Padj-values ≤ 0.001 as DECGs. Overall, 349 genes accounting for ~15% of total number of genes expressed in transcriptomes of F. psychrophilum LM-02-Fp and NCMB1947T (n = 2327) were DECGs between biofilm and planktonic states. Approximately 83 and 81% of all up- and down-regulated candidate genes in mature biofilms, respectively, were assigned to at least one gene ontology term; these were primarily associated with the molecular function term "catalytic activity." We detected a potential stress response in mature biofilms, characterized by a generalized down-regulation of DECGs with roles in the protein synthesis machinery (n = 63, primarily ribosomal proteins) and energy conservation (seven ATP synthase subunit genes), as well as an up-regulation of DECGs involved in DNA repair (ruvC, recO, phrB1, smf, and dnaQ) and oxidative stress response (cytochrome C peroxidase, probable peroxiredoxin, and a probable thioredoxin). These results support the idea of a strategic trade-off between growth-related processes and cell homeostasis to preserve biofilm structure and metabolic functioning. In addition, LDH-based cytotoxicity assays and an intraperitoneal challenge model for rainbow trout fry agreed with the transcriptomic evidence that the ability of F. psychrophilum to form biofilms could contribute to the virulence. Finally, the reported changes in gene expression, as induced by the plankton-to-biofilm transition, represent the first transcriptomic guideline to obtain insights into the F. psychrophilum biofilm lifestyle that could help understand the prevalence of this bacterium in aquaculture settings.

Cytotoxic activity of Flavobacterium psychrophilum in skeletal muscle cells of rainbow trout (Oncorhynchus mykiss).

Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), which cause significant worldwide losses in aquaculture. Juvenile rainbow trout are particularly susceptible to F. psychrophilum infection, the main external clinical signs of which are extensive necrotic myositis and ulcerative lesions. Despite the economic relevance of this pathogen in aquaculture, little is known about the molecular mechanisms underlying F. psychrophilum infection and pathogenesis. In this study, cultured skeletal muscle cells from rainbow trout (Oncorhynchus mykiss) were co-incubated with the virulent strain of F. psychrophilum JIP02/86 (ATCC 49511). Trypan blue exclusion analysis at 48h post-incubation revealed decreased cellular viability. Direct bacteria-myoblast contact was found a key factor in inducing F. psychrophilum cytotoxicity. Apoptosis was characterized by nuclear DNA fragmentation, decreased plasma membrane integrity, increased caspase activity, and the proteolytic cleavage of poly(ADP-ribose)polymerase-1 (PARP-1). Moreover, bacterial infection induced an early inhibition of NF-κB signaling, as well as a differential expression of the pro- and anti-apoptotic genes, bax and bcl-2. These findings suggest that F. psychrophilum induces rainbow trout muscle apoptosis through the modulation of the NF-κB signaling as a mechanism for nutrient acquisition and survival.

Different Phenotypes of Mature Biofilm in Flavobacterium psychrophilum Share a Potential for Virulence That Differs from Planktonic State.

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease and the rainbow trout fry syndrome in salmonid aquaculture worldwide. However, there have been few studies into the capacity of F. psychrophilum to form biofilms and how these cellular accretions differ from planktonic cells or how they affect potential virulence. We evaluated the biofilm formation by three Chilean isolates of F. psychrophilum (LM-02-Fp, LM-06-Fp, and LM-13-Fp) and two non-Chilean strains (JIP02/86 and NCMB1947T), and compared biofilm and planktonic states to obtain insights into expression differences of virulence- and biofilm-related genes (VBRGs). Our findings are based on scanning confocal laser microscopy (SCLM) and LIVE/DEAD staining, enzymatic reactions, reverse transcription-quantitative PCR (RT-qPCR) of genes encoding putative virulence factors, and transcriptomes (RNA-Seq). The LM-02-Fp and NCMB1947T strains were the strongest and weakest biofilm producers, respectively. The strong-biofilm producer showed different physiological cell states distributed in different layers of mature biofilms, whereas the NCMB1947T biofilms consisted of cells arranged in a monolayer. WGA-binding exopolysaccharides would be the main components of their corresponding extracellular matrices. Transcriptomes of F. psychrophilum NCMB1947T and LM-02-Fp were clustered by state (biofilm vs. planktonic) rather than by strain, indicating important state-dependent differences in gene expression. Analysis of differentially expressed genes between states identified putative VBRGs involved in polysaccharide biosynthesis, lateral gene transfer, membrane transport (e.g., for drugs and Fe3+), sensory mechanisms, and adhesion, and indicated that about 60-100% of VBRGs involved in these processes was significantly upregulated in the biofilm state. Conversely, upregulated motility-related genes in the biofilm state were not identified, whereas a lower fraction of proteolysis-related genes (33%) was upregulated in biofilms. In summary, F. psychrophilum strains that produce different biofilm phenotypes show global transcriptional activity in the mature biofilm state that differs significantly from their planktonic counterparts. Also, different biofilm phenotypes share a genetic potential for virulence that is transcriptionally enhanced with respect to free-living cells. Our results suggest that the F. psychrophilum biofilm lifestyle acts as a reservoir for a given set of putative virulence factors, and recommend a deeper understanding of which could help prevent recurring infections in salmonid farms.

Antimicrobial Susceptibility of Flavobacterium psychrophilum from Chilean Salmon Farms and Their Epidemiological Cut-Off Values Using Agar Dilution and Disk Diffusion Methods.

Flavobacterium psychrophilum is the most important bacterial pathogen for freshwater farmed salmonids in Chile. The aims of this study were to determine the susceptibility to antimicrobials used in fish farming of Chilean isolates and to calculate their epidemiological cut-off (COWT) values. A number of 125 Chilean isolates of F. psychrophilum were isolated from reared salmonids presenting clinical symptoms indicative of flavobacteriosis and their identities were confirmed by 16S rRNA polymerase chain reaction. Susceptibility to antibacterials was tested on diluted Mueller-Hinton by using an agar dilution MIC method and a disk diffusion method. The COWT values calculated by Normalized Resistance Interpretation (NRI) analysis allow isolates to be categorized either as wild-type fully susceptible (WT) or as manifesting reduced susceptibility (NWT). When MIC data was used, NRI analysis calculated a COWT of ≤0.125, ≤2, and ≤0.5 µg mL-1 for amoxicillin, florfenicol, and oxytetracycline, respectively. For the quinolones, the COWT were ≤1, ≤0.5, and ≤0.125 µg mL-1 for oxolinic acid, flumequine, and enrofloxacin, respectively. The disk diffusion data sets obtained in this work were extremely diverse and were spread over a wide range. For the quinolones there was a close agreement between the frequencies of NWT isolates calculated using MIC and disk data. For oxolinic acid, flumequine, and enrofloxacin the frequencies were 45, 39, and 38% using MIC data, and 42, 41, and 44%, when disk data were used. There was less agreement with the other antimicrobials, because NWT frequencies obtained using MIC and disk data, respectively, were 24 and 10% for amoxicillin, 8 and 2% for florfenicol, and 70 and 64% for oxytetracycline. Considering that the MIC data was more precise than the disk diffusion data, MIC determination would be the preferred method for susceptibility testing for this species and the NWT frequencies derived from the MIC data sets should be considered as the more authoritative. Despite the high frequency of isolates showing full susceptibility to florfenicol, the significant frequencies of isolates exhibiting reduced susceptibility to oxytetracycline and quinolones may result in treatment failures when these agents are used.

Introduction, expansion and coexistence of epidemic Flavobacterium psychrophilum lineages in Chilean fish farms.

Chile is one of the countries where the development of salmonid farming has been the most successful. The first importation of salmonids in Chile from the northern hemisphere dates back to the late 19th century and the country now ranks as the world second largest producer of farmed salmon. However, the fast increase of infections caused by the bacterium Flavobacterium psychrophilum is a growing concern for this local industry. This pathogen, also recognized as an important problem worldwide, has been first reported in Chile in 1993 and is currently affecting all three cultivated salmonid species: Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch) and rainbow trout (O. mykiss). Here we conducted a MLST (multi-locus sequence typing) analysis of the local genetic diversity of F. psychrophilum to better understand its origin and propagation in the country, and to suggest practices that could contribute to its control in the future. A total of 94 bacterial isolates, collected from the main production zones, were analyzed and compared to those of other origins already available. The data reveal the country-wide distribution of several genotypes closely related to those that are the most prevalent in European and North American fish farms, and overlapping host fish species of the different lineages. This population structure is probably the direct consequence of local fish farming practices that relied until recently on massive import of fish eggs (e.g., 78 million of eggs in 2012) and where mixed-species farms and fish transportation across the country are common.

Inhibition of Flavobacterium psychrophilum biofilm formation using a biofilm of the antagonist Pseudomonas fluorescens FF48.

The most important bacterial pathology currently occurring in Chilean freshwater salmon farming is the cold-water disease produced by the psychrotrophic bacteria Flavobacterium psychrophilum. The main aim of this study was to characterize the inhibitory activity of an antagonist strain on the formation of biofilms of a F. psychrophilum strain. The antagonistic strain Pseudomonas fluorescens FF48 was isolated from the sediment beneath the salmon cages of a freshwater Chilean salmon farm and was identified by using the 16S rRNA gene sequence analysis. The production of siderophores, mainly during the stationary phase of growth of the antagonist strain was demonstrated using the Chrome Azurol S method and through F. psychrophilum inhibition under iron saturation conditions. Subsequently, the effect of the antagonist supernatant on the formation of F. psychrophilum biofilm was tested using the crystal violet staining method observing an inhibition of the growth of F. psychrophilum, but no effect was observed when iron saturation concentrations were used. Furthermore, when the antagonist strain was previously deposited on the support, it completely inhibited the formation of F. psychrophilum biofilms, but when both bacteria were inoculated simultaneously no inhibitory effect was detected. In conclusion, it was demonstrated that FF48 strain is able to inhibit the formation of F. psychrophilum biofilms in vitro probably mediated by the siderophore production, suggesting its potential use as a biocontrol biofilm in freshwater fish rearing systems to prevent the persistence of biofilms of the fish pathogenic species F. psychrophilum.