New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides.

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

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.

Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

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.

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.

Determination of the Dissolution/Permeation and Apparent Solubility for Microencapsulated Emamectin Benzoate Using In Vitro and Ex Vivo Salmo salar Intestine Membranes.

In this work, two microencapsulation techniques were used to protect and improve the absorption of emamectin benzoate (EB), which is an antiparasitic drug used to control Caligus rogercresseyi. EB has a low aqueous solubility, which affects its absorption in the intestine of Salmo salar. Microparticles were produced by spray drying and ionic gelation, using Soluplus® (EB−SOL) and sodium alginate (EB−ALG) as polymers, respectively. Studies were conducted on dissolution/permeation, apparent permeability (Papp), apparent solubility (Sapp), and absorption using synthetic and biological membranes. Based on these results, the amount of EB in the microparticles needed to achieve a therapeutic dose was estimated. The EB−ALG microparticles outperformed both EB−SOL and free EB, for all parameters analyzed. The results show values of 0.45 mg/mL (80.2%) for dissolution/permeation, a Papp of 6.2 mg/mL in RS−L, an absorption of 7.3% in RS, and a Sapp of 53.1% in EM medium. The EB−ALG microparticles decrease the therapeutic dose necessary to control the parasite, with values of 3.0−2 mg/mL and 1.1−2 mg/mL for EB in EM and RS, respectively. The Korsmeyer−Peppas kinetic model was the best model to fit the EB−ALG and EB−SOL dissolution/permeation experiments. In addition, some of our experimental results using synthetic membranes are similar to those obtained with biological membranes, which suggests that, for some parameters, it is possible to replace biological membranes with synthetic membranes. The encapsulation of EB by ionic gelation shows it is a promising formulation to increase the absorption of the poorly soluble drug. In contrast, the spray-dried microparticles produced using Soluplus® result in even less dissolution/permeation than free EB, so the technique cannot be used to improve the solubility of EB.

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 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.

New strategy for the design, production and pre-purification of chimeric peptide with immunomodulatory activity in Salmosalar.

The intensive salmon farming is associated with massive outbreaks of infections. The use of antibiotics for their prevention and control is related to damage to the environment and human health. Antimicrobial peptides (AMPs) have been proposed as an alternative to the use of antibiotics for their antimicrobial and immunomodulatory activities. However, one of the main challenges for its massive clinical application is the high production cost and the complexity of chemical synthesis. Thus, recombinant DNA technology offers a more sustainable, scalable, and profitable option. In the present study, using an AMPs function prediction methodology, we designed a chimeric peptide consisting of sequences derived from cathelicidin fused with the immunomodulatory peptide derived from flagellin. The designed peptide, CATH-FLA was produced by recombinant expression using an easy pre-purification system. The chimeric peptide was able to induce IL-1ß and IL-8 expression in Salmo salar head kidney leukocytes, and prevented Piscirickettsia salmonis-induced cytotoxicity in SHK-1 cells. These results suggest that pre-purification of a recombinant AMP-based chimeric peptide designed in silico allow obtaining a peptide with immunomodulatory activity in vitro. This could solve the main obstacle of AMPs for massive clinical applications.

Salmonid MyD88 is a key adapter protein that activates innate effector mechanisms through the TLR5M/TLR5S signaling pathway and protects against Piscirickettsia salmonis infection.

The membrane-anchored and soluble Toll-like Receptor 5 -TLR5M and TLR5S, respectively-from teleost recognize bacterial flagellin and induce the pro-inflammatory cytokines expression in a MyD88-dependent manner such as the TLR5 mammalian orthologous receptor. However, it has not been demonstrated whether the induced signaling pathway by these receptors activate innate effector mechanisms MyD88-dependent in salmonids. Therefore, in this work we study the MyD88 dependence on the induction of TLR5M/TLR5S signaling pathway mediated by flagellin as ligand on the activation of some innate effector mechanisms. The intracellular and extracellular Reactive Oxygen Species (ROS) production and conditioned supernatants production were evaluated in RTS11 cells, while the challenge with Piscirickettsia salmonis was evaluated in SHK-1 cells. Our results demonstrate that flagellin directly stimulates ROS production and indirectly stimulates it through the production of conditioned supernatants, both in a MyD88-dependent manner. Additionally, flagellin stimulation prevents the cytotoxicity induced by infection with P. salmonis in a MyD88-dependent manner. In conclusion we demonstrate that MyD88 is an essential adapter protein in the activation of the TLR5M/TLR5S signaling pathway mediated by flagellin in salmonids, which leads downstream to the induction of innate effector mechanisms, promoting immuno-protection against a bacterial challenge with P. salmonis.

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.

Molecular survey and genetic characterization of 'Candidatus Mycoplasma haemolamae' in llamas (Lama glama) and alpacas (Vicugna pacos) from Southern Chile.

This study aimed to perform a molecular survey and identification of hemotropic Mycoplasma spp. in domestic South American Camelids from Southern Chile. Conventional PCR (cPCR) for hemotropic Mycoplasma spp. based on 16S rRNA gene (620bp fragment) was performed in 87 EDTA-blood samples taken from 48 llamas (Lama glama) and 39 and alpacas (Vicugna pacos) from to Temuco, La Araucanía region and Valdivia, Los Rios region, Southern Chile. 16S rRNA hemotropic Mycoplasma PCR-positive were sequenced for species identification, phylogenetic and haplotype analyses, and further tested by cPCR targeting a fragment (160-210 bp) of the RNaseP (rnpB) gene. Based upon 16S rRNA cPCR results, the overall hemotropic Mycoplasma spp. occurrence in Southern camelids was 9.2% (8/87 [95% CI (4.0-17.3%)]), with five positive alpacas (12.8%; 5/39 [95% CI (4.3-27.4%)]) and three llamas (6.3%; 3/48 [95% CI (1.7-17.2%)]). All 16S rRNA PCR-positive samples were negative for the rnpB gene. Obtained 16S sequences presented high identity (99-100%) by BLASTn analysis to 'Candidatus Mycoplasma haemolamae' from an alpaca in the United Kingdom. Phylogenetic and haplotype analyses of the 16s rRNA gene showed high similarity among 'Candidatus M. haemolamae' sequences of this study and the ones from North America, Europe, and Asia evidencing a low diversity of Chilean samples, with only one haplotype detected (#1). Haplotype #1 from South American Camelids in Chile was worldwide distributed and observed in North America, Europe, and Asia. 'Candidatus M. haemolamae' detected for the first time in South American camelids in Southern Chile had low diversity and was worldwide spread.

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.

Identification of Neospora caninumin aborted bovine fetuses of southern Chile

The aim of this study was to identify Neospora caninum in histologic sections of brain and optic nerve of aborted bovine fetuses. Sections of these tissues from 296 aborted bovine fetuses from Southern Chile, submitted between 2000 and 2010, to the Instituto de Patología Animal of the Universidad Austral de Chile were analyzed. Forty-four (14.9%) fetuses had microscopic lesions compatible with bovine neosporosis, including gliosis, necrosis, intralesional protozoan cysts and neuritis of optic nerves. N. caninum was identified by immunohistochemistry in brain sections of 27 cases (9.1%) with compatible lesions. Positive immunostaining was also found in the optic nerves of 13 cases (4.4%) with neuritis. N. caninum DNA was identified by PCR in brain sections in 31 cases (10.5%). In total, 36 cases (12.2%) were positive by either technique. Phylogenetic analysis of the ITS1 gene sequence of N. caninum revealed a high degree of conservation among different isolates. This is the first report of N. caninum-associated optic neuritis in cattle and also the first report of confirmed bovine abortion associated with this parasite in Chile.(AU)

Identification of Neospora caninumin aborted bovine fetuses of southern Chile

The aim of this study was to identify Neospora caninum in histologic sections of brain and optic nerve of aborted bovine fetuses. Sections of these tissues from 296 aborted bovine fetuses from Southern Chile, submitted between 2000 and 2010, to the Instituto de Patología Animal of the Universidad Austral de Chile were analyzed. Forty-four (14.9%) fetuses had microscopic lesions compatible with bovine neosporosis, including gliosis, necrosis, intralesional protozoan cysts and neuritis of optic nerves. N. caninum was identified by immunohistochemistry in brain sections of 27 cases (9.1%) with compatible lesions. Positive immunostaining was also found in the optic nerves of 13 cases (4.4%) with neuritis. N. caninum DNA was identified by PCR in brain sections in 31 cases (10.5%). In total, 36 cases (12.2%) were positive by either technique. Phylogenetic analysis of the ITS1 gene sequence of N. caninum revealed a high degree of conservation among different isolates. This is the first report of N. caninum-associated optic neuritis in cattle and also the first report of confirmed bovine abortion associated with this parasite in Chile.

Nutritional Immunity Triggers the Modulation of Iron Metabolism Genes in the Sub-Antarctic Notothenioid Eleginops maclovinus in Response to Piscirickettsia salmonis.

Iron deprivation is a nutritional immunity mechanism through which fish can limit the amount of iron available to invading bacteria. The aim of this study was to evaluate the modulation of iron metabolism genes in the liver and brain of sub-Antarctic notothenioid Eleginops maclovinus challenged with Piscirickettsia salmonis. The specimens were inoculated with two P. salmonis strains: LF-89 (ATCC® VR-1361™) and Austral-005 (antibiotic resistant). Hepatic and brain samples were collected at intervals over a period of 35 days. Gene expression (by RT-qPCR) of proteins involved in iron storage, transport, and binding were statistically modulated in infected fish when compared with control counterparts. Specifically, the expression profiles of the transferrin and hemopexin genes in the liver, as well as the expression profiles of ferritin-M, ferritin-L, and transferrin in the brain, were similar for both experimental groups. Nevertheless, the remaining genes such as ferritin-H, ceruloplasmin, hepcidin, and haptoglobin presented tissue-specific expression profiles that varied in relation to the injected bacterial strain and sampling time-point. These results suggest that nutritional immunity could be an important immune defense mechanism for E. maclovinus against P. salmonis injection. This study provides relevant information for understanding iron metabolism of a sub-Antarctic notothenioid fish.

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.

First identification of Francisella noatunensis subsp. orientalis causing mortality in Mexican tilapia Oreochromis spp.

Francisellosis, an emerging disease in tilapia Oreochromis spp., is caused by the facultative, intracellular bacterium Francisella noatunensis subsp. orientalis, which is present in various countries where tilapia farming is commercially important. We confirmed the presence of francisellosis in Mexican tilapia cultures in association with an outbreak during the second semester of 2012. Broodstock fish presented a mortality rate of approximately 40%, and disease was characterized by histologically classified granulomas, or whitish nodules, in different organs, mainly the spleen and kidney. Through DNA obtained from infected tissue and pure cultures in a cysteine heart medium supplemented with hemoglobin, F. noatunensis subsp. orientalis was initially confirmed through the amplification and analysis of the 16S rRNA gene and the internal transcribed spacer region. Phylogenetic analysis of these genes demonstrated close similarity with previously reported F. noatunensis subsp. orientalis sequences obtained from infected tilapia from various countries. The identification of this subspecies as the causative agent of the outbreak was confirmed using the iglC gene as a target sequence, which showed 99.5% identity to 2 F. noatunensis subsp. orientalis strains (Ethime-1 and Toba04). These findings represent the first documented occurrence of francisellosis in Mexican tilapia cultures, which highlights the importance of establishing preventative measures to minimize the spread of this disease within the Mexican aquaculture industry.

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.