Draft genome sequence of Vibrio lentus VLO8, recovered from the larval culture of the Chilean scallop (Argopecten purpuratus).

This announcement reports the genome of Vibrio lentus VLO8 recovered from the larval culture of Chilean scallop. The genomes of strain VLO8 have two contigs with a total length of 5,499,980 bp, an average G + C content of 44.22%, a total number of protein-coding genes of 6,439, and 170 RNAs.

First Report, Characterization and Pathogenicity of Vibrio chagasii Isolated from Diseased Reared Larvae of Chilean Scallop, Argopecten purpuratus (Lamarck, 1819).

Two Vibrio strains (VPAP36 and VPAP40) were isolated from moribund-settled larvae of the Chilean scallop Argopecten purpuratus during vibriosis outbreaks that occurred in two commercial scallop larvae hatcheries located in the Inglesa and Tongoy bays in Northern Chile. The strains were identified as Vibrio chagasii using phenotypic characterization and whole genome sequence analysis. Both strains exhibited the phenotypic properties associated with virulence, gelatin hydrolysis and ß-hemolysis, whereas only VPAP36 produced phospholipase and only VPAP40 produced caseinase. The whole genome analysis showed that the strains harbored genes encoding for the virulence factors, the EPS type II secretion system, and Quorum Sensing (auto-inductor 1 and auto-inductor 2), whereas genes encoding a metalloproteinase and a capsular polysaccharide were detected only in the VPAP40 genome. When challenge bioassays using healthy 11-day-old scallop larvae were performed, the V. chagasii VPAP36 and VPAP40 strains exhibited significant (p < 0.05) differences in their larval lethal activity, producing, after 48 h, larval mortalities of 65.51 ± 4.40% and 28.56 ± 5.35%, respectively. Otherwise, the cell-free extracellular products of the VPAP36 and VPAP40 strains produced larval mortalities of 20.86 ± 2.40% and 18.37 ± 2.40%, respectively, after 48 h of exposure. This study reports for the first time the isolation of V. chagasii from the massive larval mortalities of the farmed scallop (Argopecten purpuratus) in Chile, and demonstrates the pathogenic activity of V. chagasii towards the Chilean scallop, the second most important species for Chilean mariculture.

Role of the Vibriolysin VemA Secreted by the Emergent Pathogen Vibrio europaeus in the Colonization of Manila Clam Mucus.

Vibrio europaeus is an emergent pathogen affecting clams, oysters and scallops produced in the most important countries for bivalve aquaculture. Studies concerning virulence factors involved in the virulence of V. europaeus are very scarce despite its global significance for aquaculture. Zinc-metalloproteases have been described as a major virulence factor in some Vibrio spp., although their contribution and role in the virulence of V. europaeus is not clear. To address this, we have studied an extracellular zinc-metalloprotease (VemA) encoded by V. europaeus, which was identified as a vibriolysin, highly conserved in this species and homologous in other pathogenic and non-pathogenic species. Virulence challenge experiments demonstrated that infection processes were faster when Manila clam larvae and juveniles were infected with the wildtype rather than with a mutant defective in the vemA gene (ΔvemA). V. europaeus was able to resist the bactericidal action of mucus and displayed a chemotaxis ability favoured by VemA to colonize the body mucus of clams and form a biofilm. The overall results suggest that VemA, although it is not a major virulence factor, plays a role in the colonization of the Manila clam mucus, and thus boosts the infection process as we observed in virulence challenge experiments.

Vibrio neptunius Produces Piscibactin and Amphibactin and Both Siderophores Contribute Significantly to Virulence for Clams.

Vibrio neptunius is an inhabitant of mollusc microbiota and an opportunistic pathogen causing disease outbreaks in marine bivalve mollusc species including oysters and clams. Virulence of mollusc pathogenic vibrios is mainly associated with the production of extracellular products. However, siderophore production is a common feature in pathogenic marine bacteria but its role in fitness and virulence of mollusc pathogens remains unknown. We previously found that V. neptunius produces amphibactin, one of the most abundant siderophores in marine microbes. In this work, synthesis of the siderophore piscibactin was identified as the second siderophore produced by V. neptunius. Single and double mutants in biosynthetic genes of each siderophore system, piscibactin and amphibactin, were constructed in V. neptunius and their role in growth ability and virulence was characterized. Although the High Pathogenicity Island encoding piscibactin is a major virulence factor in vibrios pathogenic for fish, the V. neptunius wild type did not cause mortality in turbot. The results showed that amphibactin contributes more than piscibactin to bacterial fitness in vitro. However, infection challenges showed that each siderophore system contributes equally to virulence for molluscs. The V. neptunius strain unable to produce any siderophore was severely impaired to cause vibriosis in clams. Although the inactivation of one of the two siderophore systems (either amphibactin or piscibactin) significantly reduced virulence compared to the wild type strain, the ability to produce both siderophores simultaneously maximised the degree of virulence. Evaluation of the gene expression pattern of each siderophore system showed that they are simultaneously expressed when V. neptunius is cultivated under low iron availability in vitro and ex vivo. Finally, the analysis of the distribution of siderophore systems in genomes of Vibrio spp. pathogenic for molluscs showed that the gene clusters encoding amphibactin and piscibactin are widespread in the Coralliilyticus clade. Thus, siderophore production would constitute a key virulence factor for bivalve molluscs pathogenic vibrios.

The Vibriolysin-Like Protease VnpA and the Collagenase ColA Are Required for Full Virulence of the Bivalve Mollusks Pathogen Vibrio neptunius.

Vibrio neptunius is an important pathogen of bivalve mollusks worldwide. Several metalloproteases have been described as virulence factors in species of Vibrio that are pathogenic to bivalves, but little is known about the contribution of these potential virulence factors to Vibrio neptunius pathogenesis. In silico analysis of the genome of V. neptunius strain PP-145.98 led to the identification of two hitherto uncharacterized chromosomal loci encoding a probable vibriolysin-like metalloprotease and a putative collagenase, which were designated VnpA and ColA, respectively. Single defective mutants of each gene were obtained in V. neptunius PP-145.98, and the phospholipase, esterase and collagenase activities were studied and compared with those of the wild-type strain. The results showed that the single inactivation of vnpA resulted in a 3-fold reduction in phospholipase/esterase activity. Inactivation of colA reduced the collagenase activity by 50%. Finally, infection challenges performed in oyster larvae showed that ΔvnpA and ΔcolA-single mutant strains of V. neptunius-are between 2-3-fold less virulent than the wild-type strain. Thus, the present work demonstrates that the production of both VnpA and ColA is required for the full virulence of the bivalve pathogen V. neptunius.

Draft Genome Sequences of Five Vibrio neptunius Strains Isolated from Hatcheries of Bivalve Mollusks.

Vibrio neptunius is a Gram-negative bacterium that has been shown to cause disease in marine bivalve mollusk larvae. Here, we report the draft genome sequences and annotations of five V. neptunius strains isolated from larvae of European oyster (Ostrea edulis) and Manila clam (Ruditapes philippinarum) at hatcheries in Galicia, northwest Spain.

First description outside Europe of the emergent pathogen Vibrio europaeus in shellfish aquaculture.

Vibrio europaeus is an emergent pathogen affecting the most important bivalve species reared in Spanish and French hatcheries. Using a genomic approach, we identified V. europaeus outside Europe for the first time from massive larval mortalities of scallop (Argopecten purpuratus) in Chile and from seawater near a shellfish hatchery in the US West Coast. Results show the worldwide spreading and potential impact of V. europaeus for aquaculture; these four countries are among the 10 major producers of mollusks. Pathogenicity of V. europaeus was demonstrated for the first time towards scallop, the second most important species for Chilean mariculture.

The marine bivalve molluscs pathogen Vibrio neptunius produces the siderophore amphibactin, which is widespread in molluscs microbiota.

Amphiphilic siderophores, including amphibactins, are the most abundant siderophores in oceans. Genes putatively encoding the amphibactin system were proposed in some bacteria and homologues of these genes are particularly abundant in multiple bacterial lineages inhabitant of low-iron seawater. However, since no defective mutant strains in any of these genes were studied to date, their role in amphibactin synthesis or uptake was not demonstrated. In this work, an in silico analysis of the genome of the mollusc pathogen Vibrio neptunius leads us to identify a gene cluster (denoted absABDEF) that is predicted to encode an amphibactin-like siderophore and several mutant strains unable to synthesize or use siderophores were constructed. The results showed that genes absABDEF are required for amphibactin synthesis. A comparative chemical analysis of V. neptunius wild type and biosynthesis mutants allowed us to identify a mixture of nine amphibactin forms produced by this bacterium. In addition, the gene abtA is predicted to encode the ferri-amphibactin outer membrane transporter. The prevalence of the amphibactin system in bivalve hemolymph microbiota was also studied. We found that the amphibactin system is widespread in hemolymph microbiota including both commensal and pathogenic bacterial species. Thus, its contribution to bacterial fitness must be more related to environmental persistence than to pathogenicity.

Draft Genome Sequence of Vibrio ostreicida Strain PP-203, the Type Strain of a Pathogen That Infects Bivalve Larvae.

Vibrio ostreicida is a Gram-negative gammaproteobacterium that has been shown to cause disease in bivalve larvae. Presented here is the draft genome of the type strain Vibrio ostreicida strain PP-203, which was isolated from the inner surface of an Ostrea edulis (European flat oyster) spat container with recorded deaths at a hatchery in Galicia, Spain.

Encapsulation of live marine bacteria for use in aquaculture facilities and process evaluation using response surface methodology.

New strategies are being proposed in marine aquaculture to use marine bacteria as alternative to antibiotics, as nutritional additive or as immune-stimulant. These approaches are particularly promising for larval and juvenile cultures. In many cases, the bacteria are released in the seawater, where they have to be at appropriate concentrations. In addition, only low-cost technologies are sustainable for this industry, without any complex requirements for use or storage. In this work, we explore the possibilities of preservation of a potential marine probiotic bacterium (Phaeobacter PP-154) as a product suitable for use in marine aquaculture by addition to the seawater. A method which guaranteed the preservation of the viable marine bacteria in a saline medium and their rapid release in the seawater was searched for. In a previous step, classical procedures (freeze-drying and freezing) had been explored, but undesirable results of the interaction of the products obtained with natural seawater led to investigate alternatives. We report the results of the immobilization of the marine bacteria in calcium alginate beads. The final product complies the salinity which allows the requirements of the bacteria without interference with alginate in the formation of beads, and a balanced hardness to retain the bacteria and to be easily released in the marine aquaculture environment. The process was evaluated using the central composite rotatable design (CCRD), a standard response surface methodology (RSM).

Isolation and Pathogenic Characterization of Vibrio bivalvicida Associated With a Massive Larval Mortality Event in a Commercial Hatchery of Scallop Argopecten purpuratus in Chile.

The VPAP30 strain was isolated as the highly predominant bacteria from an episode of massive larval mortality occurring in a commercial culture of the Chilean scallop Argopecten purpuratus. The main aims of this study were, to characterize and identify the pathogenic strain using biochemical and molecular methods, to demonstrate its pathogenic activity on scallop larvae, to characterize its pathogenic properties and to describe the chronology of the pathology. The pathogenic strain was identified as Vibrio bivalvicida based on its phenotypic properties, the multilocus sequence analysis (MLSA) of eight housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) and different in silico genome-to-genome comparisons. When triplicate cultures of healthy 10 days old scallop larvae were challenged with 1 × 105 colony forming units (CFU) mL-1 of the VPAP30 strain, percentages of larval survival of 78.9 ± 3.3%, 34.3 ± 4.9%, and 0% were observed at 12, 2,4 and 36 h, respectively, whereas uninfected larval cultures showed survival rates of 97.4 ± 1.2% after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30 strain include the accumulation of bacteria around the scallop larvae, velum disruption and necrosis of digestive gland. The 50% lethal dose (LD50) of VPAP30 strain at 24 and 48 h was 1.3 × 104 and 1.2 × 103 CFU mL-1, respectively. The invasive pathogenic activity of the VPAP30 strain was investigated with staining of the bacterial pathogen with 5-DTAF and analyzing bacterial invasion using epifluorescence, and a complete bacterial dissemination inside the larvae at 24 h post-infection was observed. When scallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30, the larval survival rate was 59.5 ± 1.7%, significantly (P < 0.001) lower than the control group (97.4 ± 1.2%) whereas larvae treated with heat-treated ECPs exhibited a survival rate of 61.6 ± 1.8% after 48 h of exposure. V. bivalvicida VPAP30 exhibits high pathogenic activity on scallop larvae, mediated both by bacterial invasion and the production of toxigenic heat-stable compounds. This report constitutes the first isolation of V. bivalvicida out of Europe and extends the host range of this species, having demonstrated its pathogenic activity on the Chilean scallop larvae (A. purpuratus). These results supporting the pathogenic potential of V. bivalvicida to kill the larvae of a broad range of bivalve species reared in hatcheries located in the Atlantic and the Pacific coasts.

New Insights into Pathogenic Vibrios Affecting Bivalves in Hatcheries: Present and Future Prospects.

Hatcheries constitute nowadays the only viable solution to support the husbandry of bivalve molluscs due to the depletion and/or overexploitation of their natural beds. Hatchery activities include the broodstock conditioning and spawning, rearing larvae and spat, and the production of microalgae to feed all stages of the production cycle. However, outbreaks of disease continue to be the main bottleneck for successful larval and spat production, most of them caused by different representatives of the genus Vibrio. Therefore, attention must be paid on preventive and management measures that allow the control of such undesirable bacterial populations. The present review provides an updated picture of the recently characterized Vibrio species associated with disease of bivalve molluscs during early stages of development, including the controversial taxonomic affiliation of some of them and relevant advances in the knowledge of their virulence determinants. The problematic use of antibiotics, as well as its eco-friendly alternatives are also critically discussed.

Reclassification of the larval pathogen for marine bivalves Vibrio tubiashii subsp. europaeus as Vibrio europaeus sp. nov.

The Orientalis clade has a relevant significance for bivalve aquaculture since it includes the pathogens Vibrio bivalvicida, Vibrio tubiashii subsp. tubiashii and Vibrio tubiashii subsp. europaeus. However, the previous taxonomic description of the subspecies of V. tubiashii shows some incongruities that should be emended. In the genomic age, the comparison between genome assemblies is the key to clarify the taxonomic position of both subspecies. With this purpose, we have tested the ability of multilocus sequence analysis based on eight housekeeping gene sequences (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA and topA), different in silico genome-to-genome comparisons, chemotaxonomic features and phenotypic traits to reclassify the subspecies V. tubiashii subsp. europaeus within the Orientalis clade. This polyphasic approach clearly demonstrated that this subspecies is phylogenetically and phenotypically distinct from V. tubiashii and should be elevated to the rank of species as Vibrio europaeus sp. nov. This reclassification allows us to update the Orientalis clade (V. bivalvicida,V. brasiliensis, V. crosai, V. hepatarius, V. orientalis, V. sinaloensis, V. tubiashii and V. europaeus sp. nov.) and reconstruct a better phylogeny of the genus Vibrio. An emended description of V. tubiashii is provided. Finally, the proposed novel species is represented by emergent bivalve pathogens [type strain PP-638T (=CECT 8136T=DSM 27349T), PP2-843 and 07/118 T2] responsible for high mortalities in Spanish and French hatcheries.

Draft Genome Sequence of the Emerging Bivalve Pathogen Vibrio tubiashii subsp. europaeus.

Vibrio tubiashii subsp. europaeus is a bivalve pathogen isolated during episodes of mortality affecting larval cultures in different shellfish hatcheries. Here, we announce the draft genome sequence of the type strain PP-638 and describe potential virulence factors, which may provide insight into the mechanism of pathogenicity.

Draft Genome Sequence of the New Pathogen for Bivalve Larvae Vibrio bivalvicida.

Vibrio bivalvicidais a novel pathogen of bivalve larvae responsible for recent vibriosis outbreaks affecting shellfish hatcheries. Here, we announce the draft genome sequence ofV. bivalvicida605(T)and describe potential virulence factors.

N-Acyl Dehydrotyrosines, Tyrosinase Inhibitors from the Marine Bacterium Thalassotalea sp. PP2-459.

Thalassotalic acids A-C and thalassotalamides A and B are new N-acyl dehydrotyrosine derivatives produced by Thalassotalea sp. PP2-459, a Gram-negative bacterium isolated from a marine bivalve aquaculture facility. The structures were elucidated via a combination of spectroscopic analyses emphasizing two-dimensional NMR and high-resolution mass spectrometric data. Thalassotalic acid A (1) displays in vitro inhibition of the enzyme tyrosinase with an IC50 value (130 µM) that compares favorably to the commercially used control compounds kojic acid (46 µM) and arbutin (100 µM). These are the first natural products reported from a bacterium belonging to the genus Thalassotalea.

Persistence of Antibiotic Resistant Vibrio spp. in Shellfish Hatchery Environment.

The characterization of antibiotic-resistant vibrios isolated from shellfish aquaculture is necessary to elucidate the potential transfer of resistance and to establish effective strategies against vibriosis. With this aim, we analyzed a collection of bacterial isolates obtained from 15 failed hatchery larval cultures that, for the most part, had been treated experimentally with chloramphenicol to prevent vibriosis. Isolates were obtained during a 2-year study from experimental cultures of five different clam species. Among a total of 121 Vibrio isolates studied, 28 were found to be chloramphenicol resistant, suggesting that the shellfish hatchery had been using a sublethal concentration of the antibiotic. Interestingly, chloramphenicol-resistant vibrios showed also resistance to tetracycline and amoxicillin (group A; n = 19) or to streptomycin (group B; n = 9). Chloramphenicol-resistant vibrios were subjected to a PCR amplification and DNA sequencing of the chloramphenicol acetyltransferase genes (cat), and the same approach was followed to study the tetracycline resistance markers (tet). 16S ribosomal RNA (rRNA) gene sequencing revealed that chloramphenicol-resistant vibrios pertained mostly to the Splendidus clade. Conjugation assays demonstrated that various R-plasmids which harbored the cat II/tet(D) genes and cat III gene in groups A and B respectively, were transferred to E. coli and bivalve pathogenic vibrios. Most interestingly, transconjugants exhibited the antibiotic resistance patterns of the donors, despite having been selected only on the basis of chloramphenicol resistance. This is the first report carried out in a bivalve hatchery elucidating the persistence of resistant vibrios, the mechanisms of antibiotic resistance, and the transfer of different R-plasmids.

Following the infection process of vibriosis in Manila clam (Ruditapes philippinarum) larvae through GFP-tagged pathogenic Vibrio species.

Vibriosis represents the main bottleneck for the larval production process in shellfish aquaculture. While the signs of this disease in bivalve larvae are well known, the infection process by pathogenic Vibrio spp. during episodes of vibriosis has not been elucidated. To investigate the infection process in bivalves, the pathogens of larvae as V. tubiashii subsp. europaensis, V. neptunius and V. bivalvicida were tagged with green fluorescent protein (GFP). Larvae of Manila clam (Ruditapes philippinarum) were inoculated with the GFP-labeled pathogens in different infection assays and monitored by microscopy. Manila clam larvae infected by distinct GFP-tagged Vibrio spp. in different challenges showed the same progression in the infection process, defining three infection stages. GFP-tagged Vibrio spp. were filtered by the larvae through the vellum and entered in the digestive system through the esophagus and stomach and colonized the digestive gland and particularly the intestine, where they proliferated during the first 2h of contact (Stage I), suggesting a chemotactic response. Then, GFP-tagged Vibrio spp. expanded rapidly to the surrounding organs in the body cavity from the dorsal to ventral region (Stage II; 6-8h), colonizing the larvae completely at the peak of infection (Stage III) (14-24h). Results demonstrated for the first time that the vibriosis is asymptomatic in Manila clam larvae during the early infection stages. Thus, the early colonization and the rapid proliferation of Vibrio pathogens within the body cavity supported the sudden and fatal effect of the vibriosis, since the larvae exhibited the first signs of disease when the infection process is advanced. As a first step in the elucidation of the potential mechanisms of bacterial pathogenesis in bivalve larvae the enzymatic activities of the extracellular products released from the wild type V. neptunius, V. tubiashii subsp. europaensis and V. bivalvicida were determined and their cytotoxicity was demonstrated in fish and homeothermic cell lines for the first time. That activity was lost after heat treatment.

Vibrio bivalvicida sp. nov., a novel larval pathogen for bivalve molluscs reared in a hatchery.

Three isolates were obtained from cultures of carpet shell clam (Ruditapes decussatus) reared in a bivalve hatchery (Galicia, NW Spain) from different sources: healthy broodstock, moribund larvae and the seawater corresponding to the larval tank. All isolates were studied by a polyphasic approach, including a phylogenetic analysis based on concatenated sequences of the five housekeeping genes ftsZ, gyrB, pyrH, recA and rpoA. The analysis supported their inclusion in the Orientalis clade of the genus Vibrio, and they formed a tight group separated from the closest relatives: Vibrio tubiashii subsp. europaensis, Vibrio tubiashii subsp. tubiashii and Vibrio orientalis. The percentages of genomic resemblance, including average nucleotide identity, DNA-DNA hybridization and in silico genome-to-genome comparison, between the type strain and the closest relatives were below values for species delineation and confirmed the taxonomic position of the new species, which could be differentiated from the related taxa on the basis of several phenotypic and chemotaxonomic features, including FAME and MALDI-TOF-MS. The pathogenicity of the new species was demonstrated in larvae of R. decussatus, Ruditapes philippinarum, Ostrea edulis and Donax trunculus. The results demonstrated that the strains analyzed represented a novel species in the Orientalis clade of the genus Vibrio, for which the name Vibrio bivalvicida sp. nov. is proposed, with 605(T) (= CECT 8855(T)=CAIM 1904(T)) designated as the type strain.

Characterization of pathogenic vibrios isolated from bivalve hatcheries in Galicia, NW Atlantic coast of Spain. Description of Vibrio tubiashii subsp. europaeus [corrected] subsp. nov.

The taxonomic position of the bivalve pathogen PP-638 was studied together with five similar isolates. The strains were isolated from flat oyster (Ostrea edulis) and Manila clam (Venerupis philippinarum) cultures during outbreaks of disease in two shellfish hatcheries (Galicia, NW Spain). The pathogenicity, previously established for PP-638, was demonstrated with all isolates and for several bivalve species, including the original hosts. On the basis of phenotypic characterization and 16S rRNA gene sequences, a tight group was defined within the genus Vibrio. Multilocus sequence analysis (MLSA) based on concatenated sequences of the 16S rRNA gene and the five housekeeping genes recA, rpoA, pyrH, gyrB and ftsZ revealed that these strains form a cluster within the Orientalis clade, close to the species Vibrio tubiashii. The results of MLSA, the DDH rate and the phenotypic differences with the type strain of V. tubiashii supported the differentiation of the Galician isolates as a new subspecies within V. tubiashii, for which the name V. tubiashii subsp. europaeus [corrected] subsp. nov. is proposed (type strain PP-638(T)=CECT 8136(T)=DSM 7349(T)) The emended description of V. tubiashii is included. The pathogenicity assays widen the host range of V. tubiashii to add two unreported species, Venerupis decussata and Donax trunculus, and the described as relatively resistant species V. philippinarum.