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.

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.

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

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

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.

Effect of low-dose Piscirickettsia salmonis infection on haematological-biochemical blood parameters in Atlantic salmon (Salmo salar).

Piscirickettsia salmonis is the etiological agent of Piscirickettsiosis, a severe disease that affects Atlantic salmon (Salmo salar) farmed in Chile and many other areas (Norway, Scotland, Ireland, Canada and the USA). This study investigated the effects of low-dose P. salmonis infection (1 × 102 CFU/ml) on Atlantic salmon. In this study, we challenged fish with an isolated representative of the EM-90 genogroup via intraperitoneal injection for 42 days. Infected fish displayed decreased haematocrit and haemoglobin levels at day 13 post-infection, indicating erythropenia, haemolysis and haemodilution. Conversely, their white blood cell counts increased on days 13 and 21 post-infection. Additionally, their iron levels decreased from day 2 post-infection, indicating iron deficiency and an inability to retrieve stored iron before infection. Their magnesium levels also decreased at day 28 post-infection, possibly due to osmoregulatory problems. Also, we observed an increase in lactate dehydrogenase activity on days 5, 21, and 28 post-infection, suggesting early symptoms of hepatotoxicity. Later analyses determined a decrease in plasma glucose levels from day 2 post-infection. This may be attributed to the hypoxic conditions caused by P. salmonis, leading to an excess utilization of stored carbohydrates. Our results suggest that the blood parameters we studied are useful for monitoring the physiological status of Atlantic salmon infected with P. salmonis.

Antagonistic activity of endophytic actinobacteria from native potatoes (Solanum tuberosum subsp. tuberosum L.) against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum.

BACKGROUND: The native potatoes (Solanum tuberosum subsp. tuberosum L.) grown in Chile (Chiloé) represent a new, unexplored source of endophytes to find potential biological control agents for the prevention of bacterial diseases, like blackleg and soft rot, in potato crops. RESULT: The objective of this study was the selection of endophytic actinobacteria from native potatoes for antagonistic activity against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum, and their potential to suppress tissue maceration symptoms in potato tubers. This potential was determined through the quorum quenching activity using a Chromobacterium violaceaum ATCC 12472 Wild type (WT) bioassay and its colonization behavior of the potato plant root system (S. tuberosum) by means of the Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) targeting technique. The results showed that although Streptomyces sp. TP199 and Streptomyces sp. A2R31 were able to inhibit the growth of the pathogens, only the Streptomyces sp. TP199 isolate inhibited Pectobacterium sp. growth and diminished tissue maceration in tubers (p ≤ 0.05). Streptomyces sp. TP199 had metal-dependent acyl homoserine lactones (AHL) quorum quenching activity in vitro and was able to colonize the root endosphere 10 days after inoculation. CONCLUSIONS: We concluded that native potatoes from southern Chile possess endophyte actinobacteria that are potential agents for the disease management of soft rot and blackleg.

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.

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.

Survival behaviour and virulence of the fish pathogen Vibrio ordalii in seawater microcosms.

Vibrio ordalii, the causative agent of atypical vibriosis, is a Gram-negative, motile, rod-shaped bacterium that severely affects the salmonid aquaculture industry. V. ordalii has been biochemically, antigenically and genetically characterized. However, studies on the survival behaviour of this bacterium in aquatic environments are scarce, and there is no information regarding its disease transmission and infectious abilities outside of the fish host or regarding water as a possible reservoir. The present study investigated the survival behaviour of V. ordalii Vo-LM-06 and Vo-LM-18 in sterile and non-sterile seawater microcosms. After a year in sterile seawater without nutrients, 1% of both V. ordalii strains survived (~10(3) colony-forming units ml(-1)), and long-term maintenance did not affect bacterial biochemical or genetic properties. Additionally, V. ordalii maintained for 60 d in sterile seawater remained infective in rainbow trout Oncorhynchus mykiss. However, after 2 d of natural seawater exposure, this bacterium became non-culturable, indicating that autochthonous microbiota may play an important role in survival. Recuperation assays that added fresh medium to non-sterile microcosms did not favour V. ordalii recovery on solid media. Our results contribute towards a better understanding of V. ordalii survival behaviour in seawater ecosystems.

Iron assimilation and siderophore production by Vibrio ordalii strains isolated from diseased Atlantic salmon Salmo salar in Chile.

Vibrio ordalii is the causative agent of vibriosis in several cultured salmonid species worldwide. Despite its impact on aquaculture, relatively little information is available about its virulence factors. The present study demonstrates for the first time that V. ordalii possesses different systems of iron acquisition, one involving siderophore synthesis and another one that uses direct binding of heme to use iron. Using 6 strains of V. ordalii from Atlantic salmon Salmo salar and the V. ordalii type strain, we could demonstrate that all strains could grow in presence of the chelating agent 2,2'-dipyridyl and produced siderophores in solid and liquid media. Cross-feeding assays among V. ordalii strains evidenced variability in the siderophores produced. Bioassays and PCR data suggest that V. ordalii could produce a siderophore with a structure similar to piscibactin, although the production of a second siderophore in certain strains cannot be discarded. Furthermore, all strains were able to use hemin and hemoglobin as the only iron sources, although the cell yield was higher when using hemoglobin. A hemin-binding assay indicated the presence of constitutive heme-binding molecules at the cell surface of V. ordalii. Virulence tests using rainbow trout as a model of infection revealed a clear relationship between iron-uptake ability and pathogenicity in V. ordalii.

Short- and long-term responses and recovery of mussels Mytilus edulis exposed to heavy fuel oil no. 6 and styrene.

Biomarkers have the potential to be used to assess the impact of anthropogenic discharges in marine waters. We have used a suite of biomarkers spanning from enzymatic to histopathological alterations and general stress responses to assess the short- and long-term impact on mussels Mytilus edulis of heavy fuel oil no. 6 and styrene. Mussels were exposed for 5 months, with a refilling of the exposure system, to a water soluble fraction of heavy fuel and, then, kept for a month in clean water for recovery. In a second experiment, mussels were exposed to styrene for 19 days and maintained in clean water for up to 4 months. Chemical body tissue levels reflected the weathering processes of these compounds. Acyl-CoA oxidase activity was induced in oil-exposed mussels after refilling, whereas styrene inhibited it after 19 days of exposure and after 2 weeks in clean water. Gamete development and alkali-labile phosphate levels suggest that neither oil nor styrene behaved as endocrine disruptors. Neutral red retention time was lower in treated groups than in controls. Lysosomal membrane stability was significantly reduced in exposed groups and recovered after withdrawal of oil but not after removal of styrene. Neither oil nor styrene exposure affected the condition index except for the reduction seen in mussels exposed to oil for 1 month. Biomarker response index discriminated exposed mussels, which showed higher values, and returned to control levels after recovery. Results obtained from these pilot experiments can help to identify relevant monitoring tools to assess the impact of oil and chemicals in marine spill scenarios.

Breastfeeding Practice Before Bottle-Feeding: An Initiative to Increase the Rate of Breastfeeding for Preterm Infants at the Time of Neonatal Intensive Care Unit Discharge.

Preterm babies who received 72 hours of breastfeeding practice before introducing a bottle had significantly higher rates of breastfeeding at the time of neonatal intensive care unit (NICU) discharge than did babies who were introduced to bottle-feeding with or before breastfeeding during the first 72 hours of oral feeding or babies who were primarily bottle-fed. There were no statistical differences in corrected gestational age (CGA) at birth, first oral feeding, or full oral feeds, in days from first to full oral feeds, or in CGA or days of life at NICU discharge.

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.

The influence of temperature on the effects of lead and lithium in Mytilus galloprovincialis through biochemical, cell and tissue levels: Comparison between mono and multi-element exposures.

Lead (Pb) and lithium (Li) are metals which have been detected in the environment and, at high concentrations, can induce toxic effects that disturb the growth, metabolism or reproduction of organisms along the entire trophic chain. The impacts of these metals have scarcely been investigated using marine bivalves, especially when acting as a mixture. The present study aimed to investigate the influence of temperature on the ecotoxicological effects caused by Pb and Li, acting alone and as a mixture, on the mussel species Mytilus galloprovincialis after 28 days of exposure. The impacts were evaluated under actual (17 °C) and projected (+4 °C) warming conditions, to understand the influence of temperature rise on the effects of the metals (both acting alone or as a mixture). The results obtained showed that the increased temperature did not influence the accumulation of metals. However, the biomarkers evaluated showed greater responses in mussels that are exposed to metals under increased temperature (21 °C). The IBR index showed that there is a comparable toxic effect of Li and Pb separately, while exposure to a mixture of both pollutants causes a significantly higher stress response. Overall, the results obtained revealed that temperature may cause extra stress on the mussels and exposure to the metal mixture caused the greatest impacts compared to each metal acting alone.

Somatic evolution of marine transmissible leukemias in the common cockle, Cerastoderma edule.

Transmissible cancers are malignant cell lineages that spread clonally between individuals. Several such cancers, termed bivalve transmissible neoplasia (BTN), induce leukemia-like disease in marine bivalves. This is the case of BTN lineages affecting the common cockle, Cerastoderma edule, which inhabits the Atlantic coasts of Europe and northwest Africa. To investigate the evolution of cockle BTN, we collected 6,854 cockles, diagnosed 390 BTN tumors, generated a reference genome and assessed genomic variation across 61 tumors. Our analyses confirmed the existence of two BTN lineages with hemocytic origins. Mitochondrial variation revealed mitochondrial capture and host co-infection events. Mutational analyses identified lineage-specific signatures, one of which likely reflects DNA alkylation. Cytogenetic and copy number analyses uncovered pervasive genomic instability, with whole-genome duplication, oncogene amplification and alkylation-repair suppression as likely drivers. Satellite DNA distributions suggested ancient clonal origins. Our study illuminates long-term cancer evolution under the sea and reveals tolerance of extreme instability in neoplastic genomes.

Transcriptional responses of cancer-related genes in turbot Scophthalmus maximus and mussels Mytilus edulis exposed to heavy fuel oil no. 6 and styrene.

Recent spills in European waters have released polycyclic aromatic hydrocarbons, important components of heavy fuel oil, and the hydrocarbon styrene. Heavy fuel oil and styrene are classified as potentially genotoxic and carcinogenic. Here we investigate transcription of genes involved in cancer development in the liver of juvenile turbots and in the digestive gland of mussels exposed to heavy fuel oil and to styrene and after a recovery period. In turbot, oil produced a significant up-regulation of p53 and gadd45α after 14 days exposure. cyclin G1 was up-regulated after 7 days treatment with styrene. In mussels, ras was down-regulated in both treatments after the recovery periods. No mutations in ras hotspots were detected in exposed mussels. gadd45α was up-regulated after the recovery period of the styrene experiment. Overall, transcriptional responses differed in mussels compared to turbot. Turbot responded to hydrocarbon exposure by triggering cell cycle arrest (p53) and DNA repair (gadd45α).

Toxicopathic effects of lithium in mussels.

The rising use of lithium (Li) in industrial processes, modern technology and medicine has generated concerns in the scientific community, in particular its potential impact on the environment. Unfortunately, there is only scarce information concerning the toxicity of lithium in marine organisms. The objective of this study is to determine the toxicity of Li using Mytilus galloprovincialis as model organism, based on acute and sublethal toxicity tests. In the first experiment, mussels were exposed for 9 days to a range of acute concentrations of Li (0, 2, 5, 13, 34, 89, 233 and 610 mg/L Li) in order to find the median lethal concentration. In the sublethal experiment, mussels were exposed to environmentally relevant concentrations of Li (0, 0.1, 1, 10 mg/L Li) for 21 days. Digestive gland and gonad samples were taken at day 0, 1, 7 and 21 for histopathological analysis. Samples of the whole mussels were taken for chemical analysis at day 0 and after 21 days. Results showed that M. galloprovincialis had a LC50 value of 153 mg/L Li after 9 days of exposure. Lower concentrations (environmentally relevant), led to Li bioaccumulation in a dose-dependent manner and histopathological effects in a time-dependent manner. Atrophy of the digestive alveoli epithelium and degeneration of the digestive gland were observed after 21 days of exposure. These findings open new perspectives for the understanding of the toxic effects of Li on marine organisms and evidence the need for further long-term research at different levels of biological organizations.

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.

Correction: Catabolic Signaling Pathways, Atrogenes, and Ubiquitinated Proteins Are Regulated by the Nutritional Status in the Muscle of the Fine Flounder.

[This corrects the article DOI: 10.1371/journal.pone.0044256.].