Molecular confirmation of pearl formation in arctic mussels (Mytilus edulis) caused by Gymnophallus bursicola (Odhner 1900) metacercariae.

In recent field studies, suspected gymnophallid metacercariae were histologically located in the mantle of mussels from the Norwegian Sea. Mussels from the sites in which that infection was detected also presented abnormally high pearl numbers. It has been previously described that gymnophallid metacercariae could cause pearl formation processes in mussels, as a host reaction to encapsulate these metacercariae. Given the pathological host reaction these parasites elicit, a study was performed to identify gymnophallid metacercariae found in mussels collected from Tromsø at morphological and molecular level and to assess, by the use of molecular tools, the relationship between the parasite and the biological material inside the pearls. As a result, Gymnophallus bursicola metacercariae infecting Norwegian Mytilus edulis were identified according to morphological characters, along with the first 18S rDNA and COI sequences for this trematode species. In addition, parasite DNA from the core of the pearls was extracted and amplified for the first time, confirming the parasitological origin of these pearls. This procedure could allow identifying different parasitic organisms responsible for the generation of pearls in bivalves.

Thermal processing of live bivalve molluscs for controlling viruses: On the need for a risk-based design.

Norovirus (NoV) and Hepatitis A virus (HAV) are the most important viral hazards associated with human illness following consumption of contaminated bivalve molluscs. The effectiveness of the current EU criteria for heat processing of bivalve molluscs (i.e. raising the temperature of the internal mollusc flesh to at least 90°C for a minimum of 90 seconds) was evaluated using predictive microbiology. A HAV thermal inactivation model was developed based on literature data in mollusc matrices during isothermal heat treatment. Application of the developed model demonstrated that the 90°C-90 s requirement may lead to significantly different virus inactivation depending on the commercial process design. This shows the need for the establishment of a Performance Criterion for bivalve molluscs heat processing which will assure a common specified level of consumer protection. A risk-based approach is described that allows for an effective processing design providing a more transparent and objective relation between the thermal processing targets and public health. Model simulations demonstrate that the F-value is a more appropriate Process Criterion than a single time-temperature combination since it enables the food business operators to design a process that is compliant with the safety requirements while at the same time achieving a desired product quality.

Immune state correlates with histopathological level and reveals molluscan health in populations of Modiolus kurilensis by integral health index (IHI).

Quantitative analysis of the histopathological and immune parameters of bivalve Modiolus kurilensis collected from water areas with different level of ecotoxicological stress was performed. Significant differences between samples from polluted and non-polluted sites were revealed for total haemocyte count; percentage of agranulocytes; size and internal complexity of agranulocytes and granulocytes; phagocytic activity; percentage of NBT-positive cells; hemolytic activity and plasma protein concentration; percentage of the optical density of haemolymph major polypeptide bands at 55 kDa, 78 kDa, and 124 kDa; concretion coverage area in the kidney tubules; thickness of the tubular basement membrane; nephrocyte shape; and karyopyknosis of the kidneys; and hypervacuolisation; necrosis; karyopyknosis; haemocyte infiltration; fibrosis; and invasion of the digestive gland. Analysis of the global histopathological condition index based on the weighted indices also revealed that both the digestive gland and kidneys showed significantly greater histopathological changes in the bivalves collected from polluted water. Bivalve histopathology is an established tool in aquatic toxicology. However, it reflects a morphological picture of change, which, as a rule, can be clearly recorded only at the later stages of pathology, and in some cases, indicates an adaptation to stressors within the physiological norm. In this respect, a promising and highly sensitive biomarker of the functional state of bivalves, in terms of norm and pathology as well as their habitat, is the evaluation of immune status in combination with morphological changes. However, the use of different methods and scales of assessment and the diagnosis of biomarkers, characterised by different profiles of the stress response, makes it difficult to compare the results of different studies. We propose a reliable and powerful system for assessing the physiological state of bivalve molluscs, expressed in the integral health index (IHI) and based on the standardisation of the numerical values for all parameters that have significant differences between animals collected from impacted and non-impacted water areas. In our study, IHI calculated in three variants (for histopathological parameters, for immunological parameters, and in combination) showed the most significant differences in each of the cases, but the strongest difference (-4.07) was in calculating the total IHI, which included both the immune and histopathological parameters (p = 0.00005).

Preliminary Results on the Evaluation of the Occurrence of Tetrodotoxin Associated to Marine Vibrio spp. in Bivalves from the Galician Rias (Northwest of Spain).

Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio, in particular Vibrio parahaemolyticus. This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias.

Nutritional composition and total collagen content of two commercially important edible bivalve molluscs from the Sea of Japan coast.

The study aimed to evaluate the chemical composition and nutraceutical potential of two commercially significant edible bivalve mollusc species (Anadara broughtonii and Mactra chinensis). The edible parts (motor muscle, mantle and adductor) of these molluscs were analyzed for their proximate composition, collagen content, amino acid profile, chemical score and elemental constituents. Both molluscs had low fat content (2.43-6.91 g/100 g dry weight), and protein (55.36-68.01 g/100 g dry weight) and carbohydrates (11.36-20.37 g/100 g dry weight) were their main components. Total collagen content of the edible bivalve molluscs varied from 30.5 to 39 mg/g wet weight, accounting for approximately half of their total protein content. Among amino acids, glycine, glutamate, aspartic acid, alanine, leucine, lysine and arginine were present at high levels in the edible parts of both bivalve molluscs, while the major elements present were sodium, potassium, magnesium, calcium, iron, zinc and nickel. Having high-quality protein content, edible bivalve molluscs could be excellent sources of nutritive ingredients and, after further study, may find applications in nutricosmetics and functional foods.

Low prevalence of Aichi virus in molluscan shellfish samples from Galicia (NW Spain).

AIMS: The aim of this study was to detect and quantify Aichi virus (AiV) in shellfish from three estuaries in Galicia, the main producer of molluscs in Europe. METHODS AND RESULTS: A total of 249 shellfish samples were analysed using a reverse transcription-quantitative PCR procedure. AiV was detected in 15 of 249 (6·02%) samples. Ría de Ares-Betanzos showed the highest prevalence (11·1%), followed by Ría do Burgo (3·7%) and Ría de Vigo, (2·56%). AiV quantifications ranged from nonquantifiable (under the limit of quantification of the method) to 6·9 × 103 RNAc per g DT, with a mean value of 1·9 × 102 RNAc per g DT. CONCLUSION: Results obtained indicated that the prevalence of this enteric virus in the studied area is considerably lower than those of other enteric viruses, such as Norovirus, Sapovirus, HAV or HEV. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that detects the presence of AiV in shellfish from authorized harvesting areas in Spain. Further studies with clinical samples are needed to determine the potential risk of AiV for human health in Galicia.

Detection of Tetrodotoxin Shellfish Poisoning (TSP) Toxins and Causative Factors in Bivalve Molluscs from the UK.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostreagigas), native oysters (Ostreaedulis) common mussels (Mytilusedulis) and hard clams (Mercenariamercenaria), but not found in cockles (Cerastodermaedule), razors (Ensis species) or scallops (Pectenmaximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrumcordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Species identification of bivalve molluscs by pyrosequencing.

BACKGROUND: The increase in seafood consumption and the presence of different species of bivalves on the global markets has given rise to several commercial frauds based on species substitution. To prevent and detect wilful or unintentional frauds, reliable and rapid techniques are required to identify seafood species in different products. In the present work, a pyrosequencing-based technology has been used for the molecular identification of bivalve species. RESULTS: Processed and unprocessed samples of 15 species belonging to the bivalve families Pectinidae, Mytilidae, Donacidae, Ostreidae, Pharide and Veneridae were analysed and correctly identified by the developed pyrosequencing-based method according to the homology between query sequences of the 16S ribosomal RNA (16S rRNA) and cytochrome c oxidase I (COI) genes and their correspondent reference libraries. This technique exhibits great potential in automated and high-throughput processing systems, allowing the simultaneous analysis of 96 samples in shorter execution and turnaround times. CONCLUSIONS: The correct identification of all the species shows how useful this technique may prove to differentiate species from different products, providing an alternative, simple, rapid and economical tool to detect seafood substitution frauds. © 2016 Society of Chemical Industry.

Permanent culture and parasitic impact of the microalga Coccomyxa parasitica, isolated from horse mussel Modiolus kurilensis.

Animals with deformed shells and microalgal invasion have been identified in the natural population of the horse mussel species Modiolus kurilensis of Peter the Great Bay in the Sea of Japan. The haemolymph is initially infested with algae, followed by the rectum, siphons, mantles and gonads located in the posterior body areas. Mantles, which are primarily exposed to light, are major depots for algae. The microscopic analysis of algal cells has revealed the absence of flagella and pyrenoids, the presence of single chloroplast, and reproduction by autosporulation, with dispores prevailing over tetraspores. These results, together with the nearly complete sequence analysis of small subunit (SSU) 18S rDNA (1728bp), have confirmed that these cells are Coccomyxa parasitica. A newly developed method of isolating microalgae from mollusk tissues has facilitated the continuous pure - probably axenic - culture of C. parasitica, thereby providing a description of the time course of each life stage. Histological analyses have revealed significant haemocyte infiltration into the mantles, gonads, kidneys and digestive gland tissues infested with microalgae and the gill tissues, in which the intruder was not identified. Algal encapsulation with major focal areas of fibrosis and amorphic necrosis has been revealed in these infested organs. The spaces between the gonad follicles and digestive gland tubules were significantly widened as these areas were filled with a mass of algae and phagocytic haemocytes, showing acini with a thickened basement membrane. The mantles and kidneys of Modiolus displayed significant morphological deviations of different cells in epithelial, connective and muscle tissues, resulting in the dysfunction of the infested organs. Therefore, C. parasitica, which reproduces in the culture, regardless of the host, is a facultative parasite, causing major pathological alterations, such as anomalous histomorphological patterns and infested organ dysfunctions.

Occurrence of potentially pathogenic arcobacters in shellfish.

Considering that several recent cases of human gastroenteritis have been associated with species from the Arcobacter genus, and that few data are currently available about the occurrence of this genus in Italian shellfish, the aim of the present study was to evaluate the occurrence of Arcobacter spp. and the presence of virulence-associated genes. The approach consisted of cultural and biomolecular (multiplex-PCR and 16S-RFLP) methods identifying isolates, followed by PCR assays aimed at the cadF, ciaB, cjl349, irgA, hecA putative virulence genes. Arcobacter spp. was detected in 16/70 (22.8%) shellfish samples. Specifically, Arcobacter spp. was highlighted in 10/42 (23.8%) mussel and in 6/28 (21.4%) clam samples. Subsequently, biomolecular assays revealed Arcobacter butzleri in 12/16 (75%) and Arcobacter cryaerophilus 1B in 4/16 (25%) isolates. PCRs aimed at the five putative virulence genes demonstrated widespread distribution of these genes among Arcobacter isolates and some differences from the results published by other authors. Our research provides more information regarding the health risks associated with the consumption of raw bivalve molluscs and underlines the need to implement an adequate control plan by performing intensive and continuous monitoring in order to guarantee human health.

The known and unknown sources of reactive oxygen and nitrogen species in haemocytes of marine bivalve molluscs.

Reactive oxygen and nitrogen species (ROS and RNS) are naturally produced in all cells and organisms. Modifications of standard conditions alter reactive species generation and may result in oxidative stress. Because of the degradation of marine ecosystems, massive aquaculture productions, global change and pathogenic infections, oxidative stress is highly prevalent in marine bivalve molluscs. Haemocytes of bivalve molluscs produce ROS and RNS as part of their basal metabolism as well as in response to endogenous and exogenous stimuli. However, sources and pathways of reactive species production are currently poorly deciphered in marine bivalves, potentially leading to misinterpretations. Although sources and pathways of ROS and RNS productions are highly conserved between vertebrates and invertebrates, some uncommon pathways seem to only exist in marine bivalves. To understand the biology and pathobiology of ROS and RNS in haemocytes of marine bivalves, it is necessary to characterise their sources and pathways of production. The aims of the present review are to discuss the currently known and unknown intracellular sources of reactive oxygen and nitrogen species in marine bivalve molluscs, in light of terrestrial vertebrates, and to expose principal pitfalls usually encountered.

The Occurrence of Microplastics in Donax trunculus (Mollusca: Bivalvia) Collected along the Tuscany Coast (Mediterranean Sea).

Microplastics (MPs) (0.1 µm-5 mm particles) have been documented in oceans and seas. Bivalve molluscs (BMs) can accumulate MPs and transfer to humans through the food chain. BMs (especially mussels) are used to assess MPs' contamination, but the genus Donax has not been thoroughly investigated. The aim of this study was to detect and characterize MPs in D. trunculus specimens collected along the Tuscan coast (Italy), and to assess the potential risk for consumers. The samples (~10 g of tissue and intervalval liquid from 35 specimens) were digested using a solution of 10% KOH, subjected to NaCl density separation, and filtered through 5 µm pore-size filters. All items were morphologically classified and measured, and their mean abundance (MA) was calculated. Furthermore, 20% of them were analyzed by Raman spectroscopy and, based on the obtained results, the MA was recalculated (corrected MA) and the annual human exposure was estimated. In the 39 samples analyzed, 85 items fibers (n = 45; 52.94%) and fragments (n = 40; 47.06%) were found. The MA was 0.23 ± 0.17 items/grww. Additionally, 83.33% of the items were confirmed as MPs (polyethylene and polyethylene terephthalate). Based on the correct MA (0.18 MPs/grww), D. trunculus consumers could be exposed to 19.2 MPs/per capita/year. The health risk level of MPs was classified as level III (moderate).

Per- and polyfluoroalkyl substances (PFASs) in bivalve molluscs from Shandong Province, China: Occurrence, distribution, and implications for human consumption.

We examined the occurrence and levels of 19 legacy and emerging per- and polyfluoroalkyl substances (PFASs) in 7 species of marine bivalve molluscs collected from four coastal cities of Shandong Province, China. Perfluorooctanoic acid (PFOA) was the most prevalent component, accounting for 68.1 % of total PFASs. The total PFASs in bivalve molluscs ranged from 0.86 to 6.55 ng/g wet weight, with the highest concentration found in Meretrix meretrix L. The concentration of total PFASs in bivalve molluscs showed the following trend: clams > scallops > oysters > mussels. Estimation on the human intake of PFASs from consumption of bivalve molluscs resulted in hazard ratios (HR) ranging from 0.12 to 6.40. Five of the seven species had HR >1, indicating high exposure risks associated with PFASs. Therefore, the occurrence of PFASs in marine biota is particularly concerning and further investigations on the sources of PFASs in Shandong are warranted.

Polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls in bivalve molluscs. Risk to Polish consumers?

Introduction: Concentrations of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) were investigated in six species of bivalve mollusc purchased on the Polish retail market. The risk to consumers was calculated as a percentage of the tolerable weekly intake (TWI) (2 pg World Health Organization toxic equivalent (WHO-TEQ) kg-1 body weight (b.w.)). Material and Methods: Altogether 32 samples were analysed using an isotope dilution technique with high resolution gas chromatography coupled with high resolution mass spectrometry. Results: Low levels of all analysed compounds were found. The range of PCDD/Fs was 0.08-0.37 pg WHO-TEQ g-1 of wet weight (w.w.) and 0.04-0.41 pg WHO-TEQ g-1 w.w. for DL-PCBs. The highest concentrations of all analysed compounds were found in Pacific oysters, at 0.30 pg WHO-TEQ g-1 w.w. for the sum of PCDD/Fs and 0.19 pg WHO-TEQ g-1 for the sum of DL-PCBs. These concentrations were 2-4 times higher than those detected in the other analysed mollusc species. Different species-dependent congener profiles were observed for PCDD/F concentrations, while PCB congener concentration profiles were species independent. The risk to consumers was assessed relating theoretical intakes of PCDD/Fs and DL-PCBs per 25, 50 and 100 g of consumption of molluscs per week to the TWI. Conclusion: Taking into account the low consumption of molluscs in Poland and low concentrations of analysed compounds, neither adults nor children are likely to exceed the TWI by ingestion of food in this category.

Snapshot of resistome, virulome and mobilome in aquaculture.

Aquaculture environments can be hotspots for resistance genes through the surrounding environment. Our objective was to study the resistome, virulome and mobilome of Gram-negative bacteria isolated in seabream and bivalve molluscs, using a WGS approach. Sixty-six Gram-negative strains (Aeromonadaceae, Enterobacteriaceae, Hafniaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Vibrionaceae, and Yersiniaceae families) were selected for genomic characterization. The species and MLST were determined, and antibiotic/disinfectants/heavy metals resistance genes, virulence determinants, MGE, and pathogenicity to humans were investigated. Our study revealed new sequence-types (e.g. Aeromonas spp. ST879, ST880, ST881, ST882, ST883, ST887, ST888; Shewanella spp. ST40, ST57, ST58, ST60, ST61, ST62; Vibrio spp. ST206, ST205). >140 different genes were identified in the resistome of seabream and bivalve molluscs, encompassing genes associated with ß-lactams, tetracyclines, aminoglycosides, quinolones, sulfonamides, trimethoprim, phenicols, macrolides and fosfomycin resistance. Disinfectant resistance genes qacE-type, sitABCD-type and formA-type were found. Heavy metals resistance genes mdt, acr and sil stood out as the most frequent. Most resistance genes were associated with antibiotics/disinfectants/heavy metals commonly used in aquaculture settings. We also identified 25 different genes related with increased virulence, namely associated with adherence, colonization, toxins production, red blood cell lysis, iron metabolism, escape from the immune system of the host. Furthermore, 74.2 % of the strains analysed were considered pathogenic to humans. We investigated the genetic environment of several antibiotic resistance genes, including blaTEM-1B, blaFOX-18, aph(3″)-Ib, dfrA-type, aadA1, catA1-type, tet(A)/(E), qnrB19 and sul1/2. Our analysis also focused on identifying MGE in proximity to these genes (e.g. IntI1, plasmids and TnAs), which could potentially facilitate the spread of resistance among bacteria across different environments. This study provides a comprehensive examination of the diversity of resistance genes that can be transferred to both humans and the environment, with the recognition that aquaculture and the broader environment play crucial roles as intermediaries within this complex transmission network.

Application of residuals from purification of bivalve molluscs in Galician to facilitate marine ecosystem resiliency through artificial reefs with shells - One generation.

The seas and oceans of the planet provide a wide range of essential resources. However, marine ecosystems are undergoing severe degradation due to the unsustainable exploitation and consumption patterns of the linear economy. On the other hand, many economic activities linked to the sea generate a large amount of waste, leading to negative impacts, such as the cost of treating or disposing of this waste. A case in point is bivalve mollusc production: a purification process is needed to avoid the risk of diseases through faecal contamination. The present work proposes an innovative procedure to convert this waste, calcium carbonate as calcite and aragonite allotropic types, into by-products. These by-products can be used to manufacture green artificial reefs, partially replacing concrete aggregates with a sustainable alternative to the geological sources of CaCO3. By installing these reefs, marine ecosystems could be created in a sustainable way and an innovative approach based on the circular economy could be taken towards protecting them. To this end, different concrete mixtures with bivalve shells are proposed. Although this study had been carried out for Galicia (NW Spain), the methodology followed could also be valid for other regions. A physicochemical characterisation of the waste from purifying the bivalves, including oysters, mussels, clams and scallops, was performed. Statistical and multi-criteria analyses were done in order to select the best dosage. Both have provided justification for using a mixture of shells with a predominance of calcite (oyster, scallop) instead of shells with a predominance of aragonite. The multi-criteria analysis served to identify the two best alternatives with dosages in which the medium aggregates were substituted with shells mainly from oysters, with a predominance of calcite. Finally, the statistical analysis played a role in estimating the compressive strength and water absorption of each mixture from the design parameter values.

Comparison and quantification of estrogen receptor-mediated responsiveness to endocrine disruptors in bivalves by using complementary model and a novel yeast assay approach.

Endocrine disrupting chemicals (EDCs) in estuaries and coastal habitats have been widely detected over the world and caused global concern. Bivalves have been shown to be vulnerable to endocrine disruption. However, estrogen receptors (ERs) sensitivity to steroids and EDCs has long been considered to be restricted to vertebrates. In the present study, a computational simulation docking model was applied to qualitatively predict the binding behavior of two bivalve ERs to estradiol and compared the docking activity with zebra fish ERa. A novel reconstituted yeast system was constructed by using transcriptional activator GAL-4 consists of ER-expressing plasmid and ERE (estrogen responsive element)-containing plasmid. The assays showed that bivalve ER specifically activate transcription in response to tested steroids and EDCs, but the activation ability is weaker compared to zebra fish ERa. The results corroborate the presence of an active ER in bivalve molluscs and provide a promising tool for screening of marine environmental pollutants active in disturbing ERs of bivalves, as well as understanding the underlying mechanism across taxonomic groups and phyla.

Prevalence and Antimicrobial Resistance of Bacterial Foodborne Pathogens Isolated from Raw Bivalve Molluscs Subjected to Consumption in Poland during a Ten-Year Period.

The aim of this study was to evaluate the microbiological contamination of raw bivalve molluscan shellfish (BMS) available on the Polish market and determinate the antimicrobial resistance of the obtained isolates. A total of 1000 mollusc samples were tested for the presence of Salmonella spp., L. monocytogenes, V. parahaemolyticus, and S. aureus using the ISO standard methods. Additionally, the bacterial isolates' susceptibility to antimicrobials was determined using the minimum inhibitory concentration (MIC) method. The obtained results showed that Salmonella spp. was detected in 31 (3.1%) samples, and 51.6% of the bacterial isolates were classified as Salmonella Typhimurium. A total of 74.2% of the Salmonella isolates were sensitive to all antimicrobial agents, whereas three isolates were multiresistant. L. monocytogenes was isolated from 18 (1.8%) BMS, and the isolates belonged to serogroups IIa, IIb, and IVb. Most of them were resistant to ceftriaxone (77.8%) and oxacillin (55.6%). V. parahaemolyticus was present in 24.2% BMS. These isolates were mainly resistant to ampicillin (77.3%) and streptomycin (64.0%). Moreover, 15.2% of the bivalve molluscs were contaminated with S. aureus. Most isolates belonging to this species were resistant to penicillin (84.9%). A total of 60 (6.0%) bivalve molluscs were contaminated with more than one pathogen simultaneously. In addition, the tested bacteria were more likely to be identified during the warmer period (53.9%) compared to the samples analyzed in colder months (35.7%). The obtained results indicate that raw bivalve molluscs from the Polish market are frequently contaminated with bacterial foodborne pathogens, which may be resistant to antimicrobials.

First Data on Campylobacter spp. Presence in Shellfish in Croatia.

This study aimed to assess the presence of thermotolerant Campylobacter spp., as one of the most important foodborne zoonotic pathogens, in three shellfish species: mussels (Mytilus galloprovincialis), oysters (Ostrea edulis) and queen scallops (Aequipecten opercularis). The samples were collected from nine locations in the Istrian aquatory, Croatia. Isolation of Campylobacter was done according to standard ISO method, and species were identified using multiplex PCR. Isolates identified as C. jejuni and C. lari were genotyped using multilocus sequence typing (MLST) to determine the potential source of contamination. Among 108 examined samples of bivalve molluscs, mussels dominated and were the only ones found positive for the presence of Campylobacter (25.6%). In total, 19 C. lari and 1 C. jejuni strains were isolated. C. lari isolates found in this study belong to 13 sequence types (STs), and 9 of them are newly described in this paper. Two out of the four previously described C. lari STs that were found in this study were previously found in human stool. The only C. jejuni isolate was found to be sequence type 1268, which belongs to ST-1275 clonal complex that is almost exclusively found in seabirds and can sporadically cause infection in humans. Regarding the obtained results, introducing surveillance of thermotolerant Campylobacter in shellfish in the Republic of Croatia is advised as an improvement for public health safety.

Insights into the genetic diversity, antibiotic resistance and pathogenic potential of Klebsiella pneumoniae from the Norwegian marine environment using whole-genome analysis.

Klebsiella pneumoniae (Kp) can cause hospital- and community acquired infections. Although, Kp is widespread in the environment, very little is known about the genetic diversity and pathogenicity of Kp from the marine environment. The aim of our study was to understand the genetic diversity, resistome and pathogenic potential of 87 Kp isolates from the Norwegian marine environment, using whole-genome sequencing. We identified 50 sequence types, including globally disseminated sequence types associated with multidrug resistance or hypervirulence. Ten isolates carried the yersiniabactin loci. Acquired antibiotic resistance genes were identified in six Kp isolates. Heavy metal resistance genes were widespread among the isolates, with 71% carrying genes encoding resistance to copper, silver, arsenic, nickel and/or mercury. Co-occurrence of antibiotic resistance genes and heavy metal resistance genes was seen in five Kp isolates. Phylogenetic analysis revealed a close genetic relationship between Kp 2016-1200 ST25 isolated from blue mussels (Mytilus edulis) and a clinical isolate reported in Germany. To the best of our knowledge, this study provides the first comprehensive account of genetic diversity among Kp from the marine environment. Our study reveals high diversity of Kp in the Norwegian marine environment and seafood, including globally disseminated pathogenic sequence types carrying clinically relevant antibiotic resistance genes and virulence factors, as well as several heavy metal resistance genes.