Ongoing diversification of the global fish pathogen Piscirickettsia salmonis through genetic isolation and transposition bursts.

The management of bacterial pathogens remains a key challenge of aquaculture. The marine gammaproteobacterium Piscirickettsia salmonis is the etiological agent of piscirickettsiosis and causes multi-systemic infections in different salmon species, resulting in considerable mortality and substantial commercial losses. Here, we elucidate its global diversity, evolution, and selection during human interventions. Our comprehensive analysis of 73 closed, high quality genome sequences covered strains from major outbreaks and was supplemented by an analysis of all P. salmonis 16S rRNA gene sequences and metagenomic reads available in public databases. Genome comparison showed that Piscirickettsia comprises at least three distinct, genetically isolated species of which two showed evidence for continuing speciation. However, at least twice the number of species exist in marine fish or seawater. A hallmark of Piscirickettsia diversification is the unprecedented amount and diversity of transposases which are particularly active in subgroups undergoing rapid speciation and are key to the acquisition of novel genes and to pseudogenization. Several group-specific genes are involved in surface antigen synthesis and may explain the differences in virulence between strains. However, the frequent failure of antibiotic treatment of piscirickettsiosis outbreaks cannot be explained by horizontal acquisition of resistance genes which so far occurred only very rarely. Besides revealing a dynamic diversification of an important pathogen, our study also provides the data for improving its surveillance, predicting the emergence of novel lineages, and adapting aquaculture management, and thereby contributes towards the sustainability of salmon farming.

The Phagosome-Lysosome Fusion Is the Target of a Purified Quillaja saponin Extract (PQSE) in Reducing Infection of Fish Macrophages by the Bacterial Pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis, the etiological agent of Piscirickettsiosis, is a Gram-negative and facultative intracellular pathogen that has affected the Chilean salmon industry since 1989. The bacterium is highly aggressive and can survive and replicate within fish macrophages using the Dot/Icm secretion system to evade the host's immune response and spread systemically. To date, no efficient control measures have been developed for this disease; therefore, the producers use large amounts of antibiotics to control this pathogen. In this frame, this work has focused on evaluating the use of saponins from Quillaja saponaria as a new alternative to control the Piscirickettsiosis. It has been previously reported that purified extract of Q. saponaria (PQSE) displays both antimicrobial activity against pathogenic bacteria and viruses and adjuvant properties. Our results show that PQSE does not present antimicrobial activity against P. salmonis, although it reduces P. salmonis infection in an in vitro model, promoting the phagosome-lysosome fusion. Additionally, we demonstrate that PQSE modulates the expression of IL-12 and IL-10 in infected cells, promoting the immune response against the pathogen and reducing the expression of pathogen virulence genes. These results together strongly argue for specific anti-invasion and anti-intracellular replication effects induced by the PQSE in macrophages.

Expression of ssa-miR-155 during ISAV infection in vitro: Putative role as a modulator of the immune response in Salmo salar.

Multiple cellular components are involved in pathogen-host interaction during viral infection; in this context, the role of miRNAs have become highly relevant. We assessed the expression of selected miRNAs during an in vitro infection of a Salmo salar cell line with Infectious Salmon Anemia Virus (ISAV), the causative agent of a severe disease by the same name. Salmon orthologs for miRNAs that regulate antiviral responses were measured using RT-qPCR in an in vitro time-course assay. We observed a modulation of specific miRNAs expression, where ssa-miR-155-5p was differentially over-expressed. Using in silico analysis, we identified the putative mRNA targets for ssa-miR-155-5p, finding a high prevalence of hosts immune response-related genes; moreover, several mRNAs involved in the viral infective process were also identified as targets for this miRNA. Our results suggest a relevant role for miR-155-5p in Salmo salar during an ISAV infection as a regulator of the immune response to the virus.

An in vitro model mimicking the complement system to favor directed phagocytosis of unwanted cells

BACKGROUND: Opsonization, is the molecular mechanism by which target molecules promote interactions with phagocyte cell surface receptors to remove unwanted cells by induced phagocytosis. We designed an in vitro system to demonstrate that this procedure could be driven to eliminate adipocytes, using peptides mimicking regions of the complement protein C3b to promote opsonization and enhance phagocytosis. Two cell lines were used: (1) THP-1 monocytes differentiated to macrophages, expressing the C3b opsonin receptor CR1 in charge of the removal of unwanted coated complexes; (2) 3T3-L1 fibroblasts differentiated to adipocytes, expressing AQP7, to evaluate the potential of peptides to stimulate opsonization. (3) A co-culture of the two cell lines to demonstrate that phagocytosis could be driven to cell withdrawal with high efficiency and specificity. RESULTS: An array of peptides were designed and chemically synthesized p3691 and p3931 joined bound to the CR1 receptor activating phagocytosis (p < 0.033) while p3727 joined the AQP7 protein (p < 0.001) suggesting that opsonization of adipocytes could occur. In the co-culture system p3980 and p3981 increased lipid uptake to 91.2% and 89.0%, respectively, as an indicator of potential adipocyte phagocytosis. CONCLUSIONS: This in vitro model could help understand the receptor­ligand interaction in the withdrawal of unwanted macromolecules in vivo. The adipocyte-phagocytosis discussed may help to control obesity, since peptides of C3b stimulated the CR1 receptor, promoting opsonisation and phagocytosis of lipidcontaining structures, and recognition of AQP7 in the differentiated adipocytes, favored the phagocytic activity of macrophages, robustly supported by the co-culture strategy.

Expression of DC-SIGN-like C-Type Lectin Receptors in Salmo salar.

C-Type Lectin Receptors (CTLR) are involved in the activation of innate and adaptative immune responses. Among these receptors, the Dendritic Cell-Specific ICAM-3-Grabbing nonintegrin (DC-SIGN/CD209) has become a hot topic due to its ability to bind and facilitate the infections processes of several pathogens. Although well characterized in mammals, little documentation exists about the receptor in salmonid fishes. Here, we report the sequence and expression analysis of eight DC-SIGN-like genes in Salmo salar. Each receptor displays structural similarities to DC-SIGN molecules described in mammals, including internalization motifs, a neck region with heptad repeats, and a Ca+2-dependent carbohydrate recognition domain. The receptors are expressed in multiple tissues of fish, and fish cell lines, with differential expression upon infection with viral and bacterial pathogens. The identification of DC-SIGN-like receptors in Salmo salar provides new information regarding the structure of the immune system of salmon, potential markers for cell subsets, as well as insights into DC-SIGN conservation across species.

Synthetic Peptides as a Promising Alternative to Control Viral Infections in Atlantic Salmon.

Viral infections in salmonids represent an ongoing challenge for the aquaculture industry. Two RNA viruses, the infectious pancreatic necrosis virus (IPNV) and the infectious salmon anemia virus (ISAV), have become a latent risk without healing therapies available for either. In this context, antiviral peptides emerge as effective and relatively safe therapeutic molecules. Based on in silico analysis of VP2 protein from IPNV and the RNA-dependent RNA polymerase from ISAV, a set of peptides was designed and were chemically synthesized to block selected key events in their corresponding infectivity processes. The peptides were tested in fish cell lines in vitro, and four were selected for decreasing the viral load: peptide GIM182 for IPNV, and peptides GIM535, GIM538 and GIM539 for ISAV. In vivo tests with the IPNV GIM 182 peptide were carried out using Salmo salar fish, showing a significant decrease of viral load, and proving the safety of the peptide for fish. The results indicate that the use of peptides as antiviral agents in disease control might be a viable alternative to explore in aquaculture.

Pseudomonas fildesensis sp. nov., a psychrotolerant bacterium isolated from Antarctic soil of King George Island, South Shetland Islands.

The strain KG01T was isolated from a soil sample from King George Island, Antarctica. Cells of KG01T are rod-shaped and motile by means of multiple polar flagella. The absence of arginine dihydrolase activity could be a key feature to readily distinguish KG01T from its closest phylogenetic relative species. The main fatty acids of the strain include summed feature 3 (C16 : 1 ω7c and/or C15 : 0 iso 2-OH), C16 : 0 and C18 : 1 ω7c. Phylogenetic analysis based on the 16S rRNA gene sequence and on a multilocus sequence analysis (MLSA) using housekeeping genes (16S rRNA, rpoB, rpoD, gyrB) were carried out. These analyses allowed us to include the strain within the Pseudomonas fluorescens group, presenting the highest similarity of multilocus sequence with Pseudomonas veronii LMG 17761T (96.67 %). The genome of KG01T was sequenced and in silico compared with genomes of the most closely related species of the P. fluorescens group. The average nucleotide identity (ANIb) and average amino acid identity (AAI) values of the species phylogenetically closest to KG01T were less than 95-96 %, threshold currently accepted to define strain as belonging to a bacterial species, the highest scores being those to Pseudomonas veronii LMG 17761T (87.98 %) and Pseudomonas marginalis ICMP 3553T (91.90 %). Therefore, the phenotypic and genotypic analyses results, allow us to propose that KG01T represents a member of a novel species of the genus Pseudomonas, for which the name Pseudomonas fildesensis is proposed, and KG01T (=CECT 9084T;=DSM 102036T) is established as the type strain .

Molecular features associated with the adaptive evolution of Infectious Salmon Anemia Virus (ISAV) in Chile.

Infectious salmon anemia virus (ISAV) is an Orthomyxovirus challenging salmon production, with a particular impact in Chile. During 2007-2010 a devastating and of unexpected consequences epizootic event almost destroyed a blooming industry in the country. The event was caused by an aggressive variant with a distinctive deletion in Segment 6, one of the eight genomic segments of the virus. After the outburst, although the infective viral variant seemed to have disappeared, a non-infective variant, not previously reported, was discovered and is characterized by a complete, non-deleted coding segment 6, which has prevailed in the fish population until now. This variant, known as HPR0, appears to be the ancestor strain of ISAV from which novel infective variants are generated. Additional variations in segment 5 have also been associated with the virulence observed in the field, an analysis of the differences in these two protein coding segments has been performed. It appears to us that a combinatorial effect exists between the features displayed by segments 5 and 6 which modulate the intensity of viral outbursts. As a result, a theoretical integrative model is presented which explains the different degree of virulence observed in the field based only on molecular data, this could help estimating the intensity of damage a given variant might exert over a productive farm.

Phylogenetic MLSA and phenotypic analysis identification of three probable novel Pseudomonas species isolated on King George Island, South Shetland, Antarctica

ABSTRACT Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms' role in this extreme environment, the characterization and description of new species is vital.

Extended antibiotic treatment in salmon farms select multiresistant gut bacteria with a high prevalence of antibiotic resistance genes.

The high use of antibiotics for the treatment of bacterial diseases is one of the main problems in the mass production of animal protein. Salmon farming in Chile is a clear example of the above statement, where more than 5,500 tonnes of antibiotics have been used over the last 10 years. This has caused a great impact both at the production level and on the environment; however, there are still few works in relation to it. In order to demonstrate the impact of the high use of antibiotics on fish gut microbiota, we have selected four salmon farms presenting a similar amount of fish of the Atlantic salmon species (Salmo salar), ranging from 4,500 to 6,000 tonnes. All of these farms used treatments with high doses of antibiotics. Thus, 15 healthy fish were selected and euthanised in order to isolate the bacteria resistant to the antibiotics oxytetracycline and florfenicol from the gut microbiota. In total, 47 bacterial isolates resistant to florfenicol and 44 resistant to oxytetracycline were isolated, among which isolates with Minimum Inhibitory Concentrations (MIC) exceeding 2048 µg/mL for florfenicol and 1024 µg/mL for oxytetracycline were found. In addition, another six different antibiotics were tested in order to demonstrate the multiresistance phenomenon. In this regard, six isolates of 91 showed elevated resistance values for the eight tested antibiotics, including florfenicol and oxytetracycline, were found. These bacteria were called "super-resistant" bacteria. This phenotypic resistance was verified at a genotypic level since most isolates showed antibiotic resistance genes (ARGs) to florfenicol and oxytetracycline. Specifically, 77% of antibiotic resistant bacteria showed at least one gene resistant to florfenicol and 89% showed at least one gene resistant to oxytetracycline. In the present study, it was demonstrated that the high use of the antibiotics florfenicol and oxytetracycline has, as a consequence, the selection of multiresistant bacteria in the gut microbiota of farmed fish of the Salmo salar species at the seawater stage. Also, the phenotypic resistance of these bacteria can be correlated with the presence of antibiotic resistance genes.

Involvement of selected cellular miRNAs in the in vitro and in vivo infection of infectious salmon anemia virus (ISAV).

Infectious salmon anemia virus (ISAV) is the causative agent of infectious salmon anemia (ISA), a relatively novel disease primarily affecting farmed salmon species, primarily in Salmo salar specimens, causing severe outbreaks in most producer countries. Although ISAV has been extensively studied at the molecular level, not much is known about the host/cell interaction at the small RNA level. MicroRNAs (miRNAs) are small, non-coding RNA that regulate mRNA expression at the post-transcriptional level. In recent years, the putative role of these molecules in host-pathogen interactions has drawn particular attention because of their pivotal involvement as regulatory elements in a number of eukaryotic organisms. Given the importance of the salmon industry in Chile, a deep understanding of the interaction between ISAV and its hosts is of importance. In the present work, we studied the kinetic expression of selected miRNAs during ISAV infection, both in vitro and in vivo. Based on initial experimental data derived from a small RNA-Seq analysis, a group of miRNAs that were differentially expressed in infected cells were selected for analysis. As a result, two miRNAs, miR-462a-5p and miR-125 b-5p, showed increased and decreased expression, respectively, during ISAV infection.

Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis.

Draft genomes and reference transcriptomes extend the coding potential of the fish pathogen Piscirickettsia salmonis

Background: Draft and complete genome sequences from bacteria are key tools to understand genetic determinants involved in pathogenesis in several disease models. Piscirickettsia salmonis is a Gram-negative bacterium responsible for the Salmon Rickettsial Syndrome (SRS), a bacterial disease that threatens the sustainability of the Chilean salmon industry. In previous reports, complete and draft genome sequences have been generated and annotated. However, the lack of transcriptome data underestimates the genetic potential, does not provide information about transcriptional units and contributes to disseminate annotation errors. Results: Here we present the draft genome and transcriptome sequences of four P. salmonis strains. We have identified the transcriptional architecture of previously characterized virulence factors and trait-specific genes associated to cation uptake, metal efflux, antibiotic resistance, secretion systems and other virulence factors. Conclusions: This data has provided a refined genome annotation and also new insights on the transcriptional structures and coding potential of this fish pathogen.

Phylogenetic MLSA and phenotypic analysis identification of three probable novel Pseudomonas species isolated on King George Island, South Shetland, Antarctica.

Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms' role in this extreme environment, the characterization and description of new species is vital.

Identification and isolation of infective filamentous particles in Infectious Salmon Anemia Virus (ISAV).

The infectious salmon anemia virus (ISAV) is an aquatic pathogen that is a member of the Orthomyxoviridae family with lethal hemorrhagic potential. Although it affects other species of salmonid fish, ISAV only causes disease in Atlantic salmon (Salmo salar) specimens in sea water. In spite of the fact that the virus has been described as enveloped with icosahedral symmetry, viral like particles with anomalous morphology have been observed in field samples, this we have not been able to recover then in adequate quantities for full demonstration. We report a procedure to concentrate and recover these novel forms of the virus, comparing two cell lines from different origins, demonstrating that these forms were preferentially expressed in cells of epithelial origin.

Alginate overproduction and biofilm formation by psychrotolerant Pseudomonas mandelii depend on temperature in Antarctic marine sediments

Background: In recent years, Antarctica has become a key source of biotechnological resources. Native microorganisms have developed a wide range of survival strategies to adapt to the harsh Antarctic environment, including the formation of biofilms. Alginate is the principal component of the exopolysaccharide matrix in biofilms produced by Pseudomonas, and this component is highly demanded for the production of a wide variety of commercial products. There is a constant search for efficient alginate-producing organisms. Results: In this study, a novel strain of Pseudomonas mandelii isolated from Antarctica was characterized and found to overproduce alginate compared with other good alginate producers such as Pseudomonas aeruginosa and Pseudomonas fluorescens. Alginate production and expression levels of the alginate operon were highest at 4°C. It is probable that this alginate-overproducing phenotype was the result of downregulated MucA, an anti-sigma factor of AlgU. Conclusion: Because biofilm formation is an efficient bacterial strategy to overcome stressful conditions, alginate overproduction might represent the best solution for the successful adaptation of P. mandelii to the extreme temperatures of the Antarctic. Through additional research, it is possible that this novel P. mandelii strain could become an additional source for biotechnological alginate production.

An updated proposal for classification of infectious salmon anemia virus strains.

Biological databases contain a wealth of valuable information that can contribute to the enrichment of virtually any area. However, the exponential growth of information together with its dissemination through virtual networks has become a double-edged sword, promoting synonymy that leads to confusion and chaos. Organization of data is a big effort that must be accompanied by clarity, both in the deposited data and in the publications arising from them. In this report, an effort is made to organize the information related to infectious salmon anemia virus and its classification based on the variability of genomic segment 6.

Expression of flagellin and key regulatory flagellar genes in the non-motile bacterium Piscirickettsia salmonis.

The Piscirickettsia salmonis genome was screened to evaluate potential flagella-related open reading frames, as well as their genomic organization and eventual expression. A complete and organized set of flagellar genes was found for P. salmonis, although no structural flagellum has ever been reported for this bacterium. To gain further understanding, the hierarchical flagellar cascade described for Legionella pneumophila was used as a reference model for putative analysis in P. salmonis. Specifically, 5 of the most relevant genes from this cascade were chosen, including 3 regulatory genes (fleQ, triggers the cascade; fliA, regulates the σ28-coding gene; and rpoN, an RNA polymerase-dependent gene) and 2 terminal structural genes (flaA and flaB, flagellin and a flagellin-like protein, respectively). Kinetic experiments evaluated gene expressions over time, with P. salmonis assessed in 2 liquid, cell-free media and during infection of the SHK-1 fish cell line. Under all conditions, the 5 target genes were primarily expressed during early growth/infection and were differentially expressed when bacteria encountered environmental stress (i.e. a high-salt concentration). Intriguingly, the flagellin monomer was fully expressed under all growth conditions and was located near the bacterial membrane. While no structural flagellum was detected under any condition, the recombinant flagellin monomer induced a proinflammatory response in SHK-1 cells, suggesting a possible immunomodulatory function. The potential implications of these observations are discussed in the context of P. salmonis biology and pathogenic potential.

Deprotection Reagents in Fmoc Solid Phase Peptide Synthesis: Moving Away from Piperidine?

The deprotection step is crucial in order to secure a good quality product in Fmoc solid phase peptide synthesis. 9-Fluorenylmethoxycarbonyl (Fmoc) removal is achieved by a two-step mechanism reaction favored by the use of cyclic secondary amines; however, the efficiency of the reaction could be affected by side reactions and by-product formation. Several aspects have to be taken into consideration when selecting a deprotection reagent: its physicochemical behavior, basicity (pKa) and polarity, concentration, and time of reaction, toxicity and disposability of residues and, finally, availability of reagents. This report presents a comparison of the performance of three strategies for deprotection using microwave-assisted Fmoc peptide synthesis. Four peptide sequences were synthesized using Rink amide resin with a Liberty Blue™ automated synthesizer and 4-methylpiperidine (4MP), piperidine (PP), and piperazine (PZ) as Fmoc removal reagents. In the first instance all three reagents behaved similarly. A detailed analysis showed a correlation between the hydrophobicity and size of the peptide with the yield and purity of the obtained product. The three reagents are interchangeable, and replacement of piperidine could be advantageous regarding toxicity and reagent handling.

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model.

Piscirickettsia salmonis is a facultative intracellular bacterium that causes the disease called "salmon rickettsial syndrome". Attempts to control this disease have been unsuccessful, because existing vaccines have not achieved the expected effectiveness and the antibiotics used fail to completely eradicate the pathogen. This is in part the product of lack of scientific information that still lacks on the mechanisms used by this bacterium to overcome infected-cell responses and survive to induce a productive infection in macrophages. For that, this work was focused in determining if P. salmonis is able to modify the expression and the imbalance of IL-12 and IL-10 using an in vitro model. Additionally, we also evaluated the role the antimicrobial peptide hepcidin had in the control of this pathogen in infected cells. Therefore, the expression of IL-10 and IL-12 was evaluated at earlier stages of infection in the RTS11 cell line derived from Oncorhynchus mykiss macrophages. Simultaneously, the hepcidin expression and location was analyzed in the macrophages infected with the pathogen. Our results suggest that IL-10 is clearly induced at early stages of infection with values peaking at 36 hours post infection. Furthermore, infective P. salmonis downregulates the expression of antimicrobial peptide hepcidin and vesicles containing this peptide were unable to merge with the infective bacteria. Our results suggest that P. salmonis is able to manipulate the behavior of host cytokines and likely might constitute a virulence mechanism that promotes intracellular bacterial replication in leukocytes cells lines of trout and salmon. This mechanism involves the generation of an optimum environment for the microorganism and the downregulation of antimicrobial effectors like hepcidin.