Experimental evaluation of survival of Vibrio parahaemolyticus in fertilized cold-water sediment.

AIMS: This experimental study focuses on survival and consistence of Vibrio parahaemolyticus in cold-water sediments and how increasing temperature and nutritional availability can affect growth. METHODS AND RESULTS: A pathogenic strain of V. parahaemolyticus was inoculated in seawater microcosms containing bottom sediment. Gradually, during 14 days, the temperature was upregulated from 8 to 21°C. Culturable V. parahaemolyticus was only found in the sediment but declined over time and did not recover even after another 2 days at 37°C. Numbers of culturable bacteria matched the amount found by q-PCR indicating that they did not enter a dormant state, contrary to those in the water layer. After adding decaying phytoplankton as fertilizer to the microcosms of 8 and 21°C for 7 and 14 days, the culturability of the bacteria increased significantly in the sediments at both temperatures and durations of exposure. CONCLUSION: The study showed that V. parahaemolyticus can stay viable in cold-water sediment and growth was stimulated by fertilizers rather than by temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio parahaemolyticus is a common cause of seafood-borne gastroenteritis and is today recognized in connection to increasing ocean temperature. The results indicate that this pathogen should be considered a risk in well-fertilized environments, such as aquacultures, even during cold periods.

Mass spectrometry data from a quantitative analysis of protein expression in gills of immuno-challenged blue mussels (Mytilus edulis).

Here, we provide the dataset associated with our research article on the potential effects of ocean acidification on antimicrobial peptide (AMP) activity in the gills of Mytilus edulis, "Impact of ocean acidification on antimicrobial activity in gills of the blue mussel (Mytilus edulis)" [1]. Blue mussels were stimulated with lipopolysaccharides and samples were collected at different time points post injection. Protein extracts were prepared from the gills, digested using trypsin and a full in-depth proteome investigation was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Protein identification and quantification was performed using the MaxQuant 1.5.1.2 software, "MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification" [2].

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.

Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas.

The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.

The influence of temperature and dose on antibacterial peptide response against lipopolysaccharide in the blue mussel, Mytilus edulis.

Blue mussels (Mytilus edulis) were inoculated with two different doses of lipopolysaccharides (LPS) or phosphate-saline (PS) buffer under different temperature conditions (6 and 20 degrees C). The activity of the antibacterial peptide fraction, purified through reverse phase chromatography from mussel haemolyph, was compared at different time intervals after the inoculation. The activity was determined as the minimal peptide concentration that inhibited growth of the Gram-negative bacteria Escherichia coli D21, by using radial diffusion assay. The antibacterial activity for mussels inoculated with LPS changed over time, both at 6 and 20 degrees C, but those inoculated with PS-buffer did not. The response was enhanced within a time course of 3h. The higher temperature did increase the inhibitory activity and made the mussel respond at an earlier stage, in comparison to that at 6 degrees C. At 20 degrees C, mussels inoculated with 10 microg of LPS responded faster than those inoculated with 0.1 microg of LPS. In addition, cytotoxic effects of LPS on mussel haemocytes were investigated in vitro, using a colorimetric assay. The survival index (SI%) for haemocytes decreased with 76% at 6 degrees C but increased with 100% at 20 degrees C, irrespective of the dose of LPS. This indicated that LPS did not influence the viability of the haemocytes but the high temperature increased their metabolic state. Likely, antibacterial response was provoked by LPS in a dose-dependent manner and favoured by higher metabolic state of the haemocytes, elicited at higher temperature. These results provide important considerations for variability in the internal defence of mussels and consequently, also the retention of viable human pathogens in mussels.

Factors influencing bactericidal activity of blue mussel (Mytilus edulis) haemocytes against Salmonella typhimurium.

This study showed that in vitro survival of Salmonella typhimurium, after exposure to haemocytes of Mytilus edulis, was significantly affected by the lipopolysaccharides (LPS) structures expressed on the cell surface of the bacteria. Survival seemed to be affected by the surrounding temperature as well. Mussel haemocytes were in vitro exposed to mutants of S. typhimurium, expressing differences in O -antigen polysaccharide chains and core sugars of LPS on their cell surface. Surviving cells of the mutants were determined after incubation with the haemocytes at different temperatures, using a colorimetric assay. In addition, a complementary study on clearance of these mutants, inoculated into the adductor muscle of mussels, was performed at 6 and 20 degrees C. It was concluded that the survival index (SI%) measured in vitro for the mutant with complete LPS was significantly lower at 6 degrees C (c.15%) compared to that at 14 and 20 degrees C (c.70%). SI% for the other mutants was c.35-45% and was not affected by temperature. The in vivo study at 20 degrees C showed that during the first 24h, the clearance rate for the mutants with complete LPS was significantly higher than for the others. Thereafter all mutants, with exception for the most deficient, started to increase in numbers and caused death to the mussels. At 6 degrees C the mutants were slowly reduced and after 17 days, viable cells of the mutant with complete LPS were still detectable in the haemolymph. The study indicated that the mussel haemocytes responded in relation to the LPS of the mutants. However, more intact LPS also seemed to protect the bacteria from being killed. The higher temperatures favoured the growth of the mutants that managed to resist the haemocyte defence. Cell surface properties and temperature seem to affect the survival of bacteria in mussels, which consequently can affect risk assessments in regard to public health.

Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom.

Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.