Vibrio splendidus infection induces dysbiosis in the blue mussel and favors pathobiontic bacteria.

Studies on marine epizootics are often based on the identification of a single pathogen. However, the one-pathogen-one-disease paradigm is not always sufficient to explain the disease, especially since the evidences on the role of microbiota in health and disease. Vibrio splendidus strains have been associated with Mytilus edulis mortality in France. To assess the role of mussel microbiota in the infectious process, we performed experiments combining the investigation of total microflora dynamics during a realistic experimental infection by V. splendidus and the monitoring of mussel survival and the dominance of potential opportunistic bacteria after antibiotic treatment. We found that Vibrio exposure affected the structure and predictive function of the mussel microbiota. Dysbiosis was accompanied with the appearance of a pathobiont dominated by Bacteroidetes and Fusobacteria phyla. The injection of a homogenate of infected organisms increased Mytilus mortality compared to the direct injection of Vibrio while the antibiotic pretreatment reduced the effect of pathogen exposure and mortalities. The decrease of opportunistic bacteria abundance in antibiotic pretreated animals confirmed their implication in pathogenesis. Our findings suggest that mussel disease results from a collaboration between external pathogens and pathobiont bacteria. Therefore, an insight into microbiota functions is needed to a better understanding of pathosystems.

Direct Comparison of Fecal and Gut Microbiota in the Blue Mussel (Mytilus edulis) Discourages Fecal Sampling as a Proxy for Resident Gut Community.

Bivalves have ecological and economic importance but information regarding their associated microbiomes is lacking. As suspension feeders, bivalves capture and ingest a myriad of particles, and their digestive organs have a high throughput of particle-associated microbiota. To better understand the complement of transient and resident microbial communities, standard methods need to be developed. For example, fecal sampling could represent a convenient proxy for the gut microbiome and is simple, nondestructive, and allows for sampling of individuals through time. The goal of this study was to evaluate fecal sampling as a reliable proxy for gut microbiome assessment in the blue mussel (Mytilus edulis). Mussels were collected from the natural environment and placed into individual sterilized microcosms for 6 h to allow for fecal egestion. Feces and gut homogenates from the same individuals were sampled and subjected to 16S rRNA gene amplicon sequencing. Fecal communities of different mussels resembled each other but did not resemble gut communities. Fecal communities were significantly more diverse, in terms of amplicon sequence variant (ASV) richness and evenness, than gut communities. Results suggested a mostly transient nature for fecal microbiota. Nonetheless, mussels retained a distinct resident microbial community in their gut after fecal egestion that was dominated by ASVs belonging to Mycoplasma. The use of fecal sampling as a nondestructive substitute for direct sampling of the gut is strongly discouraged. Experiments that aim to study solely resident bivalve gut microbiota should employ an egestion period prior to gut sampling to allow time for voidance of transient microbes.

Aislamiento de cepas de Enterococcus hirae productoras de enterocinas a partir del contenido intestinal de mejillón patagónico (Mytilus edulis platensis) Aislamiento de Enterococcus hirae de mejillón patagónico / Isolation of enterocin-producing Enterococcus hirae strains from the intestinal content of the Patagonian mussel (Mytilus edulis platensis)

Resumen Se aislaron del contenido intestinal del mejillón patagónico dos cepas de bacterias ácido lácticas y se caracterizaron por pruebas fenotípicas y moleculares. Los aislamientos se identificaron como Enterococcus hirae y fueron denominados E. hirae 463Me y 471Me. Por técnicas de PCR se identificó el gen de la enterocina P en ambas cepas, mientras que solamente en la cepa 471Me se detectó la enterocina hiracin JM79. Ambas cepas resultaron sensibles a los antibióticos clínicamente importantes y entre los rasgos de virulencia investigados mediante amplificación por PCR solo se pudieron detectar los genes cylL l y cylL s , sin embargo, no se observó actividad hemolítica en la prueba de agar sangre. Los sobrenadantes libres de células resultaron activos contra todas las cepas de Listeria y Enterococcus ensayadas, contra Lactobacillus plantarum TwLb 5 y contra Vibrio anguilarum V10. En óptimas condiciones de crecimiento, ambas cepas mostraron actividad inhibitoria contra Listeria innocua ATCC 33090 después de 2h de incubación. E. hirae 471Me alcanzó una actividad inhibitoria máxima de 163.840UA/ml después de 6h de incubación, mientras que el mismo valor se registró para E. hirae 463Me después de 8h. En ambos casos, la actividad antagonista alcanzó su máximo antes de lograr la fase estacionaria y permaneció estable hasta las 24h de incubación. En nuestro conocimiento, este es el primer informe de aislamiento de cepas bacteriocinogénicas de E. hirae de mejillón patagónico. La alta actividad inhibitoria y la ausencia de rasgos de virulencia indican que estos microorganismos podrían aplicarse en áreas biotecnológicas como la biopreservación de alimentos o las formulaciones probióticas.

Abstract Two bacteriocin-producing lactic acid bacterial strains were isolated from the intestinal content of the Patagonian mussel and characterized by phenotypic and molecular tests. The isolates were identified as Enterococcus hirae and named E. hirae 463Me and 471Me. The presence of the enterocin P gene was identified in both strains by PCR techniques, while enterocin hiracin JM79 was detected only in the 471Me strain. Both strains were sensitive to clinically important antibiotics and among the virulence traits investigated by PCR amplification, only cylL l and cylL s could be detected; however, no hemolytic activity was observed in the blood agar test. Cell free supernatants were active against all Listeria and Enterococcus strains tested, Lactobacillus plantarum TwLb 5 and Vibrio anguilarum V10. Under optimal growth conditions, both strains displayed inhibitory activity against Listeria innocua ATCC 33090 after 2h of incubation. E. hirae 471Me achieved a maximum activity of 163840AU/ml after 6h of incubation, while the same value was recorded for E. hirae 463Me after 8h. In both cases, the antagonist activity reached its maximum before the growth achieved the stationary phase and remained stable up to 24h of incubation. To our knowledge, this is first report of the isolation of bacteriocinogenic E. hirae strains from the Patagonian mussel. The high inhibitory activity and the absence of virulence traits indicate that they could be applied in different biotechnological areas such as food biopreservation or probiotic formulations.

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum ß-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway.

BACKGROUND: Environmental surveillance of antibiotic resistance can contribute towards better understanding and management of human and environmental health. This study applied a combination of long-read Oxford Nanopore MinION and short-read Illumina MiSeq-based sequencing to obtain closed complete genome sequences of two CTX-M-producing multidrug-resistant Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway, in order to understand the potential for mobility of the detected antibiotic resistance genes (ARGs). RESULTS: The complete genome sequence of strain 631 (E. coli sequence type 38) was assembled into a circular chromosome of 5.19 Mb and five plasmids (between 98 kb and 5 kb). The majority of ARGs cluster in close proximity to each other on the chromosome within two separate multidrug-resistance determining regions (MDRs), each flanked by IS26 transposases. MDR-1 carries blaTEM-1, tmrB, aac(3)-IId, aadA5, mph(A), mrx, sul1, qacEΔ1 and dfrA17; while MDR-2 harbors aph(3″)-Ib, aph(6)-Id, blaTEM-1, catA1, tet(D) and sul2. Four identical chromosomal copies of blaCTX-M-14 are located outside these regions, flanked by ISEc9 transposases. Strain 1500 (E. coli sequence type 191) exhibited a circular chromosome of 4.73 Mb and two plasmids (91 kb and 4 kb). The 91 kb conjugative plasmid belonging to IncI1 group carries blaCTX-M-15 and blaTEM-1 genes. CONCLUSION: This study confirms the efficacy of combining Nanopore long-read and Illumina short-read sequencing for determining complete bacterial genome sequences, enabling detection and characterization of clinically important ARGs in the marine environment in Norway, with potential for further dissemination. It also highlights the need for environmental surveillance of antibiotic resistance in low prevalence settings like Norway.

[Isolation of enterocin-producing Enterococcus hirae strains from the intestinal content of the Patagonian mussel (Mytilus edulis platensis)]. / Aislamiento de cepas de Enterococcus hirae productoras de enterocinas a partir del contenido intestinal de mejillón patagónico (Mytilus edulis platensis).

Two bacteriocin-producing lactic acid bacterial strains were isolated from the intestinal content of the Patagonian mussel and characterized by phenotypic and molecular tests. The isolates were identified as Enterococcus hirae and named E. hirae 463Me and 471Me. The presence of the enterocin P gene was identified in both strains by PCR techniques, while enterocin hiracin JM79 was detected only in the 471Me strain. Both strains were sensitive to clinically important antibiotics and among the virulence traits investigated by PCR amplification, only cylLl and cylLs could be detected; however, no hemolytic activity was observed in the blood agar test. Cell free supernatants were active against all Listeria and Enterococcus strains tested, Lactobacillus plantarum TwLb 5 and Vibrio anguilarum V10. Under optimal growth conditions, both strains displayed inhibitory activity against Listeria innocua ATCC 33090 after 2h of incubation. E. hirae 471Me achieved a maximum activity of 163840AU/ml after 6h of incubation, while the same value was recorded for E. hirae 463Me after 8h. In both cases, the antagonist activity reached its maximum before the growth achieved the stationary phase and remained stable up to 24h of incubation. To our knowledge, this is first report of the isolation of bacteriocinogenic E. hirae strains from the Patagonian mussel. The high inhibitory activity and the absence of virulence traits indicate that they could be applied in different biotechnological areas such as food biopreservation or probiotic formulations.

Experimental infection of Mytilus edulis by two Vibrio splendidus-related strains: Determination of pathogenicity level of strains and influence of the origin and annual cycle of mussels on their sensitivity.

This study aimed at assessing the pathogenicity of two Vibrio splendidus-related species and evaluating the influence of the origin and annual life cycle of mussels on their sensitivity during a bacterial challenge. Thus, in vivo infection assays were made with Vibrio crassostreae 7T4_12 and Vibrio splendidus 3G1_6, over, respectively, thirteen and 9 months, on adult blue mussels from five recruitment areas in France. Two bacterial concentrations were tested: one consistent with the loads of Vibrio spp. in environment and mussel tissues (~105  CFU/ml) and another one much higher (~108  CFU/ml). The tested environmental concentration has no pathogenic effect whatever the time of year, the strain used and the origin of mussels. However, at the highest concentration, a pathogenic effect was observed only at specific moments, and one of the origins appeared to be more resistant. The physiological state of mussels-depending on the time of year-seemed significant in mussels' sensitivity, as their recruitment origin. This study is the first to test the pathogenicity of V. splendidus-related strains at concentrations close to what is found in the wild, over the annual cycle of mussels, and considering their origin.

High mortality of mussels in northern Brittany - Evaluation of the involvement of pathogens, pathological conditions and pollutants.

In 2014, a high and unusual mass mortality of mussels occurred in several important production areas along the French coasts of the Atlantic and English Channel. In the first quarter of 2016, mass mortalities hit farms on the west coast of the country once again. These heterogeneous mortality events elicited a multi-parametric study conducted during the 2017 mussel season in three sites in northern Brittany (Brest, Lannion and St. Brieuc). The objective was to assess the health status of these mussels, follow mortality and attempt to identify potential causes of the abnormal high mortality of farmed mussels in northern Brittany. Brest was the most affected site with 70% cumulative mortality, then Lannion with 40% and finally St. Brieuc with a normal value of 15%. We highlighted a temporal 'mortality window' that opened throughout the spring season, and concerned the sites affected by mortality of harmful parasites (including pathogenic bacteria), neoplasia, metal contamination, and tissue alterations. Likely, the combination of all these factors leads to a weakening of mussels that can cause death.

Gut Microbiomes of the Eastern Oyster (Crassostrea virginica) and the Blue Mussel (Mytilus edulis): Temporal Variation and the Influence of Marine Aggregate-Associated Microbial Communities.

Gut microbial community structure was evaluated for two species of bivalve molluscs, the eastern oyster (Crassostrea virginica) and the blue mussel (Mytilus edulis) collected from Long Island Sound, Connecticut, over the course of a year. These bivalves utilize a shared feeding mechanism, which may result in similar gut microbial communities. Their particle diet, marine aggregates, and surrounding environment, aggregate-free seawater (AFSW), were also collected for comparison. Due to the suspension-feeding activities of bivalves, the potential for aggregate- and AFSW-associated microbiota to influence their microbial communities may be significant. Both taxonomic and functional diversity of the samples were assessed. 16S rRNA gene amplicon sequencing indicated that oysters and mussels maintained similar, but not identical, gut microbiomes, with some temporal variation. Throughout the year, bivalve species had gut microbial community compositions that were more similar to one another than to aggregates. Within a month, bivalves shared on average a quarter of their total operational taxonomic units (OTUs) with each other and a 10th of their total OTUs with aggregates. During months with warm water temperatures, individuals within each of the four sample types had similar alpha diversity, but again, temporal variation was observed. On a functional level, bivalve gut microbial communities exhibited variation attributed to host species and season. Unlike oysters, mussel gut bacterial communities maintained high richness and evenness values throughout the year, even when values for the particle diet and AFSW were reduced. Overall, a core gut bivalve microbiome was present, and it was partially influenced by the marine aggregate microbial community.IMPORTANCE This work investigates the influence that extrinsic factors, diet, and the environment can have on the microbiomes of shellfish. Over the course of a year, the gut microbial communities of two species of bivalves, oysters and mussels, held under identical conditions in coastal marine waters were compared. While the mussels and oysters harbored gut microbial communities with similar composition, on a functional level, they exhibited species and temporal variation. These results indicate that intrinsic factors influence the bivalve microbiome, resulting in species variability, even when environmental conditions, feeding mechanism, and particle diet are constant. Seasonal and multispecies comparisons for bivalve-associated microbial communities are rare, and we believe this research represents an important contribution. The results presented here advance our understanding of the symbiotic interactions between marine invertebrates, the microbial communities they harbor, and the environment.

Does Ralstonia eutropha, rich in poly-ß hydroxybutyrate (PHB), protect blue mussel larvae against pathogenic vibrios?

The natural amorphous polymer poly-ß-hydroxybutyrate (PHB-A: lyophilized Ralstonia eutropha containing 75% PHB) was used as a biological agent to control bacterial pathogens of blue mussel (Mytilus edulis) larvae. The larvae were supplied with PHB-A at a concentration of 1 or 10 mg/L for 6 or 24 hr, followed by exposure to either the rifampicin-resistant pathogen Vibrio splendidus or Vibrio coralliilyticus at a concentration of 105 CFU/ml. Larvae pretreated 6 hr with PHB-A (1 mg/L) survived a Vibrio challenge better relative to 24 hr pretreatment. After 96 hr of pathogen exposure, the survival of PHB-A-treated mussel larvae was 1.41- and 1.76-fold higher than the non-treated larvae when challenged with V. splendidus and V. coralliilyticus, respectively. Growth inhibition of the two pathogens at four concentrations of the monomer ß-HB (1, 5, 25 and 125 mM) was tested in vitro in LB35 medium, buffered at two different pH values (pH 7 and pH 8). The highest concentration of 125 mM significantly inhibited the pathogen growth in comparison to the lower levels. The effect of ß-HB on the production of virulence factors in the tested pathogenic Vibrios revealed a variable pattern of responses.

Combined genotyping, microbial diversity and metabolite profiling studies on farmed Mytilus spp. from Kiel Fjord.

The blue mussel Mytilus is a popular food source with high economical value. Species of the M. edulis complex (M. edulis, M. galloprovincialis and M. trossulus) hybridise whenever their geographic ranges overlap posing difficulties to species discrimination, which is important for blue mussel aquaculture. The aim of this study was to determine the genetic structure of farmed blue mussels in Kiel Fjord. Microbial and metabolic profile patterns were studied to investigate a possible dependency on the genotype of the bivalves. Genotyping confirmed the complex genetic structure of the Baltic Sea hybrid zone and revealed an unexpected dominance of M. trossulus alleles being in contrast to the predominance of M. edulis alleles described for wild Baltic blue mussels. Culture-dependent and -independent microbial community analyses indicated the presence of a diverse Mytilus-associated microbiota, while an LC-MS/MS-based metabolome study identified 76 major compounds dominated by pigments, alkaloids and polyketides in the whole tissue extracts. Analysis of mussel microbiota and metabolome did not indicate genotypic dependence, but demonstrated high intraspecific variability of farmed mussel individuals. We hypothesise that individual differences in microbial and metabolite patterns may be caused by high individual plasticity and might be enhanced by e.g. nutritional condition, age and gender.

Bacicyclin, a new antibacterial cyclic hexapeptide from Bacillus sp. strain BC028 isolated from Mytilus edulis.

A new cyclic hexapeptide, cyclo-(Gly-Leu-Val-IIe-Ala-Phe), named bacicyclin (1), was isolated from a marine Bacillus sp. strain associated with Mytilus edulis. The sequences of the amino acid building blocks of the cyclic peptide and its structure were determined by 1D- and 2D-NMR techniques. Marfey's analysis showed that the amino acid building blocks had L-configuration in all cases except for alanine and phenylalanine, which had D-configuration. Bacicyclin (1) exhibited antibacterial activity against the clinically relevant strains Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentration values of 8 and 12 µM, respectively. These results demonstrate the potential of marine bacteria as a promising source for the discovery of new antibiotics.

The immune response of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain: A transcriptomic attempt at identifying molecular actors.

The marine mussel Mytilus edulis, tolerant to a wide range of environmental changes, combines a key role as a sentinel species for environmental monitoring programs and a significant economic importance. Mortality events caused by infective agents and parasites have not been described in mussels, which suggests an efficient immune system. This study aims at identifying the molecular mechanisms involved in the early immune responses M. edulis' hemocytes challenged with Vibrio splendidus LGP32 strain during 2, 4 and 6 h. A total of 149,296 assembled sequences has been annotated and compared to KEGG reference pathways. Several immune related sequences were identified such as Toll-Like receptors (TLRs), transcription factors, cytokines, protease inhibitors, stress proteins and sequences encoding for proteins involved in cell adhesion, phagocytosis, oxidative stress, apoptosis and autophagy. Differential gene expression clustered 10 different groups of transcripts according to kinetics of transcript occurrence. Sequences were assigned to biological process gene ontology categories. Sequences encoding for galectins, fibrinogen-related proteins, TLRs, MyD88, some antimicrobial peptides, lysosomal hydrolases, heat shock proteins and protease inhibitors, as well as proteins of oxidative stress and apoptosis were identified as differently regulated during the exposure to V. splendidus LGP32. The levels of candidate transcripts were quantified in M. edulis' hemocytes exposed to V. splendidus LGP32 and 7SHRW by using branched DNA technology. Transcripts encoding for inhibitor kappa B, inhibitor of apoptosis proteins, tumor protein D54, serine/threonine-proteine kinase SIK2 were identified as up-regulated in hemocytes exposed to both strains.

Infection dynamics of a V. splendidus strain pathogenic to Mytilus edulis: In vivo and in vitro interactions with hemocytes.

The pathogenic strain V. splendidus 10/068 1T1 has previously been reported for its virulence to the blue mussel and for its capacity to alter immune responses. In this study, we expanded the knowledge on hemocyte-pathogen interactions by using in vitro and in vivo assays. V. splendidus 10/068 1T1 severely inhibited cell adhesion and acidic vacuole formation unlike the innocuous phylogenetically related V. splendidus 12/056 M24T1 which had no effect on these cell functions. Furthermore, the virulent bacteria decreased hemocyte viability (59% of viability after 24 h). Infection dynamics were explored by using a model based on water tank cohabitation with septic mussels infected by GFP-tagged V. splendidus 10/068 1T1. Experimental infections were successfully produced (16.6% and 45% mortalities in 3 days and 6 days). The amount of GFP Vibrio in seawater decreased during the experiment suggesting its horizontal transfer from diseased animals to healthy ones. At the same time periods, bacteria were detected in hemocytes and in various organs and caused necrosis especially in gills. Total hemocyte count and viability were affected. Taken together, our results indicate that the pathogen V. splendidus 10/068 1T1 colonizes its host both by bypassing external defense barriers and impairing hemocyte defense activities.

Fate and stability of polyamide-associated bacterial assemblages after their passage through the digestive tract of the blue mussel Mytilus edulis.

We examined whether bacterial assemblages inhabiting the synthetic polymer polyamide are selectively modified during their passage through the gut of Mytilus edulis in comparison to the biopolymer chitin with focus on potential pathogens. Specifically, we asked whether bacterial biofilms remained stable over a prolonged period of time and whether polyamide could thus serve as a vector for potential pathogenic bacteria. Bacterial diversity and identity were analysed by 16S rRNA gene fingerprints and sequencing of abundant bands. The experiments revealed that egested particles were rapidly colonised by bacteria from the environment, but the taxonomic composition of the biofilms on polyamide and chitin did not differ. No potential pathogens could be detected exclusively on polyamide. However, after 7days of incubation of the biofilms in seawater, the species richness of the polyamide assemblage was lower than that of the chitin assemblage, with yet unknown impacts on the functioning of the biofilm community.

Isolation of Vibrionaceae from wild blue mussel (Mytilus edulis) adults and their impact on blue mussel larviculture.

The blue mussel (Mytilus edulis) is known as a robust bivalve species, although its larviculture appears to be highly susceptible to diseases. In this study, we isolated 17 strains from induced mortality events in healthy wild-caught blue mussel adults and demonstrated that they caused between 17% and 98% mortality in blue mussel larvae in a newly developed, highly controlled immersion challenge test model. Eight of the isolates belong to the Splendidus clade of vibrios, while the other isolates belong to the genus Photobacterium. The genomes of the most virulent Vibrio isolate and the most virulent Photobacterium isolate were sequenced and contained several genes encoding factors that have previously been linked to virulence towards bivalves. In vitro tests confirmed that all 17 isolates were positive for these virulence factors. The sequenced genomes also contained a remarkably high number of multidrug resistance genes. We therefore assessed the sensitivity of all isolates to a broad range of antibiotics and found that there were indeed many strong positive correlations between the sensitivities of the isolates to different antibiotics. Our data provide an ecological insight into mass mortality in blue mussels as they indicate that wild mussels contain a reservoir of pathogenic bacteria.

Oxidation-reduction potential and lipid oxidation in ready-to-eat blue mussels in red sauce: criteria for package design.

BACKGROUND: Ready-to-eat in-package pasteurized blue mussels in red sauce requires refrigerated storage or in combination with an aerobic environment to prevent the growth of anaerobes. A low barrier packaging may create an aerobic environment; however, it causes lipid oxidation in mussels. Thus, evaluation of the oxidation-reduction potential (Eh) (aerobic/anaerobic nature of food) and lipid oxidation is essential. Three packaging materials with oxygen transmission rate (OTR) of 62 (F-62), 40 (F-40) and 3 (F-3) cm3 m-2 day-1 were selected for this study. Lipid oxidation was measured by color changes in thiobarbituric acid reactive substances (TBARS) at 532 nm (TBARS@532) and 450 nm (TBARS@450). RESULTS: Significantly higher (P < 0.05) TBARS@532 was found in mussels packaged in higher OTR film. TBARS@450 in mussels packaged with F-62 and F-40 gradually increased during refrigerated storage (3.5 ± 0.5 °C), but remained constant after 20 days of storage for mussels packaged with F-3. The Eh of pasteurized sauce was not significantly affected (P > 0.05) by OTR and remained negative (< -80 mV) during storage. Negative Eh values can support the growth of anaerobes such as Clostridium botulinum. The headspace oxygen concentration was reduced by about 50% from its initial value during pasteurization, and then further declined during storage. The headspace oxygen concentration was higher in trays packaged with higher OTR film. CONCLUSION: Mussels packed with high OTR film showed higher lipid oxidation, indicating that high barrier film is required for packaging of mussels. Pasteurized mussels must be kept in refrigerated storage to prevent growth of anaerobic proteolytic C. botulinum spores under temperature abuse. © 2016 Society of Chemical Industry.

Colwellia mytili sp. nov., isolated from mussel Mytilus edulis.

A Gram-stain-negative, aerobic, motile and rod-shaped bacterial strain, designated RA2-7T, was isolated from a mussel (Mytilus edulis) collected from the South Sea, South Korea, and subjected to a taxonomic study using a polyphasic approach. Strain RA2-7T grew optimally at 20 °C, at pH 7.0-8.0 and in the presence of 2.0-3.0 % (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that strain RA2-7T belonged to the genus Colwellia. Strain RA2-7T exhibited 16S rRNA gene sequence similarity values of 98.3, 98.0 and 97.5 % to the type strains of Colwellia sediminilitoris, Colwellia aestuarii and Colwellia polaris, respectively, and of 94.5-96.5 % to the type strains of the other species of the genus Colwellia. Strain RA2-7T contained Q-8 as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0 as the major fatty acids. The major polar lipids detected in strain RA2-7T were phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content of strain RA2-7T was 39.0±0.04 mol% and its DNA-DNA relatedness values with the type strains of C. sediminilitoris, C. aestuarii and C. polaris were 14-19 %. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain RA2-7T is separated from recognized species of the genus Colwellia. On the basis of the data presented, strain RA2-7T is considered to represent a novel species of the genus Colwellia, for which the name Colwellia mytili sp. nov. is proposed. The type strain is RA2-7T (=KCTC 52417T=NBRC 112381T).

Toxic dinoflagellates and Vibrio spp. act independently in bivalve larvae.

Harmful algal blooms (HABs) and marine pathogens - like Vibrio spp. - are increasingly common due to climate change. These stressors affect the growth, viability and development of bivalve larvae. Little is known, however, about the potential for interactions between these two concurrent stressors. While some mixed exposures have been performed with adult bivalves, no such work has been done with larvae which are generally more sensitive. This study examines whether dinoflagellates and bacteria may interactively affect the viability and immunological resilience of blue mussel Mytilus edulis larvae. Embryos were exposed to environmentally relevant concentrations (100, 500, 2500 & 12,500 cells ml(-1)) of a dinoflagellate (Alexandrium minutum, Alexandrium ostenfeldii, Karenia mikimotoi, Protoceratium reticulatum, Prorocentrum cordatum, P. lima or P. micans), a known pathogen (Vibrio coralliilyticus/neptunius-like isolate or Vibrio splendidus; 10(5) CFU ml(-1)), or both. After five days of exposure, significant (p < 0.05) adverse effects on larval viability and larval development were found for all dinoflagellates (except P. cordatum) and V. splendidus. Yet, despite the individual effect of each stressor, no significant interactions were found between the pathogens and harmful algae. The larval viability and the phenoloxidase innate immune system responded independently to each stressor. This independence may be related to a differential timing of the effects of HABs and pathogens.

Bioaccumulation and persistence of faecal bacterial and viral indicators in Mytilus edulis and Crassostrea gigas.

This study investigated the response of two shellfish species - mussels (Mytilus edulis) and oysters (Crassostrea gigas) to microbial contamination in order to gain a better understanding of the bioaccumulation and persistence of microorganisms under controlled conditions. M. edulis and C. gigas were placed in sterile laboratory-prepared artificial seawater and initial tests were carried out to ensure both faecal indicator bacteria (FIB) and bacteriophages were below detection limits. FIB and phages were isolated, purified and dosed into experimental tanks containing the shellfish species. The GB124 phages were bioaccumulated to the highest concentration in M. edulis (1.88Log10) and C. gigas (1.27Log10) after 24h. Somatic coliphages were bioaccumulated to the highest concentration in both M. edulis (4.84log10) and C. gigas (1.73log10) after 48h. F-RNA phages were bioaccumulated to the highest concentration in M. edulis (3.51log10) after 6h but were below detection limit in C. gigas throughout the exposure period. Escherichia coli, faecal coliforms and intestinal enterococci were bioaccumulated to the highest concentrations in M. edulis (5.05log10, 5.06log10 and 3.98log10, respectively) after 48h. In C. gigas, E. coli reached a maximum concentration (5.47log10) after 6h, faecal coliforms (5.19log10) after 12h and intestinal enterococci (3.23log10) after 24h.M. edulis bioaccumulated phages to a greater extent than the faecal bacteria, and in both shellfish species, faecal bacteria persisted for longer periods over 48h than the phages. This study highlights significant variation in the levels and rate of accumulation and persistence with respect to both shellfish species and the indicators used to assess risk. The results suggest that phage-based indicators could help elucidate risks to human health associated with pathogenic organisms.

Impact of ocean acidification on antimicrobial activity in gills of the blue mussel (Mytilus edulis).

Here, we aimed to investigate potential effects of ocean acidification on antimicrobial peptide (AMP) activity in the gills of Mytilus edulis, as gills are directly facing seawater and the changing pH (predicted to be reduced from ∼8.1 to ∼7.7 by 2100). The AMP activity of gill and haemocyte extracts was compared at pH 6.0, 7.7 and 8.1, with a radial diffusion assay against Escherichia coli. The activity of the gill extracts was not affected by pH, while it was significantly reduced with increasing pH in the haemocyte extracts. Gill extracts were also tested against different species of Vibrio (V. parahaemolyticus, V. tubiashii, V. splendidus, V. alginolyticus) at pH 7.7 and 8.1. The metabolic activity of the bacteria decreased by ∼65-90%, depending on species of bacteria, but was, as in the radial diffusion assay, not affected by pH. The results indicated that AMPs from gills are efficient in a broad pH-range. However, when mussels were pre-exposed for pH 7.7 for four month the gill extracts presented significantly lower inhibit of bacterial growth. A full in-depth proteome investigation of gill extracts, using LC-Orbitrap MS/MS technique, showed that among previously described AMPs from haemocytes of Mytilus, myticin A was found up-regulated in response to lipopolysaccharide, 3 h post injection. Sporadic occurrence of other immune related peptides/proteins also pointed to a rapid response (0.5-3 h p.i.). Altogether, our results indicate that the gills of blue mussels constitute an important first line defence adapted to act at the pH of seawater. The antimicrobial activity of the gills is however modulated when mussels are under the pressure of ocean acidification, which may give future advantages for invading pathogens.