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.

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.

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.

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.

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.

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.

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.

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.

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.

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.].

The Fish Pathogen Vibrio ordalii Under Iron Deprivation Produces the Siderophore Piscibactin.

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using ß-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in V. ordalii. Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of V. ordalii cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like Photobacterium damselae subsp. piscicida and V. anguillarum, which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.

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.

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.

Hepatic gene transcription profiles in turbot (Scophthalmus maximus) experimentally exposed to heavy fuel oil nº 6 and to styrene.

Oil and chemical spills in the marine environment, although sporadic, are highly dangerous to biota inhabiting coastal and estuarine areas. Effects of spilled compounds in exposed organisms occur at different biological organization levels: from molecular, cellular or tissue levels to the physiological one. The present study aims to determine the specific hepatic gene transcription profiles observed in turbot juveniles under exposure to fuel oil n °6 and styrene vs controls using an immune enriched turbot (Scophthalmus maximus) oligo-microarray containing 2716 specific gene probes. After 3 days of exposure, fuel oil specifically induced aryl hydrocarbon receptor mediated transcriptional response through up-regulation of genes, such as ahrr and cyp1a1. More gene transcripts were regulated after 14 days of exposure involved in ribosomal biosynthesis, immune modulation, and oxidative response among the most significantly regulated functional pathways. On the contrary, gene transcription alterations caused by styrene did not highlight any significantly regulated molecular or metabolic pathway. This was also previously reported at cell and tissue level where no apparent responses were distinguishable. For the fuel oil experiment, obtained specific gene profiles could be related to changes in cell-tissue organization in the same individuals, such as increased hepatocyte vacuolization, decrease in melano-macrophage centers and the regulation of leukocyte numbers. In conclusion, the mode of action reflected by gene transcription profiles analyzed hereby in turbot livers could be linked with the responses previously reported at higher biological organization levels. Molecular alterations described hereby could be preceding observed alterations at cell and tissue levels.