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International audience; Francisella noatunensis subsp. orientalis (Fno) is a Gram-negative bacterium that causes granulomatous infections in fish reared at low water temperatures; it has been responsible for a large number of deaths in tilapia fish farms. Fno has been reported in many countries in the last decade. Studies on phylogenomic relationships among isolates are needed because of the increasing importance of this disease. Here, we present the complete genome of the Fno F1 strain isolated in 2015 from a tilapia farm in Sao Paulo state, Brazil. The genome is a circular chromosome with a length of 1,854,333 bp, containing 32.26% G+C DNA content, 1,448 protein-coding genes and 393 pseudogenes. In addition, a prediction of conserved vaccine targets was made between the two subspecies of F. noatunensis that cause disease in tropical and cold-water fish species. Five proteins conserved between Fno and Francisella noatunensis subsp. noatunensis were predicted to be good vaccine candidates for the development of a recombinant vaccine against francisellosis. This genome also provides useful data to help understand the pathogen evolution and epidemiology of this disease in Brazil.

Elastin is an important proteinaceous component of vertebrate connective tissues (e.g., blood vessels, lung, and skin), to which it confers elasticity. Elastases have been identified in a number of pathogenic bacteria. They are thought to be required for tissue penetration and dissemination, acting as “spreading factors.” Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonid fish (salmon and trout) that is responsible for severe economic losses worldwide. This pathogen displays strong proteolytic activities. Using a variety of techniques, including genome comparisons, we identified a gene encoding a novel elastase in F. psychrophilum. The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. In addition, this elastase likely belongs to a new family of proteases that seems to be present only in some members of this important group of bacteria., Hydrolytic extracellular enzymes degrading host tissues potentially play a role in bacterial pathogenesis. Flavobacterium psychrophilum is an important bacterial pathogen of salmonid fish reared in freshwater throughout the world. Diversity among isolates has been described at the phenotypic, serological, and genomic levels, but the links between these various traits remain poorly understood. Using a genome-wide association study, we identified a gene encoding a novel elastinolytic enzyme in F. psychrophilum. To formally demonstrate enzymatic activity, this gene (FP0506 from strain JIP 02/86) was expressed in the elastinolysis-deficient strain OSU THCO2-90, resulting in proficient elastin-degrading cells. The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. FP0506 might belong to the zincin tribe and gluzincin clan of metalloproteases, and this new elastase-encoding gene seems to be present only in some members of the family Flavobacteriaceae. IMPORTANCE Elastin is an important proteinaceous component of vertebrate connective tissues (e.g., blood vessels, lung, and skin), to which it confers elasticity. Elastases have been identified in a number of pathogenic bacteria. They are thought to be required for tissue penetration and dissemination, acting as “spreading factors.” Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonid fish (salmon and trout) that is responsible for severe economic losses worldwide. This pathogen displays strong proteolytic activities. Using a variety of techniques, including genome comparisons, we identified a gene encoding a novel elastase in F. psychrophilum. The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. In addition, this elastase likely belongs to a new family of proteases that seems to be present only in some members of this important group of bacteria.

A novel Citrobacter species was isolated from the kidney of diseased rainbow trout (Oncorhynchus mykiss) reared on a trout farm. Biochemical characterization and phylogenetic analysis were performed for bacterial identification. Sequencing of the 16S rRNA gene and five housekeeping genes indicated that the strain belongs to the Citrobacter genus. However, multilocus sequence analysis, a comparison of average nucleotide identity, and genome-to-genome distance values revealed that strain SNU WT2 is distinct and forms a separate clade from other Citrobacter species. Additionally, the phenotype characteristics of the strain differed from those of other Citrobacter species. Quinone analysis indicated that the predominant isoprenoid quinone is Q-10. Furthermore, strain virulence was determined by a rainbow trout challenge trial, and the strain showed resistance to diverse antibiotics including &beta, lactams, quinolone, and aminoglycosides. The complete genome of strain SNU WT2 is 4,840,504 bp with a DNA G + C content of 51.94% and 106,068-bp plasmid. Genome analysis revealed that the strain carries virulence factors on its chromosome and antibiotic resistance genes on its plasmid. This strain represents a novel species in the genus Citrobacter for which the name C. tructae has been proposed, with SNU WT2 (=KCTC 72517 = JCM 33612) as the type strain.

The use of cleaner fish as biological controls of salmon lice (Lepeophtheirus salmonis) has increased exponentially in the last decade in Norwegian Atlantic salmon (Salmo salar) production. This alternative to chemical treatments has resulted in the emergence of lumpsucker (Cyclopterus lumpus) hatcheries and culture facilities in Norway. It has been shown that the use of lumpsuckers can be an effective, biological approach for the removal of salmon lice, but it has also been shown that there are a number of biological challenges (i.e. parasites and bacteria) with the production and use of these fish. This study describes the first case of isolation of Tenacibaculum maritimum, a significant fish pathogen worldwide, in cultured juvenile lumpsuckers in Norway. The fish were lethargic and showed skin lesions characterised by increased mucus production and presence of whitish necrotic tissue especially in the head region. Skin scrapings revealed large amounts of bacteria dominated by rod-shaped Tenacibaculum-like bacteria, which were shown to be closely related to T. maritimum type strain through genetic and phenotypic characterisation. Histopathological analysis showed that the bacteria was closely associated with the pathology and therefore could be contributing to the disease and/or mortality. Statement of relevance This is the first isolation of Tenacibaculum maritimum in Norway and in lumpsuckers, a major aquaculture pathogen worldwide. There is a need for increased knowledge of the biological challenges facing cultured lumpsuckers, as this species is being used in increasing number by the Norwegian salmon industry.

Cyclamen L. is a member of Primulaceae family, a geophyte, which have been utilized for their biological activities in folk medicine. The aim of this study is to investigate the antibacterial activity of three Cyclamen species on some fish pathogens as well as their radical scavenging activity potentials. C. cilicium Boiss. & Heldr., C. pseudibericum Hildebr. and C. hederifolium Aiton were collected from different regions in Turkey. Cyclamen tubers were extracted in ethanol and then DPPH, ABTS and NO methods were utilized for the assurance of radical scavenging activities of the extracts. Antibacterial activity of the extracts were determined in vitro against five bacterial fish pathogens, namely, Vagococcus salmoninarum, Staphylococcus epidermidis, Lactococcus garvieae, Vibrio anguillarum and Yersinia ruckeri. All the tested tuber extracts exhibited moderate antibacterial activity against four bacteria and the radical scavenging activity in the order of potency: C. cilicium>C. pseudibericum>C. hederifolium. In conclusion, this study showed that the Cyclamen species have moderate antibacterial activity against fish pathogenic bacteria except V. anguillarum. Sıklamen (Cyclamen L.) Primulaceae familyasının bir üyesi olup, geofit bitki olarak biyolojik aktivitelerinden dolayı halk arasında tıbbi amaçlı kullanılmaktadır. Bu çalışmanın amacı, üç sıklamen türünden elde edilen ekstraktların balık patojenleri üzerindeki antibakteriyel aktivitelerini ve radikal süpürme aktivite potensiyellerini belirlemektir. Bu çalışmada kullanılan türler; C. cilicium Boiss. & Heldr., C. pseudibericum Hildebr. ve C. hederifolium Aiton, Türkiye’nin farklı bölgelerinden toplanmıştır. Sıklamen yumruları etanolde ekstrakte edilmiş ve daha sonra ekstraktlardaki radikal süpürme aktivitelerinin belirlenmesi için DPPH, ABTS ve NO metodları kullanılmıştır. Sıklamen bitki ekstraktlarının antibakteriyel aktivitesi in vitro yöntemle beş bakteriyel balık patojenine (Vagococcus salmoninarum, Staphylococcus epidermidis, Lactococcus garvieae, Vibrio anguillarum ve Yersinia ruckeri) karşı belirlenmiştir. Yumrulardan elde edilen ekstraktlarının test edilen tüm bakterilere karşı V. anguillarum hariç antibakteriyel aktivite sergilediğini ve radikal süpürme aktivitesinin en yüksekten en düşüğe sırasıyla C. cilicium>C. pseudibericum>C. hederifolium olduğu belirlenmiştir. Sonuç olarak bu çalışma, test edilen Sıklamen türlerinin su ürünleri yetiştiriciliğinde görülen patojen bakteriler üzerinde V. anguillarum hariç orta düzeyde antibakteriyel etkinliğe sahip olduğunu göstermiştir.

This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for Yersinia ruckeri infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any Y. ruckeri strain may rapidly be placed in a global epizootiological context., A multilocus variable-number tandem-repeat analysis (MLVA) assay was developed for epizootiological study of the internationally significant fish pathogen Yersinia ruckeri, which causes yersiniosis in salmonids. The assay involves amplification of 10 variable-number tandem-repeat (VNTR) loci in two five-plex PCRs, followed by capillary electrophoresis. A collection of 484 Y. ruckeri isolates, originating from various biological sources and collected from four continents over 7 decades, was analyzed. Minimum-spanning-tree cluster analysis of MLVA profiles separated the studied population into nine major clonal complexes and a number of minor clusters and singletons. The major clonal complexes could be associated with host species, geographic origin, and serotype. A single large clonal complex of serotype O1 isolates dominating the yersiniosis situation in international rainbow trout farming suggests anthropogenic spread of this clone, possibly related to transport of fish. Moreover, subclustering within this clonal complex indicates putative transmission routes and multiple biotype shift events. In contrast to the situation in rainbow trout, Y. ruckeri strains associated with disease in Atlantic salmon appear as more or less geographically isolated clonal complexes. A single complex of serotype O1 exclusive to Norway was found to be responsible for almost all major yersiniosis outbreaks in modern Norwegian salmon farming, and site-specific subclustering further indicates persistent colonization of freshwater farms in Norway. Identification of genetically diverse Y. ruckeri isolates from clinically healthy fish and environmental sources also suggests the widespread existence of less-virulent or avirulent strains. IMPORTANCE This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for Yersinia ruckeri infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any Y. ruckeri strain may rapidly be placed in a global epizootiological context.

This paper presents a validated protocol, using a novel, specifically formulated medium, to perform broth microdilution antimicrobial susceptibility assays of the salmonid bacterial pathogen Piscirickettsia salmonis. The minimum inhibitory concentrations (MIC) for florfenicol and oxytetracycline against 58 P. salmonis isolates recovered from various outbreaks occurred in Chilean salmonid farms were determined using this protocol. Normalized resistance interpretation (NRI) analysis was applied to these data to calculate appropriate protocol-specific epidemiological cut-off values. These cut-off values allow the isolates to be categorized as either fully susceptible wild type (WT) members of this species, or as manifesting reduced susceptibility non-wild type (NWT). The distribution of MIC values of florfenicol was bimodal and the distribution of the normalized values for the putative WT observation had a standard deviation of 0.896 log2 μg mL-1. This analysis calculated a cut-off value of ≤0.25 μg mL-1 and categorized 33 (56%) of the isolates as manifesting reduced susceptibility to florfenicol. For the oxytetracycline MIC data the NRI analysis also treated the distribution as bimodal. The distribution of the normalized values for the putative WT observation had a standard deviation of 0.951 log2 μg mL-1. This analysis gave a cut-off value of ≤0.5 μg mL-1 and categorized five isolates (9%) as manifesting reduced susceptibility to oxytetracycline. The susceptibility testing protocol developed in this study was capable of generating MIC data from all the isolates tested. On the basis of the precision of the data it generated, and the degree of separation of values for WT and NWT it achieved, it is argued that this protocol has the performance characteristics necessary for it to be considered as a standard protocol.

Background. Previous reports emphasized exo-enzyme production and pathogen inhibition as major criteria to select putative probiotics in carps. However, adhesion ability to the gut epithelium could be one of the decisive factors. The presently reported study was aimed to determine the probiotic potential of autochthonous bacteria isolated from gastrointestinal (GI) tract of rohu, Labeo rohita (Hamilton, 1822). Apart from characterization of functional probiotic attributes and bio-safety, the presently reported study utilized in vitro model system for preliminary selection of potentially adherent strains. Materials and methods. Altogether, 126 exo-enzyme producing bacteria were isolated from the proximal (PI) and distal (DI) segment of the GI tract and evaluated for enzyme-producing ability (viz., amylase, protease, lipase, cellulase, phytase, xylanase). Pathogen inhibition was tested by cross-streaking and double-layer method. Based on the cumulative results, isolates LR3H1A and LR3F3P were selected and identified by phenotypic and 16S rRNA partial gene sequence analyses. Both the strains were tested for their ability to grow in fish mucus, tolerance to fish bile, and bio-safety. Cell surface characteristics of the strains were analysed by aggregation assays and bio-film forming ability was determined through adherence to glass and polystyrene surfaces. Results. Seven strains (PI-4, DI-3) were primarily selected as efficient exo-enzyme producers, of which 3 strains (PI-2, DI-1) were found to be antagonistic against ≥1 of the 6 tested fish pathogens. Partial characterization of the cell-free supernatant revealed that the antagonistic compound was proteinaceous, showing its maximum efficacy at 30°C and pH 7. Isolates LR3H1A and LR3F3P were identified as Bacillus subtilis subsp. spizizenii (KF623286) and Bacillus tequilensis (KF623287), respectively. Both the strains grew well in fish mucus, tolerated diluted bile juice, and showed evidence of bio-safety. Both the strains were categorized as a moderate bio-film producer, although LR3F3P was noticed to possess stronger cell surface hydrophobicity, auto‐aggregation and co‐aggregation capacity than LR3H1A. Conclusion. Owing to better colonization potential, presently reported study indicated B. tequilensis LR3F3P as a putative probiotic for feed application.

The marine bacterium Photobacterium damselae subsp. damselae (Pdd) is a generalist and facultative pathogen that causes disease in a wide range of marine animals including fish species of importance in aquaculture. Disease outbreaks in fish farms have been correlated with an increased water temperature during summer months. In this study, we have used RNA sequencing to analyze the transcriptome of Pdd RM-71 cultured at two different temperatures, which simulated temperature conditions experienced during free swimming lifestyle at mid latitudes in winter months (15˚C) and during outbreaks in aquaculture in warm summer months (25˚C). The enhanced bacterial growth of Pdd observed at 25˚C in comparison to 15˚C suggests that an elevated seawater temperature contributes to the build-up of a sufficient bacterial population to cause disease. In comparison to growth at 15˚C, growth at 25˚C resulted in the upregulation of genes involved in DNA synthesis, nutrient uptake, chemotaxis, flagellar motility, secretion systems and antimicrobial resistance. Plasmid-encoded virulence factors, which include a putative adhesin/invasin OmpU, a transferrin receptor and a serum resistance protein, were also upregulated. Transcription factor RpoS, genes involved in cold shock response, modulation of cell envelope and amino acid metabolism, as well as genes of yet unknown function were downregulated at 25˚C. Notably, the gene encoding damselysin cytotoxin (Dly) was among the most highly transcribed genes at the two assayed temperatures, at levels comparable to the most highly expressed housekeeping genes. This study contributes to our understanding of the regulatory networks and biology of a generalist marine bacterial pathogen, and provides evidence that temperature regulates multiple physiological and virulence-related functions in Pdd This work has been supported by grant AGL2016-79738-R (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, and co-funded by the FEDER Programme from the European Union, to CRO. The support of Xunta de Galicia (Spain) with grant GRC-2014/007 to CRO and XMM is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funders websites: http://www.ciencia.gob.es/, http://www.edu.xunta.gal/portal/ SI

Vibrio toranzoniae is a Gram-negative bacterium of the Splendidus clade within the Vibrio genus. V. toranzoniae was first isolated from healthy clams in Galicia (Spain) but recently was also identified associated to disease outbreaks of red conger eel in Chile. Experimental challenges showed that the Chilean isolates were able to produce fish mortalities but not the strains isolated from clams. The aim of the present study was to determine the differences at the genomic level between the type strain of the species (CECT 7225T) and the strain R17, isolated from red conger eel in Chile, which could explain their different virulent capacity. The genome-based comparison showed high homology between both strains but differences were observed in certain gene clusters that include some virulence factors. Among these, we found that iron acquisition systems and capsule synthesis genes were the main differential features between both genomes that could explain the differences in the pathogenicity of the strains. Besides, the studied genomes presented genomic islands and toxins, and the R17 strain presented CRISPR sequences that are absent on the type strain. Taken together, this analysis provided important insights into virulence factors of V. toranzoniae that will lead to a better understanding of the pathogenic process This research was supported in part by grants AGL2013-42628R and AGL2016-77539R from Ministerio de Economia y Competitividad (Spain) SI

Bacteria from the family Flavobacteriaceae often show low susceptibility to antibiotics. With the exception of two Chryseobacteriumspp. isolates that were positive for the florfenicol resistance gene floR, no clinical resistance genes were identified by microarray in 36 Flavobacteriaceae isolates from salmonid fish that could grow in ≥ 4 mg/L florfenicol. Whole genome sequence analysis of the floRpositive isolates revealed the presence of a region that contained the antimicrobial resistance genes floR, a tet(X) tetracycline resistance gene, a streptothricin resistance gene and a chloramphenicol acetyltransferase gene. In silicoanalysis of 377 published genomes for Flavobacteriaceae isolates from a range of sources confirmed that well-characterised resistance gene cassettes were not widely distributed in bacteria from this group. Efflux pump-mediated decreased susceptibility to a range of antimicrobials was confirmed in both floRpositive isolates using an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide) assay. The floRisolates possessed putative virulence factors, including production of siderophores and haemolysins, and were mildly pathogenic in rainbow trout. Results support the suggestion that, despite the detection of floR, susceptibility to antimicrobials in Flavobacteriaceae is mostly mediated via intrinsic mechanisms rather than the horizontally acquired resistance genes more normally associated with Gram-negative bacterial pathogens such as Enterobacteriaceae.

International audience; Tenacibaculum maritimum is a devastating bacterial pathogen of wild and farmed marine fish with a broad host range and a worldwide distribution. We report here the complete genome sequence of the T. maritimum type strain NCIMB 2154 T. The genome consists of a 3,435,971-base pair circular chromosome with 2,866 predicted protein-coding genes. Genes encoding the biosynthesis of exopolysaccharides, the type IX secretion system, iron uptake systems, adhesins, hemolysins, proteases, and glycoside hydrolases were identified. They are likely involved in the virulence process including immune escape, invasion, colonization, destruction of host tissues, and nutrient scavenging. Among the predicted virulence factors, type IX secretion-mediated and cell-surface exposed proteins were identified including an atypical sialidase, a sphingomyelinase and a chondroitin AC lyase which activities were demonstrated in vitro.

International audience; Tenacibaculum maritimum is a devastating bacterial pathogen of wild and farmed marine fish with a broad host range and a worldwide distribution. We report here the complete genome sequence of the T. maritimum type strain NCIMB 2154 T. The genome consists of a 3,435,971-base pair circular chromosome with 2,866 predicted protein-coding genes. Genes encoding the biosynthesis of exopolysaccharides, the type IX secretion system, iron uptake systems, adhesins, hemolysins, proteases, and glycoside hydrolases were identified. They are likely involved in the virulence process including immune escape, invasion, colonization, destruction of host tissues, and nutrient scavenging. Among the predicted virulence factors, type IX secretion-mediated and cell-surface exposed proteins were identified including an atypical sialidase, a sphingomyelinase and a chondroitin AC lyase which activities were demonstrated in vitro.

Citrobacter freundii is a worldwide emerging nosocomial pathogen with escalating incidence of multidrug resistance. Citrobacter freundii exists in natural environment, especially in health care settings and is difficult to eradicate. Phage therapy is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a virulent bacteriophage LK1 capable of specifically infecting Citrobacter freundii. A virulent bacteriophage LK1, specific for Citrobacter freundii was isolated from sewage water sample. TEM showed that phage Lk1 has an icosahedral head 70 nm in diameter and short tail of 17 nm, and can be classified as a member of the Podoviridae family. Restriction analysis indicated that phage LK1 was a dsDNA virus with an approximate genome size of 20-23 kb. Proteomic pattern generated by SDS PAGE using purified LK1 phage particles, revealed three major and six minor protein bands with molecular weight ranging from 25 to 80 kDa. Adsorption rate of LK1 relative to the host bacterium was also determined which showed significant improvement in adsorption with the addition of CaCl2. In a single step growth experiment, LK1 exhibited a latent period of 24 min and burst size of 801 particle/cell. Moreover, pH and thermal stability of phage LK1 demonstrated a pH range of 5.0-6.0 and phage viability decreased to 0% at 65 degrees C. When LK1 was used to infect six other clinically isolated pathogenic strains, it showed relatively narrow host range. LK1 was capable of eliciting efficient lysis of Citrobacter freundii, revealing its potential as a non-toxic sanitizer for controlling Citrobacter freundii infection and contamination in both hospital and other public environments.

Chemical analyses, mass spectrometry, and NMR spectroscopy were applied to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas veronii strain Bs19, serotype O16. ESI-MS revealed that the most abundant LPS glycoforms have tetra-acylated or hexa-acylated lipid A species, consisting of a bisphosphorylated GlcN disaccharide with an AraN residue as a non-stoichiometric substituent, and a core oligosaccharide composed of Hep5Hex3HexN1Kdo1P1. Sugar and methylation analysis together with 1D and 2D 1H and 13C NMR spectroscopy were the main methods used, and revealed that the O-specific polysaccharide (OPS) of A. veronii Bs19 was built up of tetrasaccharide repeating units with the structure: →4)-α-d-Quip3NAc-(1→3)-α-l-Rhap-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→. This composition was confirmed by mass spectrometry. The charge-deconvoluted ESI FT-ICR MS recorded for the LPS preparations identified mass peaks of SR- and R-form LPS species, that differed by Δm = 698.27 u, a value corresponding to the calculated molecular mass of one OPS repeating unit (6dHexNAc6dHexHexHexNAc-H2O). Moreover, unspecific fragmentation spectra confirmed the sequence of the sugar residues in the OPS and allowed to assume that the elucidated structure also represented the biological repeating unit.

Objective: To evaluate the antibacterial activity and immunostimulant of red seaweed Chondrococcus hornemanni (C. hornemanni) (Kuetzing, 1847) against marine ornamental fish pathogens. Methods: In the present study, seaweed extract of C. hornemanni showed significant antimicrobial activity against two fish pathogens and the partially purified potential compound was characterized by GC-MS. Purified seaweed extract was injected to the clownfish, Amphiprion sebae to study the innate immune response of these fishes. Results: The extracts was found effective and had more than 80% inhibitory activity against Aeromonas hydrophila (20 mm) and Vibrio parahaemolyticus (19 mm) at a concentration (25 mg/mL) shown higher antimicrobial activity. The white blood cell count and respirator burst activity was significantly increased in the experimental tanks (E1) and (E2) when compared with control. Conclusions: Hence, it is concluded that the seaweed extracts of C. hornemanni had potential bioactive compounds and act an immunostimulant and improve the immune response to fish.

Streptococcus agalactiae (Lancefield group B; GBS) is the causative agent of meningoencephalitis in fish, mastitis in cows, and neonatal sepsis in humans. Meningoencephalitis is a major health problem for tilapia farming and is responsible for high economic losses worldwide. Despite its importance, the genomic characteristics and the main molecular mechanisms involved in virulence of S. agalactiae isolated from fish are still poorly understood. Here, we present the genomic features of the 1,820,886 bp long complete genome sequence of S. agalactiae SA20-06 isolated from a meningoencephalitis outbreak in Nile tilapia (Oreochromis niloticus) from Brazil, and its annotation, consisting of 1,710 protein-coding genes (excluding pseudogenes), 7 rRNA operons, 79 tRNA genes and 62 pseudogenes.

Chemical analyses and mass spectrometry were used to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas bestiarum strain K296, serotype O18. ESI-MS revealed that the most abundant A. bestiarum LPS glycoforms have a hexa-acylated or tetra-acylated lipid A with conserved architecture of the backbone, consisting of a 1,4′-bisphosphorylated β-(1→6)-linked d-GlcN disaccharide with an AraN residue as a non-stoichiometric substituent and a core oligosaccharide composed of Kdo1Hep6Hex1HexN1P1. 1D and 2D NMR spectroscopy revealed that the O-specific polysaccharide (OPS) of A. bestiarum K296 consists of a branched tetrasaccharide repeating unit containing two 6-deoxy-l-talose (6dTalp), one Manp and one GalpNAc residues; thus, it is similar to that of the OPS of A. hydrophila AH-3 (serotype O34) in both the sugar composition and the glycosylation pattern. Moreover, 3-substituted 6dTalp was 2-O-acetylated and additional O-acetyl groups were identified at O-2 and O-4 (or O-3) positions of the terminal 6dTalp. Western blots with polyclonal rabbit sera showed that serotypes O18 and O34 share some epitopes in the LPS. The very weak reaction of the anti-O34 serum with the O-deacylated LPS of A. bestiarum K296 might have been due to the different O-acetylation pattern of the terminal 6dTalp. The latter suggestion was further confirmed by NMR.

Here we report the draft genome sequence and annotation of A. veronii strain Ae52 isolated from the kidney of a morbund, septicaemic gold fish (Carassius auratus) in Sri Lanka. This clinical isolate showed resistance to multiple antimicrobials; amoxicillin, neomycin, trimethoprim-sulphonamide, chloramphenicol, tetracycline, enrofloxacin, erythromycin and nitrofurantoin. The size of the draft genome is 4.56 Mbp with 58.66% of G + C content consisting 4328 coding sequences. It harbors a repertoire of putative antibiotic resistant determinants that explains the genetic basis of its resistance to various classes of antibiotics. The genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession numbers BDGY01000001-BDGY01000080.

Flavobacterium psychrophilum is the most important bacterial pathogen for freshwater farmed salmonids in Chile. The aims of this study were to determine the susceptibility to antimicrobials used in fish farming of Chilean isolates and to calculate their epidemiological cut-off (COWT) values. A number of 125 Chilean isolates of F. psychrophilum were isolated from reared salmonids presenting clinical symptoms indicative of flavobacteriosis and their identities were confirmed by 16S rRNA polymerase chain reaction. Susceptibility to antibacterials was tested on diluted Mueller-Hinton by using an agar dilution MIC method and a disk diffusion method. The COWT values calculated by Normalized Resistance Interpretation (NRI) analysis allow isolates to be categorized either as wild-type fully susceptible (WT) or as manifesting reduced susceptibility (NWT). When MIC data was used, NRI analysis calculated a COWT of ≤0.125, ≤2, and ≤0.5 μg mL-1 for amoxicillin, florfenicol, and oxytetracycline, respectively. For the quinolones, the COWT were ≤1, ≤0.5, and ≤0.125 μg mL-1 for oxolinic acid, flumequine, and enrofloxacin, respectively. The disk diffusion data sets obtained in this work were extremely diverse and were spread over a wide range. For the quinolones there was a close agreement between the frequencies of NWT isolates calculated using MIC and disk data. For oxolinic acid, flumequine, and enrofloxacin the frequencies were 45, 39, and 38% using MIC data, and 42, 41, and 44%, when disk data were used. There was less agreement with the other antimicrobials, because NWT frequencies obtained using MIC and disk data, respectively, were 24 and 10% for amoxicillin, 8 and 2% for florfenicol, and 70 and 64% for oxytetracycline. Considering that the MIC data was more precise than the disk diffusion data, MIC determination would be the preferred method for susceptibility testing for this species and the NWT frequencies derived from the MIC data sets should be considered as the more authoritative. Despite the high frequency of isolates showing full susceptibility to florfenicol, the significant frequencies of isolates exhibiting reduced susceptibility to oxytetracycline and quinolones may result in treatment failures when these agents are used.

A series of novel oxyalkylchalcones substituted with alkyl groups were designed and synthesized, and the antioomycete activity of the series was evaluated in vitro against Saprolegnia strains. All tested O-alkylchalcones were synthesized by means of nucleophilic substitution from the natural compound 2′,4′-dihydroxychalcone (1) and the respective alkyl bromide. The natural chalcone (1) and 10 synthetic oxyalkylchalcones (2–11) were tested against Saprolegnia parasitica and Saprolegnia australis. Among synthetic analogs, 2-hydroxy,4-farnesyloxychalcone (11) showed the most potent activity against Saprolegnia sp., with MIC and MOC values of 125 µg/mL (similar to bronopol at 150 µg/mL) and 175 µg/mL, respectively; however, 2′,4′-dihydroxychalcone (1) was the strongest and most active molecule, with MIC and MOC values of 6.25 µg/mL and 12.5 µg/mL.

The genus Francisella is composed of Gram-negative, pleomorphic, strictly aerobic and non-motile bacteria, which are capable of infecting a variety of terrestrial and aquatic animals, among which Francisella noatunensis subsp. orientalis stands out as the causative agent of pyogranulomatous and granulomatous infections in fish. Accordingly, F. noatunensis subsp. orientalis is responsible for high mortality rates in freshwater fish, especially Nile Tilapia. In the current study, we present the genome sequences of F. noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190. The genomes include one circular chromosome of 1,859,720 bp, consisting of 32 % GC content, 1538 coded proteins and 363 pseudogenes for FNO12; one circular chromosome of 1,862,322 bp, consisting of 32 % GC content, 1537 coded proteins and 365 pseudogenes for FNO24; and one circular chromosome of 1,859,595 bp, consisting of 32 % GC content, 1539 coded proteins and 362 pseudogenes for FNO190. All genomes have similar genetic content, implicating a clonal-like behavior for this species. Electronic supplementary material The online version of this article (doi:10.1186/s40793-016-0151-0) contains supplementary material, which is available to authorized users.

Teleost fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal axis to release glucocorticoids, in particular cortisol, into the blood. For the majority of bacterial fish pathogens, stress is considered a key factor in disease outbreaks. Based upon studies in mammals, there is considerable evidence to suggest that, besides impairing the immune system, cortisol can have a direct effect on bacterial cells. Hitherto, this intriguing field of microbial endocrinology has remained largely unexplored in aquatic diseases. The present study investigated in vitro the impact of cortisol on phenotypic traits of the fresh water fish pathogen Flavobacterium columnare. Colonies obtained from the highly virulent (HV) isolates resulted in significantly larger and more spreading colonies compared to those from the low virulent (LV) isolates. High cortisol doses added displayed a direct effect on the bacterial cells and induced a significant decrease in colony size. An additional intriguing finding was the inverse relationship between cortisol concentrations added to the broth and the spreading character of colonies retrieved, with higher cortisol doses resulting in less rhizoid to rough and even smooth colony formation (the latter only in the LV trout isolate), suggesting a dose–response effect. The loss of the rhizoid appearance of the F. columnare colonies upon administration of cortisol, and hence the loss of motility, might indicate a phenotypic change to the biofilm state. These findings form the basis for further research on the impact of glucocorticoids on other virulence factors and biofilm formation of F. columnare. Electronic supplementary material The online version of this article (doi:10.1186/s13567-016-0370-9) contains supplementary material, which is available to authorized users.

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. While their mammalian counterparts have been extensively characterized in the last two decades, data on fish cathelicidins have only recently been gathered. This review covers the available recent insights on fish cathelicidins regarding their evolution, protein structures, gene expression, antimicrobial, and immunomodulatory properties, highlighting the similarity and the differences with the mammalian ones. The picture that emerges is that of a group of immune effectors that has been subjected to intense pressure for variation in single species of salmonids, cods, and a few other families of bony fish to address the great variety of associated microorganisms and pathogens. Genes of fish cathelicidins have been shown to be highly responsive to bacteria; moreover, experimental data suggest both direct antibacterial activity and modulation of other immune effectors. These functions, which still need to be fully characterized, indicate cathelicidins as an important and effective defense line in fish.

International audience; The study of the fish pathogen Flavobacterium psychrophilum has been drastically hampered by the difficulty to perform genetic manipulation of this organism. Although recent publications described the successful transfer of genetic material into this bacterium by transformation and conjugation, additional tools are still needed. This paper reports the construction of vector pCP23-G, which permits for the first time to monitor transcriptional regulation in this pathogen by using a promoterless gfpmut3 gene as a reporter. Additionally, use of pCP23-G enabled the trancriptional analysis of three putative promoter regions of F. psychrophilum, corresponding to genes fpp2- fpp1, pdhB and gldj, under different growth conditions. Overall, the construction of pCP23-G facilitates genetic analysis in F. psychrophilum, by enabling the determination of gene expression both in vitro and in vivo. Furthermore, this would also open the possibility for studies on the location of this bacterium in the fish tissues. (C) 2012 Elsevier B.V. All rights reserved.

Members of the genus Nocardia are widespread in diverse environments; a wide range of Nocardia species are known to cause nocardiosis in several animals, including cat, dog, fish, and humans. Of the pathogenic Nocardia species, N. seriolae is known to cause disease in cultured fish, resulting in major economic loss. We isolated two N. seriolae strains, CK‐14008 and EM15050, from diseased fish and sequenced their genomes using the PacBio sequencing platform. To identify their genomic features, we compared their genomes with those of other Nocardia species. Phylogenetic analysis showed that N. seriolae shares a common ancestor with a putative human pathogenic Nocardia species. Moreover, N. seriolae strains were phylogenetically divided into four clusters according to host fish families. Through genome comparison, we observed that the putative pathogenic Nocardia strains had additional genes for iron acquisition. Dozens of antibiotic resistance genes were detected in the genomes of N. seriolae strains; most of the antibiotics were involved in the inhibition of the biosynthesis of proteins or cell walls. Our results demonstrated the virulence features and antibiotic resistance of fish pathogenic N. seriolae strains at the genomic level. These results may be useful to develop strategies for the prevention of fish nocardiosis.