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We identified areas with high individual densities of the pen shell, Pinna nobilis, in two areas along the Croatian Adriatic coast. The surveys carried out in 2018 and 2019 showed population densities of approximately 9 to 13 individuals/100 m 2 . However, in 2019 a mass mortality event (MME) causing 36% to 100% mortality of this bivalve species was observed in the surveyed Croatian bays. The parasite Haplosporidium pinnae was identified by histological and molecular methods in all affected sampled individuals, while Mycobacterium sp. and Gram negative bacilli were detected in some affected and live bivalves. This finding constitutes the first record of these pathogens affecting P. nobilis in the middle Adriatic, confirming the continuous spread of the disease. Previously, the Adriatic water body was considered to be a natural shelter against the MME caused by pathogens in pen shell populations because of its distinct ecological features. The Adriatic Sea is a semi-closed water body with the largest continental shelf in the Mediterranean Sea, and due to its geomorphology and bathymetry, it is a sea with distinct characteristics. Monitoring plans and further studies in the Adriatic bays are now a priority for mitigating the high risk of extinction and working toward the conservation of this protected species., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mycobacterium sp. and Haplosporidium pinnae constitute invasive parasite species of bivalves, reported for the first time in the present study in the Aegean Sea and Thermaikos Gulf, respectively. During the last years, the endangered fan mussel (Pinna nobilis) experienced several mortality events in the Mediterranean Sea that caused deaths to 90% or more of their populations and have been attributed to infections by these pathogens. In Greece, two mass mortality events have been recently reported, namely in the Gulf of Kalloni and in Limnos island. In the present study we investigated the presence of both pathogens in P. nobilis from these marine areas as well as from Thermaikos Gulf using both histopathological microscopy and molecular markers. The detected parasite DNA was further quantified in the three populations utilizing a real time qPCR. Histopathological results indicated the presence of a Mycobacterium species alongside with the existence of the Haplosporidian parasite, which was identified in all mortality events in the Mediterranean Sea. The parasite was present in different phases mostly on the digestive gland epithelium. Phylogenetic analysis confirmed the taxonomy of the Haplosporidian parasite as the recently described Haplosporidium pinnae, whereas it failed to identify the Mycobacteria parasite at species level. While Mycobacterium sp. was detected in all examined specimens, H. pinnae was not detected in all diseased fan mussels. Interestingly, monitoring of P. nobilis population from Thermaikos Gulf, an estuary of extremely high importance for bivalve production, revealed the presence of both pathogens in a few specimens in higher quantity but with no symptoms of the disease. Besides, all the specimens from Thermaikos Gulf had inflammatory responses similarly to moribund specimens from mortality events., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Calich Lagoon is a Mediterranean coastal lagoon located along the northwestern coast of Sardinia (Italy). The connection to marine and fresh water determines the high productivity of this coastal lagoon. Despite its great potential and the presence of natural beds of bivalve mollusks ( Mytilus galloprovincialis ), the lagoon has not yet been classified for shellfish production. In this study, through a multidisciplinary approach, the presence of several bacterial pathogens ( Escherichia coli, Salmonella spp., and Vibrio spp.) and viral pathogens (hepatitis A virus and norovirus genogroups I and II) was evaluated from March 2017 to February 2018. In addition, phytoplankton composition in lagoon waters and associated algal biotoxins (paralytic and diarrhetic shellfish poisoning) in mussels were also monitored. The aim of this study was to provide useful data to improve knowledge about their seasonal presence and to assess the potential risk for public health, as well as to provide input for future conservation and management strategies. In mussels, Salmonella spp. were found in spring, along with E. coli, but Salmonella spp. were not found in autumn or winter, even though E. coli was detected in these seasons. Vibrio parahaemolyticus was found in autumn and winter, but not in spring. Norovirus genogroups I and II were found in winter samples. None of the bacteria were found in summer. Algal biotoxins have never been detected in mussel samples. Among potentially harmful phytoplankton, only Pseudo-nitzschia spp. were present, mainly in summer. The results showed that a possible bacterial and viral contamination, together with the presence of potentially toxic microalgae, is a real problem. Therefore, the development of natural resource management strategies is necessary to ensure the good quality of waters and guarantee the protection of consumers.

Disease is an increasing threat for marine bivalves worldwide. Recently, a mass mortality event (MME) impacting the bivalve Pinna nobilis was detected across a wide geographical area of the Spanish Mediterranean Sea and linked to a haplosporidian parasite. In 2017-2018, mass mortality events affecting the pen shell Pinna nobilis were recorded in two different regions of Italy, Campania and Sicily, in the Tyrrhenian Sea (Mediterranean Sea). Histopathological and molecular examinations of specimens showed the presence of Haplosporidium sp. in only one specimen in one area. Conversely, in all of the surveyed moribund animals, strong inflammatory lesions at the level of connective tissue surrounding the digestive system and gonads and linked to the presence of intracellular Zhiel-Neelsen-positive bacteria were observed. Molecular analysis of all of the diseased specimens (13) confirmed the presence of a Mycobacterium. Blast analysis of the sequences from all of the areas revealed that they were grouped together with the human mycobacterium M. sherrisii close to the group including M. shigaense, M. lentiflavum and M. simiae. Based on pathological and molecular findings, it is proposed that a mycobacterial disease is associated with the mortality episodes of Pinna nobilis, indicating that, at this time, Haplosporidium sp. is not responsible for these events in Campanian and Sicilian waters.

The application of natural zeolite for water and wastewater treatment has been carried out and is still a promising technique in environmental cleaning processes. Natural zeolite can be used to improve the purification process of clams (Ruditapes decussatus). Thus, our study aimed at improving the clam purification system in order to reduce Escherichia coli and eliminate Salmonella in samples artificially contaminated with this bacterium using a natural zeolite to replace the biological filter. The results showed that zeolite used in a depuration system improved the clam purification process. Moreover, natural zeolite exhibited high performance in the adsorption of bacteria and allowed to reduce the Escherichia coli abundance in 24 h, thus ensuring purified clams conformity with the ISO 16649-3 standard. These results indicate the beneficial effects of using zeolite in the adsorption of bacteria and the reduction in the abundance of Escherichia coli and set the Salmonella from marine organisms.

A mechanism of mercury detoxification has been suggested by a previous study on Hg bioaccumulation in Manila clams (Ruditapes philippinarum) in the polluted Marano and Grado lagoons and in this study we demonstrate that this event could be partly related to the detoxifying activities of Hg-resistant bacteria (MRB) harbored in clam soft tissues. Therefore, natural clams were collected in six stations during two different periods (winter and spring) from Marano and Grado Lagoons. Siphons, gills and hepatopancreas from acclimatized clams were sterile dissected to isolate MRB. These anatomical parts were glass homogenized or used for whole, and they were lying on a solid medium containing 5mgl(-1) HgCl2 and incubated at 30°C. A total of fourteen bacterial strains were isolated and were identified by 16S rDNA sequencing and analysis, revealing that strains were representative of eight bacterial genera, four of which were Gram-positive (Enterococcus, Bacillus, Jeotgalicoccus and Staphylococcus) and other four were Gram-negative (Stenotrophomonas, Vibrio, Raoultella and Enterobacter). Plasmids and merA genes were found and their sequences determined. Fluorescence in situ hybridization (FISH) technique shows the presence of Firmicutes, Actinobacteria and Gammaproteobacteria by using different molecular probes in siphon and gills. Bacterial clumps inside clam flesh were observed and even a Gram-negative endosymbiont was disclosed by transmission electronic microscope inside clam cells. Bacteria harbored in cavities of soft tissue have mercury detoxifying activity. This feature was confirmed by the determination of mercuric reductase in glass-homogenized siphons and gills., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Thyasiridae are one of the less studied groups of chemosymbiotic bivalves. Here, we investigated symbioses in three different thyasirid species collected at three cold seeps from the Atlantic and Mediterranean. Phylogenetic analysis of bacterial 16S ribosomal RNA gene sequences demonstrated that each thyasirid species harbours a single phylotype of symbiont that belongs to a distinct lineage of putative sulphur-oxidizing Gammaproteobacteria. This result is confirmed by other marker genes (encoding 23S rRNA and APS reductase) and fluorescence in situ hybridization. This work highlights the diversity of bacteria involved in symbiosis with thyasirids and underlines the relevance of this group as a target for future symbiosis studies.

Heterogeneity among ribosomal operons in Vibrio vulnificus is purported as a probabilistic indicator of strain virulence and classifies V. vulnificus strains as 16S rRNA genes type A and B. In this study, 16S rRNA genes typing of V. vulnificus strains isolated from the Valencia city coast, in the western Mediterranean, showed that 24 out of 30 isolates were type A, one was type B and five could not be typed. Single strand conformation polymorphism (SSCP) analysis of this gene region revealed complex patterns indicative of intragenomic ribosomal operon sequence heterogeneity. The 16S rRNA genes of three untypeable isolates C27, C30, and C34, along with type A (ATCC 27562) and B (C7184) reference strains, were amplified, cloned and sequenced. The number of unique 16S rRNA gene sequences was 4, 3, and 4 for the environmental isolates. The type strain of the species (ATCC 27562) presented only two 16S rRNA gene types, while the reference isolate C7184 of clinical origin had only one 16S rRNA gene type. Sequences differed from five to 35 bp (99.6% to 97.6% sequence similarity). Areas of variability concentrated in helices 10, 18, and 37 and included variants with short intervening sequences in helix 10. Most of the substitutions showed compensatory mutations suggesting ancient sequence divergence generated by lateral gene transfer.

This study was carried out seasonally, throughout a year, to evaluate the filtering activity on bacteria of Mytilus galloprovincialis. Six microbiological parameters were researched in the water and mussels samples collected along the coastal area of the Northern Ionian Sea in three stations, S. Vito, Lido Gandoli and Lido Silvana. We detected the densities of culturable heterotrophic bacteria by spread plate on Marine Agar, total culturable bacteria at 37 degrees C on Plate Count Agar and vibrios abundance on thiosulphate-citrate-bile-sucrose-salt (TCBS) agar. Total and fecal coliforms as well as fecal streptococci were determined by the Most Probable Number. Bacterial concentrations at 20 and 37 degrees C as well as vibrios concentrations were higher in the mussel samples compared to the corresponding seawater throughout the year. The results obtained could contribute to improve the information relatively either to the natural processes existing between bacteria and mussels or to the risk of human infections related to the consumption of mussels.

A new Vibrio species, Vibrio ponticus, is proposed to accommodate four marine bacteria isolated from sea water, mussels and diseased sea bream (Sparus aurata), at the Mediterranean coast of Spain. Strains are Gram negative, slightly halophilic bacteria that require Na+ ion for growth, oxidase and catalase positive, negative for arginine dihydrolase and ornithine decarboxylase but positive for lysine decarboxylase and indole, and utilize beta-hydroxybutyrate as a sole carbon source. Phylogenetic analysis locate these marine bacteria in the vicinity of the V. fluvialis-V. furnissii clade, sharing with these two species 16S rDNA sequence similarities slightly above 97% (97.1 and 97.3%, respectively). DNA-DNA hybridisation values confirm that the four strains form a genospecies and represent a new species in the genus Vibrio. We propose strain 369T (CECT 5869T, DSM 16217T) as the type strain.

The presence of fecal coliforms or Escherichia coli in baby clams (Chamelea gallina L.) is considered an indicator related to their safety because they can be correlated with the presence of pathogenic bacteria. For this reason the Italian regulation has defined limits for these microorganisms. The presence of these microbial indicators is dependent on various environmental variables. In this work all the variables considered are categorical and, consequently, the traditional approach of predictive microbiology was not applicable. The data were summarized by means of a cross-tabulation and analyzed using the log-linear model technique. This statistical technique is widely used in social and economic studies but only partially developed in food microbiology. The suitability of the log-linear model to analyse microbiological data in relation to environmental variables was evaluated. In particular, the microbiological quality of baby clams harvested in five different areas of the Adriatic Sea coast in Emilia Romagna (Italy) was considered. The influence of the season and geographical origin on microbiological standards was assessed. A logit model was developed to predict the frequencies, depending on geographical origin and season, of samples with concentrations of the indicator organisms below or above the legal standards provided by Italian regulation.

Sixty-two samples of Mytilus galloprovincialis (mussels) harvested from approved shellfish waters in the Adriatic Sea were examined for the presence of Vibrio, Salmonella, Campylobacter, and verocytotoxin producing Escherichia coli. Vibrio spp. were isolated from 48.4% of samples; the species most frequently found were V. alginolyticus (32.2%) and V. vulnificus (17.7%), followed by V. cincinnatiensis (3.2%), V. parahaemolyticus (1.6%), V. fluvialis (1.6%) and V. cholerae non-O1 (1.6%). V. parahaemolyticus resulted negative to Kanagawa-phenomenon and to PCR amplification of tdh gene. V. cholerae resulted negative to PCR amplification of sto gene. No Salmonella, Campylobacter, or E. coli verocytotoxin-producing strains were isolated. The results of this study suggest the potential risk of ingesting raw or undercooked mussels due to the frequent presence of potentially pathogenic Vibrio species.