In vitro effects of phytogenic feed additive on Piscirickettsia salmonis growth and biofilm formation.

Piscirickettsiosis is the main cause of mortality in salmonids of commercial importance in Chile, which is caused by Piscirickettsia salmonis, a Gram-negative, γ-proteobacteria that can produce biofilm as one of its virulence factors. The Chilean salmon industry uses large amounts of antibiotics to control piscirickettsiosis outbreaks, which has raised concern about its environmental impact and the potential to induce antibiotic resistance. Thus, the use of phytogenic feed additives (PFA) with antibacterial activity emerges as an interesting alternative to antimicrobials. Our study describes the antimicrobial action of an Andrographis paniculate-extracted PFA on P. salmonis planktonic growth and biofilm formation. We observed complete inhibition of planktonic and biofilm growth with 500 and 400 µg/mL of PFA for P. salmonis LF-89 and EM-90-like strains, respectively. Furthermore, 500 µg/mL of PFA was bactericidal for both evaluated bacterial strains. Sub-inhibitory doses of PFA increase the transcript levels of stress (groEL), biofilm (pslD), and efflux pump (acrB) genes for both P. salmonis strains in planktonic and sessile conditions. In conclusion, our results demonstrate the antibacterial effect of PFA against P. salmonis in vitro, highlighting the potential of PFA as an alternative to control Piscirickettsiosis.

Effect of florfenicol on Piscirickettsia salmonis biofilm formed in materials used in salmonid nets, nylon and high-density polyethylene.

Piscirickettsiosis is the most prevalent bacterial disease affecting seawater salmon in Chilean salmon industry. Antibiotic therapy is the first alternative to counteract infections caused by Piscirickettsia salmonis. The presence of bacterial biofilms on materials commonly used in salmon farming may be critical for understanding the bacterial persistence in the environment. In the present study, the CDC Biofilm Reactor® was used to investigate the effect of sub- and over-MIC of florfenicol on both the pre-formed biofilm and the biofilm formation by P. salmonis under the antibiotic stimuli on Nylon and high-density polyethylene (HDPE) surfaces. This study demonstrated that FLO, at sub- and over-MIC doses, decreases biofilm-embedded live bacteria in the P. salmonis isolates evaluated. However, it was shown that in the P. salmonis Ps007 strain the presence of sub-MIC of FLO reduced its biofilm formation on HDPE surfaces; however, biofilm persists on Nylon surfaces. These results demonstrated that P. salmonis isolates behave differently against FLO and also, depending on the surface materials. Therefore, it remains a challenge to find an effective strategy to control the biofilm formation of P. salmonis, and certainly other marine pathogens that affect the sustainability of the Chilean salmon industry.

Outer membrane vesicles from Piscirickettsia salmonis induce the expression of inflammatory genes and production of IgM in Atlantic salmon Salmo salar.

Piscirickettsiosis outbreaks due to Piscirickettsia salmonis occur globally in the Chilean salmon aquaculture generating significant monetary losses in the industry. P. salmonis secretes outer membrane vesicles (OMVs) which are naturally non-replicating and highly immunogenic spherical nanoparticles. P. salmonis OMVs has been shown to induce immune response in zebrafish; however, the immune response induced by these vesicles in salmonids has not been evaluated. In this study, we inoculated Atlantic salmon with 10 and 30 µg doses of P. salmonis OMVs and took samples for 12 days. qPCR analysis indicated an inflammatory response. Thus, the inflammatory genes evaluated were up- or down-regulated at several times in liver, head kidney and spleen. In addition, the liver was the organ most immune-induced, mainly in the 30 µg-dose. Interestingly, co-expression of pro- and anti-inflammatory cytokines was evidenced by the prominent expression of il-10 at day 1 in spleen and also in head kidney on days 3, 6 and 12, while il-10 and tgf-ß were up-regulated on days 3, 6 and 12 in liver. Importantly, we detected the production of IgM against proteins of P. salmonis in the serum collected from immunized fish after 14 days. Thus, 40 and 400 µg OMVs induced the production of highest IgM levels; however, no statistical difference in the immunoglobulin levels produced by these OMVs doses were detected. The current study provides evidence that OMVs released by P. salmonis induced a pro-inflammatory responses and IgM production in S. salar, while regulatory genes were induced in order to regulate their effects and achieve the balance of the inflammatory response.

Live and inactivated Piscirickettsia salmonis activated nutritional immunity in Atlantic salmon (Salmo salar).

Nutritional immunity regulates the homeostasis of micronutrients such as iron, manganese, and zinc at the systemic and cellular levels, preventing the invading microorganisms from gaining access and thereby limiting their growth. Therefore, the objective of this study was to evaluate the activation of nutritional immunity in specimens of Atlantic salmon (Salmo salar) that are intraperitoneally stimulated with both live and inactivated Piscirickettsia salmonis. The study used liver tissue and blood/plasma samples on days 3, 7, and 14 post-injections (dpi) for the analysis. Genetic material (DNA) of P. salmonis was detected in the liver tissue of fish stimulated with both live and inactivated P. salmonis at 14 dpi. Additionally, the hematocrit percentage decreased at 3 and 7 dpi in fish stimulated with live P. salmonis, unchanged in fish challenged with inactivated P. salmonis. On the other hand, plasma iron content decreased during the experimental course in fish stimulated with both live and inactivated P. salmonis, although this decrease was statistically significant only at 3 dpi. Regarding the immune-nutritional markers such as tfr1, dmt1, and ireg1 were modulated in the two experimental conditions, compared to zip8, ft-h, and hamp, which were down-regulated in fish stimulated with live and inactivated P. salmonis during the course experimental. Finally, the intracellular iron content in the liver increased at 7 and 14 dpi in fish stimulated with live and inactivated P. salmonis, while the zinc content decreased at 14 dpi under both experimental conditions. However, stimulation with live and inactivated P. salmonis did not alter the manganese content in the fish. The results suggest that nutritional immunity does not distinguish between live and inactivated P. salmonis and elicits a similar immune response. Probably, this immune mechanism would be self-activated with the detection of PAMPs, instead of a sequestration and/or competition of micronutrients by the living microorganism.

Subinhibitory concentrations of florfenicol increase the biofilm formation of Piscirickettsia salmonis.

Public health is facing a new challenge due to the increased bacterial resistance to most of the conventional antibacterial agents. Inadequate use of antibiotics in the Chilean aquaculture industry leads to the generation of multidrug resistance bacteria. Many fish pathogenic bacteria produce biofilm upon various sources of stress such as antibiotics, which provides several survival advantages for the bacterial life in community and can constitute a reservoir of pathogens in the marine environment. Being florfenicol a broad-spectrum antibiotic commonly used to treat infections in aquaculture, the aim of this study was to assess whether this antibiotic modulates in vitro the biofilm formation in several isolates of Piscirickettsia salmonis. Standard antibiotic-micro broth 96-flat well plates were used to determinate the minimal inhibitory concentration of florfenicol in eight different P. salmonis isolates. In vitro findings, with P. salmonis growing in the presence and absence of the antibiotic, exhibited a statistically significantly increase (p < .05) in biofilm formation in all the bacterial isolates cultivated with sub-MIC (defined as the half of the minimal inhibitory concentration in the presence of antibiotic) of florfenicol compared with controls (antibiotic-free broth). In conclusion, sub-MIC of florfenicol induced an increased biofilm formation in all P. salmonis isolates tested.

Piscirickettsia salmonis forms a biofilm on nylon surface using a CDC Biofilm Reactor.

Research into Piscirickettsia salmonis biofilms on materials commonly used in salmon farming is crucial for understanding its persistence and virulence. We used the CDC Biofilm Reactor to investigate P. salmonis (LF-89 and EM-90) biofilm formation on Nylon, Stainless steel (316L), Polycarbonate and High-Density Polyethylene (HDPE) surfaces. After 144 h of biofilm visualization by scanning confocal laser microscopy under batch growth conditions, Nylon coupons generated the greatest biofilm formation and coverage compared to Stainless steel (316L), Polycarbonate and HDPE. Additionally, P. salmonis biofilm formation on Nylon was significantly greater (p ≤ .01) than Stainless steel (316L), Polycarbonate and HDPE at 288 h. We used Nylon coupons to determine the kinetic parameters of the planktonic and biofilm phases of P. salmonis. The two strains had similar latencies in the planktonic phase; however, LF-89 maximum growth was 2.5 orders of magnitude higher (Log cell ml-1 ). Additionally, LF-89 had a specified growth rate (µmax) of 0.0177 ± 0.006 h-1 and a generation time of 39.2 h. This study contributes to a deeper understanding of the biofilm formation by P. salmonis and elucidates the impact of the biofilm on aquaculture systems.

PAMPs of Piscirickettsia salmonis Trigger the Transcription of Genes Involved in Nutritional Immunity in a Salmon Macrophage-Like Cell Line.

The innate immune system can limit the growth of invading pathogens by depleting micronutrients at a cellular and tissue level. However, it is not known whether nutrient depletion mechanisms discriminate between living pathogens (which require nutrients) and pathogen-associated molecular patterns (PAMPs) (which do not). We stimulated SHK-1 cells with different PAMPs (outer membrane vesicles of Piscirickettsia salmonis "OMVs", protein extract of P. salmonis "TP" and lipopolysaccharides of P. salmonis "LPS") isolated from P. salmonis and evaluated transcriptional changes in nutritional immunity associated genes. Our experimental treatments were: Control (SHK-1 stimulated with bacterial culture medium), OMVs (SHK-1 stimulated with 1µg of outer membrane vesicles), TP (SHK-1 stimulated with 1µg of total protein extract) and LPS (SHK-1 stimulated with 1µg of lipopolysaccharides). Cells were sampled at 15-, 30-, 60- and 120-minutes post-stimulation. We detected increased transcription of zip8, zip14, irp1, irp2 and tfr1 in all three experimental conditions and increased transcription of dmt1 in cells stimulated with OMVs and TP, but not LPS. Additionally, we observed generally increased transcription of ireg-1, il-6, hamp, irp1, ft-h and ft-m in all three experimental conditions, but we also detected decreased transcription of these markers in cells stimulated with TP and LPS at specific time points. Our results demonstrate that SHK-1 cells stimulated with P. salmonis PAMPs increase transcription of markers involved in the transport, uptake, storage and regulation of micronutrients such as iron, manganese and zinc.

Piscirickettsia salmonis Produces a N-Acetyl-L-Homoserine Lactone as a Bacterial Quorum Sensing System-Related Molecule.

Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, the most prevalent disease in salmonid species in Chilean salmonids farms. Many bacteria produce N-acyl-homoserine lactones (AHLs) as a quorum-sensing signal molecule to regulate gene expression in a cell density-dependent manner, and thus modulate physiological characteristics and several bacterial mechanisms. In this study, a fluorescent biosensor system method and gas chromatography-tandem mass spectrometry (GC/MS) were combined to detect AHLs produced by P. salmonis. These analyses revealed an emitted fluorescence signal when the biosensor P. putida EL106 (RPL4cep) was co-cultured with both, P. salmonis LF-89 type strain and an EM-90-like strain Ps007, respectively. Furthermore, the production of an AHL-type molecule was confirmed by GC/MS by both P. salmonis strains, which identified the presence of a N-acetyl-L-homoserine Lactone in the supernatant extract. However, It is suggested that an alternate pathway could synthesizes AHLs, which should be address in future experiments in order to elucidate this important bacterial process. To the best of our knowledge, the present report is the first to describe the type of AHLs produced by P. salmonis.

Protein-Based Vaccine Protect Against Piscirickettsia salmonis in Atlantic Salmon (Salmo salar).

An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for sustainable salmon farming and to reduce disease-related economic losses. Consequently, the aquaculture industry urgently needs to investigate efficient prophylactic measures. Three protein-based vaccine prototypes against Piscirickettsia salmonis were prepared from a highly pathogenic Chilean isolate. Only one vaccine effectively protected Atlantic salmon (Salmo salar), in correlation with the induction of Piscirickettsia-specific IgM antibodies and a high induction of transcripts encoding pro-inflammatory cytokines (i.e., Il-1ß and TNF-α). In addition, we studied the proteome fraction protein of P. salmonis strain Austral-005 using multidimensional protein identification technology. The analyzes identified 87 proteins of different subcellular origins, such as the cytoplasmic and membrane compartment, where many of them have virulence functions. The other two prototypes activated only the innate immune responses, but did not protect Salmo salar against P. salmonis. These results suggest that the knowledge of the formulation of vaccines based on P. salmonis proteins is useful as an effective therapy, this demonstrates the importance of the different research tools to improve the study of the different immune responses, resistance to diseases in the Atlantic salmon. We suggest that this vaccine can help prevent widespread infection by P. salmonis, in addition to being able to be used as a booster after a primary vaccine to maintain high levels of circulating protective antibodies, greatly helping to reduce the economic losses caused by the pathogen.

Full recombinant flagellin B from Vibrio anguillarum (rFLA) and its recombinant D1 domain (rND1) promote a pro-inflammatory state and improve vaccination against P. salmonis in Atlantic salmon (S. salar).

Flagellin is the major component of the flagellum, and a ligand for Toll-like receptor 5. As reported, recombinant flagellin (rFLA) from Vibrio anguillarum and its D1 domain (rND1) are able to promote in vitro an upregulation of pro-inflammatory genes in gilthead seabream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss) macrophages. This study evaluated the in vitro and in vivo stimulatory/adjuvant effect for rFLA and rND1 during P. salmonis vaccination in Atlantic salmon (Salmo salar). We demonstrated that rFLA and rND1 are molecules able to generate an acute upregulation of pro-inflammatory cytokines (IL-1ß, IL-8, IL-12ß), allowing the expression of genes associated with T-cell activation (IL-2, CD4, CD8ß), and differentiation (IFNγ, IL-4/13, T-bet, Eomes, GATA3), in a differential manner, tissue/time dependent way. Altogether, our results suggest that rFLA and rND1 are valid candidates to be used as an immuno-stimulant or adjuvants with existing vaccines in farmed salmon.

Biofilm Produced In Vitro by Piscirickettsia salmonis Generates Differential Cytotoxicity Levels and Expression Patterns of Immune Genes in the Atlantic Salmon Cell Line SHK-1.

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with a high economic impact on the Chilean salmonid aquaculture industry. This bacterium produces biofilm as a potential resistance and persistence strategy against stressful environmental stimuli. However, the in vitro culture conditions that modulate biofilm formation as well as the effect of sessile bacteria on virulence and immune gene expression in host cells have not been described for P. salmonis. Therefore, this study aimed to analyze the biofilm formation by P. salmonis isolates under several NaCl and iron concentrations and to evaluate the virulence of planktonic and sessile bacteria, together with the immune gene expression induced by these bacterial conditions in an Atlantic salmon macrophage cell line. Our results showed that NaCl and Fe significantly increased biofilm production in the LF-89 type strain and EM-90-like isolates. Additionally, the planktonic EM-90 isolate and sessile LF-89 generated the highest virulence levels, associated with differential expression of il-1ß, il-8, nf-κb, and iκb-α genes in SHK-1 cells. These results suggest that there is no single virulence pattern or gene expression profile induced by the planktonic or sessile condition of P. salmonis, which are dependent on each strain and bacterial condition used.

Salmo salar glucocorticoid receptors analyses of alternative splicing variants under stress conditions.

Cortisol is the main corticosteroid in teleosts, exerting multiple functions by activating glucocorticoid receptors (GR). Most teleost species have two GR genes, gr-1 and gr-2. Some teleost also presents two splice variants for gr-1; gr-1a and gr-1b. In this study, we report for first time the presence of 2 homeologous genes for gr-1 and gr-2, located on chromosomes 4q-13q (gr-1) and 5p-9q (gr-2) of the Salmo salar genome. Furthermore, our results describe gr-1 splice variants derived from chromosome 4 and 13, sharing typical teleost GR elements such as the 9 amino acid insertion in the DNA binding domain (DBD) and variations in the length of the ligand binding domain (LBD). Three splice variants were predicted for the gr-2 homeologous gene in chromosome 5, with differences of a 5 amino acid insertion in the DBD. We also identified an uncommon truncated gr-2 gene in chromosome 9 in salmon, which lacked the DBD and LBD domains. Finally, by designing specific primers for each predicted splice variant, we validated and evaluated the expression of their transcripts in S. salar subjected to stress caused by stocking density. Differences were observed in the expression of all identified mRNAs, revealing that gr-1 and gr-2 splice variants were upregulated in head kidney and gills of post-stressed fish. In conclusion, our findings suggest that from specific salmonid genomic duplication (125 MYA), two gene copies of each GR receptor were generated in S. salar. The identified splice variants could contribute to the variability of GR receptor complex modulation expression during stressful events, leading to variations in physiological responses in fish.

Subcellular Location of Piscirickettsia salmonis Heat Shock Protein 60 (Hsp60) Chaperone by Using Immunogold Labeling and Proteomic Analysis.

Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by the T4SS, T3SS, and T6SS, with negative results. These data support the hypothesis that small amounts of Hsp60 must reach the bacterial cell surface in a manner probably not mediated by currently characterized secretion systems, and that they remain biologically active during P. salmonis infection, possibly mediating adherence and (or) invasion.

In vitro genomic and proteomic evidence of a type IV pili-like structure in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is an intracellular γ-proteobacteria and the etiological agent of piscirickettsiosis, which causes massive economic losses in the Chilean salmon industry. The type IV pili (T4P) play an important role in adherence to host cell surfaces and bacterial pathogenicity. T4P contains a variable number of components, as predicted in P. salmonis genomes. However, no studies have determined if P. salmonis possesses T4P. The aims of this investigation were to identify T4P components in the P. salmonis type strain LF-89T, evaluate respective transcript expressions, and analyze the main putative T4P proteins using bioinformatics and proteomic approaches. Two main clusters of P. salmonis T4P genes were found. Expression of the pilA gene was upregulated at 4 h post-infection (hpi), while pilQ was upregulated 4 days post-infection. At 16 hpi, pilB and pilD were strongly upregulated. The PilA amino acid sequence analysis showed a conserved N-terminal domain and sequence motifs critical for T4P biosynthesis. MudPIT analysis revealed PilA in the P. salmonis LF-89T proteome, and TEM showed pili-like filamentous structures on the P. salmonis surface. These results strongly suggest the presence of a T4P-like structure in P. salmonis.

Comparative Pan-Genome Analysis of Piscirickettsia salmonis Reveals Genomic Divergences within Genogroups.

Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, a disease that seriously affects the salmonid industry. Despite efforts to genomically characterize P. salmonis, functional information on the life cycle, pathogenesis mechanisms, diagnosis, treatment, and control of this fish pathogen remain lacking. To address this knowledge gap, the present study conducted an in silico pan-genome analysis of 19 P. salmonis strains from distinct geographic locations and genogroups. Results revealed an expected open pan-genome of 3,463 genes and a core-genome of 1,732 genes. Two marked genogroups were identified, as confirmed by phylogenetic and phylogenomic relationships to the LF-89 and EM-90 reference strains, as well as by assessments of genomic structures. Different structural configurations were found for the six identified copies of the ribosomal operon in the P. salmonis genome, indicating translocation throughout the genetic material. Chromosomal divergences in genomic localization and quantity of genetic cassettes were also found for the Dot/Icm type IVB secretion system. To determine divergences between core-genomes, additional pan-genome descriptions were compiled for the so-termed LF and EM genogroups. Open pan-genomes composed of 2,924 and 2,778 genes and core-genomes composed of 2,170 and 2,228 genes were respectively found for the LF and EM genogroups. The core-genomes were functionally annotated using the Gene Ontology, KEGG, and Virulence Factor databases, revealing the presence of several shared groups of genes related to basic function of intracellular survival and bacterial pathogenesis. Additionally, the specific pan-genomes for the LF and EM genogroups were defined, resulting in the identification of 148 and 273 exclusive proteins, respectively. Notably, specific virulence factors linked to adherence, colonization, invasion factors, and endotoxins were established. The obtained data suggest that these genes could be directly associated with inter-genogroup differences in pathogenesis and host-pathogen interactions, information that could be useful in designing novel strategies for diagnosing and controlling P. salmonis infection.

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

In vitro expression of Sec-dependent pathway and type 4B secretion system in Piscirickettsia salmonis.

Piscirickettsia salmonis is an intracellular bacterium and the causative agent of Piscirickettsiosis, a disease responsible for considerable mortalities in the Chilean salmon farming industry. Currently, P. salmonis protein translocation across the membrane and the mechanisms by which virulence factors are delivered to host cells are poorly understood. However, it is known that Gram-negative bacteria possess several mechanisms that transport proteins to the periplasmic and extracellular compartments. The aim of this study was to evaluate the expressional changes of several genes in the P. salmonis Sec-dependent pathway and type 4B secretion system during in vitro infection. Genes homologous and the main proteins belonging to Sec-dependent pathway and Type 4 Dot/Icm secretion system were found in the genome and proteome of P. salmonis AUSTRAL-005 strain. Additionally, several genes of these protein transport mechanisms were overexpressed during in vitro P. salmonis infection in SHK-1 cell line. The obtained data indicate that the Sec-dependent pathway and Type 4B secretion system are biologically active during P. salmonis infection. These mechanisms could contribute to the recycling of proteins into the inner and outer bacterial membrane and in translocate virulence factors to infected cell, which would favor the structural integrity and virulence of this bacterium.

Membrane vesicles from Piscirickettsia salmonis induce protective immunity and reduce development of salmonid rickettsial septicemia in an adult zebrafish model.

Infections caused by the facultative intracellular bacterial pathogen Piscirickettsia salmonis remains an unsolved problem for the aquaculture as no efficient treatments have been developed. As a result, substantial amounts of antibiotic have been used to limit salmonid rickettsial septicemia (SRS) disease outbreaks. The antibiotic usage has not reduced the occurrence, but lead to an increase in resistant strains, underlining the need for new treatment strategies. P. salmonis produce membrane vesicles (MVs); small spherical structures know to contain a variety of bacterial components, including proteins, lipopolysaccharides (LPS), DNA and RNA. MVs mimics' in many aspects their mother cell, and has been reported as alternative vaccine candidates. Here, MVs from P. salmonis was isolated and evaluated as a vaccine candidate against SRS in an adult zebrafish infection model. When zebrafish was immunized with MVs they were protected from subsequent challenge with a lethal dose of P. salmonis. Histological analysis showed a reduced bacterial load upon challenge in the MV immunized group, and the mRNA expression levels of several immune related genes altered, including mpeg1.1, tnfα, il1b, il10 and il6. The MVs induced the secretion of IgM upon immunization, indicating an immunogenic effect of the vesicles. Taken together, the data demonstrate a vaccine potential of MVs against P. salmonis.

Identification of chemotaxis operon cheYZA and cheA gene expression under stressful conditions in Piscirickettsia salmonis.

Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, which, as the main systemic disease in the Chilean salmon industry, causes significant economic losses. This bacterium can produce biofilm as a persistence and survival strategy in adverse conditions. In other bacteria, cheA is a key gene for modulating the onset of bacterial chemotaxis, as well as having a secondary role in biofilm production. Notwithstanding this association, the potential relationships between biofilm formation and genes involved in P. salmonis chemotaxis are poorly understood. This study aimed to determine P. salmonis cheA gene expression when grown in different culture media known to induce biofilm production. Piscirickettsia salmonis AUSTRAL-005 produced moderate/high biofilm levels after 144 h of incubation in the AUSTRAL-SRS and marine broths. In contrast, LF-89 biofilm production was weak/nonexistent in the aforementioned broths. Both assessed P. salmonis strains contained the cheYZA operon. Additionally, AUSTRAL-005 cheA transcripts increased in both culture media. In conclusion, these results suggest potential relationships between biofilm formation and genes related to chemotaxis in the fish pathogen P. salmonis.

Resistance-nodulation-division efflux pump acrAB is modulated by florfenicol and contributes to drug resistance in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is a fastidious intracellular pathogen responsible for high mortality rates in farmed salmonids, with serious economic consequences for the Chilean aquaculture industry. Oxytetracycline and florfenicol are the most frequently used antibiotics against P. salmonis, but routine use could contribute to drug resistance. This study identified differentiated florfenicol susceptibilities in two P. salmonis strains, LF-89 and AUSTRAL-005. The less susceptible isolate, AUSTRAL-005, also showed a high ethidium bromide efflux rate, indicating a higher activity of general efflux pump genes than LF-89. The P. salmonis genome presented resistance nodulation division (RND) family members, a family containing typical multidrug resistance-related efflux pumps in Gram-negative bacteria. Additionally, efflux pump acrAB genes were overexpressed in AUSTRAL-005 following exposure to the tolerated maximal concentration of florfenicol, in contrast to LF-89. These results indicate that tolerated maximum concentrations of florfenicol can modulate RND gene expression and increase efflux pump activity. We propose that the acrAB efflux pump is essential for P. salmonis survival at critical florfenicol concentrations and for the generation of antibiotic-resistant bacterial strains.