Multidrug resistant Vibrio spp. identified from mussels farmed for human consumption in Central Italy.

AIMS: This study investigated phenotypic and genotypic antimicrobial resistance profiles of Vibrio strains identified from Mytilus galloprovincialis farmed for human consumption in the Adriatic Sea Central Italy. METHODS AND RESULTS: A total of 475 mussels (M. galloprovincialis) were involved in the present study, and culture-dependent microbiological methods permitted to identify a total of 50 Vibrio strains that were tested for antibiotic susceptibility followed by the genetic determinant detections. Antibiograms showed resistance against ampicillin (36.0%), amoxicillin-clavulanic acid (30.0%), gentamycin (14.0%), and imipenem (18.0%). Biomolecular assays amplified a total of 264 antibiotic resistance genes harbored by both susceptible and resistant Vibrio species. Among resistance genes, aacC2 (62.0%) and aadA (58.0%) for aminoglycosides, blaTEM (54.0%) for beta-lactams, qnrS (24.0%) for quinolones, tetD (66.0%) for tetracyclines, and vanB (60.0%) for glycopeptides were mainly amplified by PCR assays. CONCLUSIONS: Vibrio genus is involved in the antibiotic resistance phenomenon diffusion in the aquatic environments, as demonstrated by the harboring of many genetic determinants representing a kind of genetic "dark world".

Understanding the role of Francisella halioticida in mussel mortalities in France: an integrative approach.

Since 2014, mass mortalities of mussels Mytilus spp. have occurred in production areas on the Atlantic coast of France. The aetiology of these outbreaks remained unknown until the bacterium Francisella halioticida was detected in some mussel mortality cases. This retrospective study was conducted to assess the association between F. halioticida and these mussel mortalities. Mussel batches (n = 45) from the Atlantic coast and English Channel were selected from archived individual samples (n = 863) collected either during or outside of mortality events between 2014 and 2017. All mussels were analysed by real-time PCR assays targeting F. halioticida; in addition, 185 were analysed using histological analysis and 178 by 16S rRNA metabarcoding. F. halioticida DNA was detected by real-time PCR and 16S rRNA metabarcoding in 282 and 34 mussels, respectively. Among these individuals, 82% (real-time PCR analysis) and 76% (16S rRNA metabarcoding analysis) were sampled during a mortality event. Histological analyses showed that moribund individuals had lesions mainly characterized by necrosis, haemocyte infiltration and granulomas. Risk factor analysis showed that mussel batches with more than 20% of PCR-positive individuals were more likely to have been sampled during a mortality event, and positive 16S rRNA metabarcoding batches increased the strength of the association with mortality by 11.6 times. The role of F. halioticida in mussel mortalities was determined by reviewing the available evidence. To this end, a causation criteria grid, tailored to marine diseases and molecular pathogen detection tools, allowed more evidence to be gathered on the causal role of this bacterium in mussel mortalities.

Polyester Microfibers Exposure Modulates Mytilus galloprovincialis Hemolymph Microbiome.

Microplastic (MP) contamination in the aquatic environment is a cause of concern worldwide since MP can be taken up by different organisms, altering different biological functions. In particular, evidence is accumulating that MP can affect the relationship between the host and its associated microbial communities (the microbiome), with potentially negative health consequences. Synthetic microfibers (MFs) represent one of the main MPs in the marine environment, which can be accumulated by filter-feeding invertebrates, such as bivalves, with consequent negative effects and transfer through the food chain. In the mussel Mytilus galloprovincialis, polyethylene terephthalate (PET) MFs, with a size distribution resembling that of an MF released from textile washing, have been previously shown to induce multiple stress responses. In this work, in the same experimental conditions, the effects of exposure to PET-MF (96 h, 10, and 100 µg/L) on mussel hemolymph microbiome were evaluated by 16S rRNA gene amplification and sequencing. The results show that PET-MF affects the composition of bacterial communities at the phylum, family and genus level, with stronger effects at the lowest concentration tested. The relationship between MF-induced changes in hemolymph microbial communities and responses observed at the whole organism level are discussed.

Host Species and Environment Shape the Gut Microbiota of Cohabiting Marine Bivalves.

Pacific oysters (Crassostrea gigas) and Mediterranean mussels (Mytilus galloprovincialis) are commercially important marine bivalves that frequently coexist and have overlapping feeding ecologies. Like other invertebrates, their gut microbiota is thought to play an important role in supporting their health and nutrition. Yet, little is known regarding the role of the host and environment in driving these communities. Here, bacterial assemblages were surveyed from seawater and gut aspirates of farmed C. gigas and co-occurring wild M. galloprovincialis in summer and winter using Illumina 16S rRNA gene sequencing. Unlike seawater, which was dominated by Pseudomonadata, bivalve samples largely consisted of Mycoplasmatota (Mollicutes) and accounted for >50% of the total OTU abundance. Despite large numbers of common (core) bacterial taxa, bivalve-specific species (OTUs) were also evident and predominantly associated with Mycoplasmataceae (notably Mycoplasma). An increase in diversity (though with varied taxonomic evenness) was observed in winter for both bivalves and was associated with changes in the abundance of core and bivalve-specific taxa, including several representing host-associated and environmental (free-living or particle-diet associated) organisms. Our findings highlight the contribution of the environment and the host in defining the composition of the gut microbiota in cohabiting, intergeneric bivalve populations.

Pathology, epidemiology, and phylogeny of mussel egg disease due to the microsporidian Steinhausia mytilovum (Field, 1924) in the Mediterranean mussel (Mytilus galloprovincialis).

Microsporidia are well known fungal pathogens of aquatic animals. However, the taxonomy of microsporidia is generally poorly resolved, which has consequently constrained our understanding of their pathology and epidemiology in marine animals. To date, microsporidia have been reported in both bivalves and gastropods, and microsporidia from mollusks have been classified in different genera. Despite ongoing work to better describe these genera, including detailed microscopic and ultrastructural images, so far we lack information on microsporidian phylogeny and pathogenicity of species within these genera. Here we investigate the microsporidian parasite Steinhausia mytilovum associated with the mussel, Mytilus galloprovincialis, in natural beds and farms along coast of southern Italy. A survey of M. galloprovincialis was conducted in 13 mussel farms and one natural bed between 2009 and 2020. We found the presence of S. mytilovum in 10 of the investigated farms, with a prevalence ranging between 14 and 100% of female mussels, depending on the population and season in which they were sampled. The parasite developed in the oocytes within a sporophorous vesicle (SV) where it produced 1-3 spores per cell, both in the cytoplasm and in the nucleus. Stenhausia mytilovum elicited an infiltrative (24.8%) or a strong capsular inflammatory response (43.4%) at gonadal follicles and surrounding vesicular connective tissue, in some cases accompanied by gonadal atresia (24.8%), leading to loss of gonadal architecture. In 7% of cases no reaction was observed. Ultrastructural observations revealed a mitochondrial re-organization to interact with all the phases of parasite development; the mitochondria were arranged outside the parasitophorous vesicle (PV) or directly interacting with the spore inside vesicle. There are five taxonomic clades of microsporidians as identified by SSU ribosomal gene sequence data. Maximum likelihood analysis assigned S. mytilovum within the Clade IV, defined as the Class Terresporidia, with closest genetic relationship (83.6% identity) to an undetermined invertebrate ovarian microsporidian. The constant presence, prevalence, and severity of S. mytilovum in coastline populations of M. galloprovincialis populations in southern Italy may indirectly reflect immunocompetence at both individual and population levels.

Two-Component System Response Regulator ompR Regulates Mussel Settlement through Exopolysaccharides.

The outer membrane protein (OMP) is a kind of biofilm matrix component that widely exists in Gram-negative bacteria. However, the mechanism of OMP involved in the settlement of molluscs is still unclear. In this study, the mussel Mytilus coruscus was selected as a model to explore the function of ompR, a two-component system response regulator, on Pseudoalteromonas marina biofilm-forming capacity and the mussel settlement. The motility of the ΔompR strain was increased, the biofilm-forming capacity was decreased, and the inducing activity of the ΔompR biofilms in plantigrades decreased significantly (p < 0.05). The extracellular α-polysaccharide and ß-polysaccharide of the ΔompR strain decreased by 57.27% and 62.63%, respectively. The inactivation of the ompR gene decreased the ompW gene expression and had no impact on envZ expression or c-di-GMP levels. Adding recombinant OmpW protein caused the recovery of biofilm-inducing activities, accompanied by the upregulation of exopolysaccharides. The findings deepen the understanding of the regulatory mechanism of bacterial two-component systems and the settlement of benthic animals.

Vibrio ulleungensis sp. nov., isolated from Mytilus coruscus.

A Gram-stain-negative, rod-shaped, motile via polar flagellum, facultatively aerobic, light-yellow, bacterium (designated 188UL20-2T) was isolated from a mussel sample of Mytilus coruscus collected on Ulleung Island, Ulleung-gun, Gyeongsangbuk-do, Republic of Korea. On the basis of 16S rRNA gene sequencing results, strain 188UL20-2T clustered with species of the genus Vibrio and appeared closely related to Vibrio marisflavi DSM 23086T (96.59%), Vibrio variabilis DSM 26147T (96.57%), Vibrio penaeicida DSM 14398T (96.37%) and Vibrio litoralis DSM 17657T (95.97%). The average nucleotide identity and digital DNA-DNA hybridization values between strain 188UL20-2T and its closest related strain were 71.3 and 16.4%, indicating that 188UL20-2T represents a novel species of the genus Vibrio. Growth occurred at 18-37 °C on MA medium in the presence of 1-4% NaCl (w/v) and at pH 5.0-10.0. The DNA G+C content of the genomic DNA was 45.4 mol%, and ubiquinone-8 (Q-8) was the major respiratory quinone. The major cellular fatty acids (>5%) were C16:1 ω6c and/or C16:1 ω7c (summed feature 3), C18:1 ω7c and/or C18:1 ω6c (summed feature 8), C16:0, C16:0 iso, C14:0, C14:0 iso and C12:0. The polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids, unidentified aminophospholipid, unidentified glycolipid and seven unidentified lipids. Physiological and biochemical characteristics indicated that strain 188UL20-2T represents a novel species of the genus Vibrio, for which the name Vibrio ulleungensis sp. nov. is proposed. The type strain is 188UL20-2T (=KACC 22258T=LMG 32202T).

Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg-1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018-2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8-26 µg kg-1) and 3 (2%) contained levels above the EFSA safety threshold (61-76 µg kg-1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

Development of a semi-quantitative PCR assay for the detection of Francisella halioticida and its application to field samples.

The current study describes the development and application of a TaqMan® real-time PCR assay for the detection of the bacterium Francisella halioticida. Previously, detection of F. halioticida is relied on bacterial culture and conventional PCR; however, the real-time PCR provides many advantages because it is faster, less labour-intensive and reduces the risk of cross-contamination. DNA samples from mussels collected in April 2020 from seven sites in northern Brittany (France) were tested using the newly developed real-time PCR assay. The objective was to screen for the presence of F. halioticida during spring mortality events. The bacterium was detected in 71.4% of the samples tested and was present at all sites except for Saint-Brieuc and Mont-Saint-Michel, two sites which were not concerned by mortality at the time of sampling. Less than a month later, Saint-Brieuc was affected by unusual mortalities and F. halioticida was detected in almost all mussels (81.25%). The findings from this study provide further evidence indicating that F. halioticida may be contributing to mussel mortalities; however, a direct causal relationship has not yet been established. The real-time PCR assay developed in this study allows for rapid, specific and sensitive detection of F. halioticida which should prove useful for future studies concerning the involvement of this bacterium with shellfish mortalities.

Vibrio-bivalve interactions in health and disease.

In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections. Thus, Vibrio species are associated with the main diseases affecting the worldwide oyster production. Here, we review the current knowledge on Vibrio-bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with their bivalve hosts as well as technical issues limiting the monitoring of these bacteria in bivalve health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defences of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators.

Immune-responsive gene 1 (IRG1) and dimethyl itaconate are involved in the mussel immune response.

Immune-responsive gene 1 (irg1) is a gene that is well-conserved among different taxa and is highly expressed in the mussel Mytilus galloprovincialis at the constitutive level. The expression of this gene increases after a bacterial infection, primarily in haemocytes. irg1 catalyses the production of itaconic acid from cis-aconitic acid in the Krebs cycle. Recently, itaconate has been revealed as an immune metabolite involved in macrophage polarization. In this work, we studied the effects of exogenous dimethyl itaconate (DI) on mussels in vitro and in vivo at relevant previously described endogenous concentrations and in mussels infected with Vibrio splendidus. DI did not have adverse effects on the haemocytes viability, apoptotic cells, proliferation and phagocytic activity; however, haemocyte size, velocity and accumulated distance were decreased. The antibacterial activity of DI in vitro and in vivo was observed with high concentrations of DI, that is, 30 and 50 mM, respectively. Furthermore, DI inhibited total ROS, increased mitochondrial ROS and modulated antioxidant genes, such as SOD and CAT, related to Nrf2 activation. In this research, we have demonstrated some important pathways in haemocytes in which itaconate can be involved after its production in a bacterial infection.

Species identification, virulence markers and antimicrobial resistance profiles of Aeromonas sp. isolated from marketed hard-shelled mussel (Mytilus coruscus) in Korea.

Hard-shelled mussel (Mytilus coruscus) is a popular seafood in Korea. This study aimed to determine the virulence markers and antimicrobial resistance patterns of 33 Aeromonas strains isolated from mussels. The isolates were identified as A. salmonicida (n = 14), A. veronii (n = 9), A. enteropelogenes (n = 4), A. caviae (n = 3), A. allosaccharophila (n = 2) and A. bivalvium (n = 1) by gyrB gene sequencing. The sequence divergence between and within the species ranged from 3·70 to 10·40% and 0-1·50% respectively. Every species formed a distinct group in a neighbour-joining phylogenetic tree. The DNase, gelatinase, caseinase, ß-haemolysis, biofilm and lipase activities were observed in 33 (100·00%), 31 (93·93%), 30 (90·90%), 27 (81·81%), 21 (63·63%) and 17 (51·51%) isolates respectively. The virulence genes were detected by PCR in the following frequencies: fla (90·09%), aer (87·88%), hlyA (87·88%), ahyB (81·19%), gcaT (75·76%), ser (69·70%), lip (66·67%), alt (57·58%), ast (51·51%) and act (21·21%). Every isolate was resistant to at least three of 18 antimicrobials in the disk diffusion test. The multiple antimicrobial resistance index values ranged from 0·11 to 0·44 among the isolates. Our study suggests that mussels can be a potential reservoir of virulent and multidrug-resistant Aeromonas sp. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas sp. are known as common pathogenic bacteria isolated from seafood. The virulence factors and antimicrobial resistance profiles of mussel-borne Aeromonas sp. are poorly understood. This study demonstrated for the first time the existence of virulence markers and antimicrobial resistance of Aeromonas sp. from mussels in Korea. Majority of the isolates were positive for phenotypic virulence characteristics and harboured several virulence genes which reveal the potential virulence of mussel-borne Aeromonas sp. Multiple antimicrobial resistance was also observed among the isolates. Our study highlights the importance of food safety standards in mussel consumption.

Vibrio parahaemolyticus control in mussels by a Halobacteriovorax isolated from the Adriatic sea, Italy.

This study evaluated the application of a Halobacteriovorax isolated from water of the Adriatic Sea (Italy) in controlling V. parahaemolyticus in mussels (Mytilus galloprovincialis). Two 72 h laboratory-scale V. parahaemolyticus decontamination experiments of mussels were performed. The test microcosm of experiment 1 was prepared using predator/prey free mussels experimentally contaminated with Halobacteriovorax/V. parahaemolyticus at a ratio of 103 PFU/105 CFU per ml, while that of experiment 2 using mussels naturally harbouring Halobacteriovorax that were experimentally contaminated with 105 CFU per ml of V. parahaemolyticus. For experiment 1, was also tested a control microcosm only contaminated with 105 CFU per ml of V. parahaemolyticus.. Double layer agar plating and pour plate techniques were used to enumerate Halobacteriovorax and V. parahaemolyticus, respectively. 16 S rRNA analysis was used to identify Halobacteriovorax. For both experiments in the test microcosm the concentration of prey remained at the same level as that experimentally added, i.e. 5 log for the entire analysis period. In experiment 1, V. parahaemolyticus counts in mussels were significantly lower in the test microcosm than the control with the maximum difference of 2.2 log at 24 h. Results demonstrate that Halobacteriovorax can modulate V. parahaemolyticus level in the mussels. The public impact of V. parahaemolyticus in bivalves is relevant and current decontamination processes are not always effective. Halobacteriovorax is a suitable candidate in the development of a biological approach to the purification of V. parahaemolyticus in mussels.

First report of signs of infection by Coccomyxa-like algae in wild blue mussels, Mytilus spp., in the Gulf of Maine (USA, Maine).

In August 2019, visual inspection of intertidal zones of the Gulf of Maine (ME, USA) revealed young and adult wild blue mussels, Mytilus spp., in Alley Bay (Jonesport area) with the distinctive L-shaped shell deformity (LSSD) and green spots (GS) in the mantle and adductor muscle. LSSD is a characteristic sign of current or previous mussel infection by photosynthetic unicellular alga from the group Coccomyxa, while GS are algal colonies. Based on these findings, this study represents the first report of infection signs by pathogenic Coccomyxa-like algae in mussels from the coastal waters of the Northeastern United States, providing a base for future large scale monitoring of the alga in the region.

The Flagellar Gene Regulates Biofilm Formation and Mussel Larval Settlement and Metamorphosis.

Biofilms are critical components of most marine systems and provide biochemical cues that can significantly impact overall community composition. Although progress has been made in the bacteria-animal interaction, the molecular basis of modulation of settlement and metamorphosis in most marine animals by bacteria is poorly understood. Here, Pseudoalteromonas marina showing inducing activity on mussel settlement and metamorphosis was chosen as a model to clarify the mechanism that regulates the bacteria-mussel interaction. We constructed a flagellin synthetic protein gene fliP deletion mutant of P. marina and checked whether deficiency of fliP gene will impact inducing activity, motility, and extracellular polymeric substances of biofilms. Furthermore, we examined the effect of flagellar proteins extracted from bacteria on larval settlement and metamorphosis. The deletion of the fliP gene caused the loss of the flagella structure and motility of the ∆fliP strain. Deficiency of the fliP gene promoted the biofilm formation and changed biofilm matrix by reducing ß-polysaccharides and increasing extracellular proteins and finally reduced biofilm-inducing activities. Flagellar protein extract promoted mussel metamorphosis, and ∆fliP biofilms combined with additional flagellar proteins induced similar settlement and metamorphosis rate compared to that of the wild-type strain. These findings provide novel insight on the molecular interactions between bacteria and mussels.

Clonal relationship among Vibrio parahaemolyticus isolated from Mediterranean mussels (Mytilus galloprovincialis) and grooved carpet shells (Ruditapes decussatus) harvested in Sardinia (Italy).

The aim of the present study was to investigate the genetic variability of Vibrio parahaemolyticus strains isolated from naturally contaminated Mediterranean mussels (Mytilus galloprovincialis) and Grooved carpet shells (Ruditapes decussatus) from three harvesting areas of Sardinia (Italy) using a combination of different typing methods: traditional phenotypic systems and molecular techniques. Ninety-nine putative V. parahaemolyticus strains isolated from shellfish collected before and after purification were included in the study. Seventy-two isolates were confirmed as V. parahaemolyticus and were submitted to REP, ERIC and BOX PCRs. The combined dendrogram showed the similarity of the data set of the three typing methods and demonstrates how the different techniques grouped the strains in two clusters in accordance with each singular dendrogram. Several strains rendered a unique pattern regardless of the typing method, which indicates the high discriminatory power of the methods. Moreover, the use of multiple typing methods allowed a more accurate characterization of the genetic profiles of isolates and the identification of clones hardly revealed through the common techniques. The intraspecific typing of environmental V. parahaemolyticus can be of great interest in order to recognize clonal relationships between environmental contamination, foodborne disease, and geographical/temporal distribution of this pathogen. The comparative analysis focusing on the obtained genetic profiles supports the possibility for typing methods to discriminate strains with similar phenotypic profile, identifying the level of genetic correlation among the strains and the presence of genetic clones.

Effects of soluble copper and copper oxide nanoparticle exposure on the immune system of mussels, Mytilus galloprovincialis.

Copper and copper oxide nanomaterials (nCuO) can enter the marine environment negatively impacting mussels, an environmental and commercially relevant organism. We analyzed the effects on the immune system of adult mussels exposed to soluble copper (CuSO4 , 20-50 µg/L) or nCuO (100-450 µg/L). CuSO4 caused significant copper accumulation in gills and cell-free hemolymph, while nCuO caused cell damage to gills and significant copper accumulation in hemocytes, the most abundant cells in the hemolymph. Both sources of copper caused cellular toxicity in hemocytes by increasing reactive oxygen species production and lysosome abundance, and decreasing multi-drug resistance transporter activity. Though hemocyte abundance was not affected, their in-vitro phagocytic activity decreased, explaining the slight (but not statistically significant) increase in bacterial proliferation in mussels exposed to the pathogenic bacteria Vibrio tubiashii following copper exposure. Thus, exposure to non-lethal concentrations of CuSO4 or nCuO can potentially increase mussel susceptibility to bacterial infections.

Comparative 16SrDNA Gene-Based Microbiota Profiles of the Pacific Oyster (Crassostrea gigas) and the Mediterranean Mussel (Mytilus galloprovincialis) from a Shellfish Farm (Ligurian Sea, Italy).

The pacific oyster Crassostrea gigas and the Mediterranean mussel Mytilus galloprovincialis are two widely farmed bivalve species which show contrasting behaviour in relation to microbial diseases, with C. gigas being more susceptible and M. galloprovincialis being generally resistant. In a recent study, we showed that different susceptibility to infection exhibited by these two bivalve species may depend on their different capability to kill invading pathogens (e.g., Vibrio spp.) through the action of haemolymph components. Specific microbial-host interactions may also impact bivalve microbiome structure and further influence susceptibility/resistance to microbial diseases. To further investigate this concept, a comparative study of haemolymph and digestive gland 16SrDNA gene-based bacterial microbiota profiles in C. gigas and M. galloprovincialis co-cultivated at the same aquaculture site was carried out using pyrosequencing. Bacterial communities associated with bivalve tissues (hemolymph and digestive gland) were significantly different from those of seawater, and were dominated by relatively few genera such as Vibrio and Pseudoalteromonas. In general, Vibrio accounted for a larger fraction of the microbiota in C. gigas (on average 1.7-fold in the haemolymph) compared to M. galloprovincialis, suggesting that C. gigas may provide better conditions for survival for these bacteria, including potential pathogenic species such as V. aestuarianus. Vibrios appeared to be important members of C. gigas and M. galloprovincialis microbiota and might play a contrasting role in health and disease of bivalve species. Accordingly, microbiome analyses performed on bivalve specimens subjected to commercial depuration highlighted the ineffectiveness of such practice in removing Vibrio species from bivalve tissues.

Assessing the health status of farmed mussels (Mytilus galloprovincialis) through histological, microbiological and biomarker analyses.

The Gulf of La Spezia (northern Tyrrhenian Sea, Italy) is a commercially important area both as a shipping port and for mussel farming. Recently, there has been increased concern over environmental disturbances caused by anthropogenic activities such as ship traffic and dredging and the effects they have on the health of farmed mussels. This paper reports the results of microbiological and histological analyses, as well as of measurement of several biomarkers which were performed to assess the health status of mussels (Mytilus galloprovincialis) from four rearing sites in the Gulf of La Spezia. Mussels were collected between October 2015 and September 2016 and histological analyses (including gonadal maturation stage), as well as the presence of pathogenic bacteria (Vibrio splendidus clade, V. aestuarianus and V. harveyi), viruses (Herpes virus and ostreid Herpes virus 1) and protozoa (Marteilia spp., in the summer season only) were carried out on a monthly basis. Conversely, biomarker responses in haemocyte/haemolymph (total haemocyte count, haemocyte diameter and volume, lysozyme and lactate dehydrogenase activities in cell-free haemolymph, and micronuclei frequency) and in gills and digestive gland (cortisol-like steroids and lipid peroxidation levels), were evaluated bimonthly. Microbiological data indicated that mussels contain a reservoir of potentially pathogenic bacteria, viruses and protozoa that in certain environmental conditions may cause a weakening of the immune system of animals leading to mortality episodes. The percentage of parasites detected in the mussels was generally low (9.6% for Steinhausia mytilovum, that is 17 samples out of 177 examined females; 3.4% for Proctoeces maculatus; 0.9% for Mytilicola intestinalis and 2% for ciliated protozoa), while symbiont loads were higher (31% for Eugymnanthea inquilina and Urastoma cyprinae). Interestingly, a previously undescribed haplosporidian was detected in a single mussel sample (0.2%) and was confirmed by in situ hybridization. Cells morphologically similar to Perkinsus sp. trophozoites were observed in 0.7% of the mussels analysed; however, infection with Perkinsus spp. could neither be confirmed by ISH nor by PCR. Different pathological aspects, such as host defence responses and regressive/progressive changes were detected in the gills, digestive glands, gonads and mantle. Only one single case of disseminated neoplasia (0.2%) was observed. As for the biomarker evaluation, the MANOVA analysis revealed the statistically significant effect that the variable "sampling site" had on the biological parameter measured, thus suggesting that the multibiomarker approach was able to differentiate the rearing sites.

Evaluation of short purification cycles in naturally contaminated Mediterranean mussels (Mytilus galloprovincialis) harvested in Sardinia (Italy).

The aim of the present study was to investigate the effect of short purification cycles on the safety of naturally contaminated Mytilus galloprovincialis from harvesting areas of the Gulf of Olbia (Sardinia, Italy). Samples from ten batches of mussels were collected before, during and after purification treatment at two purification centres (A-B). All the samples were analysed for Escherichia coli and Salmonella spp according to Council Regulation (EC) 2285/2015. Detection and enumeration of Vibrio spp were performed according to previously published methods. Presumptive identification of Vibrio spp isolates were performed by means of conventional biochemical tests and polymerase chain reaction. The presence of Hepatitis A virus was detected by nested reverse transcriptase-polymerase chain reaction. Environmental parameters (water temperature and salinity) were also recorded. The results of Escherichia coli counts showed the overall efficacy of the short purification cycles; a purification cycle of 8 h led to a rapid decline in the concentration. The decrease in Escherichia coli counts does not correlate with the presence of naturally occurring vibrios, the decline of which occurs at an even slower rate. The average contamination levels for Vibrio spp before purification were 8.20 ±â€¯0.47 and 7.99 ±â€¯0.62 Log10 CFU/g in samples collected at purification plants A and B, respectively. After purification, the average contamination levels were 8.10 ±â€¯0.60 Log10 CFU/g at purification plant A and 7.85 ±â€¯0.57 Log10 CFU/g at purification plant B. The contaminated samples revealed the presence of Vibrio alginolyticus (n=21), Vibrio fluvialis (n=12), Vibrio cholerae (n=4), Vibrio parahaemolyticus (n=2) and Vibrio vulnificus (n=1). The Vibrio parahaemolyticus isolates carried the tdh or the trh genes. None of the isolates was tdh+/trh+. Salmonella spp and Hepatitis A virus were not detected. The adoption of short purification cycles for Mytilus galloprovincialis in the presence of pathogenic vibrios might not be sufficient to guarantee the safety of consumers.