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Seven citrus essential oils (CEOs) and fourterpenes were tested againstelevenbacterial pathogenic strains isolated from food to evaluate the antimicrobial effects. All CEOs presented antibacterial activities. In particular, CEOs were very active against StaphylococcusEpidermidis, which was found to be the most sensitive toward all oils, showing high growth inhibition.

Prokaryotic microorganisms are at the base of marine food web and are sensitive to climate changes. In the Mediterranean Sea, an important link between surface water warming and microbial activities is hypothesized to exist on a seasonal and interannual scale, with high amounts of organic matter utilized by prokaryotes and re-cycled in warm periods. To test this hypothesis, from 1998 to 2013, the prokaryotic abundance and microbial activities (enzymatic activities, prokaryotic heterotrophic production and respiration), together with trophic parameters (chlorophyll, particulate organic carbon and nitrogen), were measured in the euphotic layer of the Ionian Sea (Central Mediterranean). A statistical simultaneous analysis of the environmental and prokaryotic data was performed with the aim of depicting the evolution of microbial activities and their implications in the biogeochemical cycles. Results showed that despite the large spatial and temporal variability, Temperature was the main factor affecting the variability of microbial abundance and metabolism with significant differences on both seasonal and interannual scales. Particularly interesting is the increase of production in summer and of the respiration in winter is very interesting. In spite of low levels of chlorophyll and organic carbon, an increase of prokaryotic heterotrophic production, coupled with that of alkaline phosphatase, was found. As a consequence, the increase of prokaryotic biomass was bottom-up sustained. Most of prokaryotic activities and respiration rates were significantly correlated to organic carbon pools; the process of organic matter decomposition was frequently enhanced at higher temperature, and both prokaryotic production and growth efficiency increased in summer. In view of climate changes, the rise in temperature was associated to the increase of heterotrophic activities and this trend can have a predictive value to describe the microbial processes in the Central Mediterranean Sea.

Several studies have indicated that plants are good sources of antimicrobial agents and pharmaceuticals. The present study is focused to evaluate the antimicrobial po-tential of 11 citrus essential oils (EOs) and terpenes (T), against 18 bacterial pathogenic strains and 4 Candida sp.strains. Volatile compounds present in the citrus Eos were determined by a gaschromatography analysis, allowing the identification of 79 components. Results highlights that lemon,bergamot oil and terpene were effective against more than 54.5% of tested bacteria. Candida spp. Strains resulted very sensitive to all terpenes and EOs. Gram negative bacteria showed lower sensitivity than Gram positive ones. Among Gram negative, fish pathogens were sensitive tooils, so we suggested their use as possible alternatives in aquaculture farms to prevent bacterial fish diseases. Moreover, results suggested that citrus oils could be a useful tool in the food industry to prolong the shelf-life.

The aim of this study was the development of antimicrobial polyvinylchloride (PVC) blends loaded with 0.1–10% (w/w) of the ILs 1-hexadecyl-3-methylimidazolium 1,3-dimethyl 5-sulfoisophthalate (HdmimDMSIP) and 1-octyloximethyl-3-methylimidazolium hexafluorophosphate (OOMmimPF6). The synthetized ILs were characterized by 1HNMR, MALDI-TOF, DSC and TGA. PVC/ILs films were obtained by solvent casting.Thermal and mechanical properties (tensile stress TS and elongation at break EB), morphology by SEM, surface wettability, antimicrobial activity, cytotoxicity and ILs release in sterile water from PVC/ILs film blends were determined. Results demonstrated that the presence of both ILs in PVC formulation slightly affected thermal and mechanical properties of blends. The loading of both ILs into PVC matrix made PVC/ILs films hydrophilic, especially at the highest concentration of HdmimDMSIP. The PVC/ILs blends displayed antibacterial activity up to ILs lowest concentrations (0.1–0.5%). The inhibition of Escherichia coli growth was lower than that showed toward Staphylococcus epidermidis. The addition of 10% ILs concentration resulted excessive as demonstrated by accumulation of ILs on film surfaces (SEM) and ILs high release from PVC/ILs blends during the first day of water immersion. Biocompatibility studies highlighted that the addition of low amounts of both ILs into PVC matrix is not cytotoxic for mouse fibroblast cells (L929), supporting their potential use for biomedical porposes.

In most of the aquatic ecosystems, microorganisms are major players in the biogeochemical and nutrients cycles (Carbon Nitrogen, Phosphorus), through their enzymatic activities (leucine aminopeptidase, alkaline phosphatase and beta-glucosidase) on organic polymers such as polypeptides, organophosphate esters and polysaccharides, respectively. The small monomers released by decomposition are metabolised by microbes, supporting their growth. Most of the extracellular enzymes are adaptative and their synthesis and activity is strongly affected by environmental factors, consequently the relative importance of leucine aminopeptidase, alkaline phosphatase and beta-glucosidase reflects differences in the composition of organic matter and assume a different meaning. Since more than two decades, at the CNR the influence of climate changes, seasonal variability, depth and coastal input on the patterns of enzymatic activities in the Mediterranean Sea have been studied. Its particular characteristics of a semi-closed basin, high summer evaporation and the occurrence of important water dynamics, make this ecosystem particularly suitable as a model site for climate changes-related observations. The present paper reviews the current information of environmental changes on extracellular enzymatic activity obtained in the Mediterranean areas with the aim of evaluating the effects of environmental changes on the microbial activities. The obtained results revealed significant variations in the rates of hydrolytic activities in relation to space and time, with the highest levels generally found in the epipelagic layer (0–100m) and in coastal zones during warm periods. In the Central Mediterranean Sea their relationship with temperature changes was demonstrated. Spatial variations in the relative enzyme activities also suggested a modulation in the metabolic profiles of the prokaryotic communities, with biogeochemical implications in nutrient regeneration. Long term studies on microbial activity and abundances in relation with rising temperatures can have a predictive value to describe the evolutionary scenario of microbial processes and the response of microbial metabolism to climate changes in the Mediterranean Sea.

Microbial contamination represents a severe problem in many sectors, including medical, industrial, food and domestic fields. Moreover, the increasingly widespread of antibiotic resistance is becoming a great concern for public health, in particular for hospitals. In order to avoid the excessive use of antibiotics, researches on alternative biocides are increasing. Among alternatives, ionic liquids (ILs), low temperature molten salts, and in particular imidazolium ILs have been studied for a wide range of biological applications to produce surfactants, oxygen transport membranes, scaffold for biomimetic applications, plasticizers, antimicrobial and anti-inflammatory agents [1]. The aim of this study was the realization of antimicrobial compounds based on Polyvinyl Chloride (PVC), Styrene-Butylene-Ethylene-Styrene (SEBS) and Low-Density Polyethylene (LDPE), loaded with different concentrations (1, 5 and 10%) of the ionic liquid (IL) 1-hexadecyl-3-methyl imidazolium 1,3-dimethyl 5-sulfoisophthalate (Hdmim DMSIP). The antibacterial activity of compounds was evaluated against two human pathogenic bacteria, Staphylococcus epidermidis and Escherichia coli. The IL was synthetized according to Colonna et al. [2], with slight modifications, and characterized by 1HNMR, MALDI TOF, TGA and DSC. The antimicrobial compounds were obtained by mixing polymer matrices with the IL in a Brabender mixer, adopting suitable operative conditions for each polymer. Film compounds were obtained by compression molding at 150 °C for PVC/IL, whereas those of LDPE/IL and SEBS/IL were realized at 180 °C. The antibacterial activity of film samples were determined by disk diffusion test. Results showed an excellent antimicrobial activity of neat IL vs S. epidermidis, with MIC value of 10 µg/ml, whereas its activity vs E. coli indicated a lower sensitivity of this strain (MIC of 100 µg/ml). The introduction, at different concentrations, of this IL into polymer matrices, highlighted different behaviors depending on structure and physical-chemical properties of the tested polymers, which may facilitate its release and diffusion in TSA. Both PVC/IL and SEBS/IL compounds showed a good antimicrobial activity against S. epidermidis growth at all concentration of IL loaded, whereas the LDPE/IL compound exhibited antibacterial activity against S. epidermidis only at the highest concentrations (5-10%). In all experiments, the antimicrobial activity of the compounds against E. coli growth was lower than that displayed against S. epidermidis, confirming a higher sensitivity of Gram-positive bacteria towards imidazolium based ionic liquids with long alkyl chain.

Planktonic communities hold a relevant role within the EU Marine Strategy Framework Directive (MSFD). In view of reaching the Good Environmental Status of marine ecosystems, within this Directive the phyto- and zooplanktonic communities have received great attention, while Prokaryotes (Bacteria, Archaea, and Cyanobacteria) have been neglected. Indeed, the relevance of microbes (particularly of the faecal pollution indicators) as water quality indicators, the role that microorganisms play within the biogeochemical fluxes and in the whole ecosystem functioning, are all important features that deserve to be focused within the MSFD. The present study aims at reviewing the main issues where prokaryotic variables find useful application as descriptors of environmental status, trying to develop a panel of prokaryotic indicators suitable for the environmental quality assessment. From a survey of scientific literature and on field datasets, prokaryotic abundance and activities (enzymatic hydrolysis, prokaryotic production, and respiratory rates) are here chosen as early warning sentinels to detect changes in environmental quality, using an integrated approach with trophic (chlorophyll-a, particulate and dissolved organic matter) measurements.

As a part of a wider project on fisheries ecology, several biological and environmental parameters were monitored during two oceanographic cruises (BANSIC 2012 and NOVESAR 2013) in the Sicily Channel, which connects the Western and Eastern Mediterranean basins. The prokaryotic abundances and biomass as well as hydrolysis rates on organic matter were investigated in the euphotic layer of a retention area for fish larval stages including anchovy (Engraulis encrasicolus, Linnaeus, 1758) with the aim to investigate the different biogeochemical signatures in two seasonal conditions. The environmental parameters, particulate organic carbon and nitrogen together with heterotrophic production were also measured. Results showed significant increases for most of the studied parameters with increasing temperature during summer. This had effects on the Carbon cycle and recycling of nutrients; in fact total prokaryotic abundance and biomass, as well as carbon hydrolyzed by two enzymes (Leucine aminopeptidase and β-glucosidase), increased significantly during summer. Conversely Alkaline phosphatase activity, Chlorophyll concentration and Oxygen increased during winter. The same environmental parameters affected also the presence of fish eggs. Moreover high percentages of free enzymes (i.e., enzymes not associated with cells) were measured, accounting for percentages variable from 12 to 95 % of the total enzymatic activity, with values generally higher in summer than in winter. In this oligotrophic environment, the prokaryotic biomass was supported by the C hydrolyzed by enzymatic activities. The ratio between the hydrolyzed C and prokaryotic biomass was higher in winter than in summer, indicating that alkaline phosphatase activity contribute to an efficient incorporation of C into biomass in winter.

Ferrocenyl(benzyl) imidazole (FcIm) and two related methyl-imidazolium salts (FcMImI and FcMImPF) were synthesized for their incorporation into plasticized PVC by solvent casting technique. The obtained materials were investigated for their thermal stability and, compared to pure polymer, films containing ionic ferrocene derivatives in 0.5% w/w loading were found slightly more stable. The pure ferrocene compounds showed antibacterial activity against Staphylococcus epidermis, but not against Escherichia coli, with a maximum for FcMImPF salt. After incorporation into PVC polymer, antibacterial activity against S. epidermis was observed (by disk diffusion test) only for PVC/FcMImPF (5% w/w) film, from which a release of 14.6% of the ferrocenilimidazolium cation in aqueous medium was measured after 24 hr.

The Ross Sea, one of the most productive seas of the South Ocean, includes polynyas, marginal ice zones, coastal areas and open sea that contribute differently to biological processes. To identifying responses of plankton compartment to climate change, an oceanographic PNRA campaign (P-ROSE Project) was here carried out in January-February 2017. Two polynyas occurred, a small near the Terra Nova Bay (TNB) and a larger one between the Ross Ice Shelf and Cape Adare. The variability of extracellular enzymatic activity (a proxy of organic matter degradation) in relation to environmental conditions (temperature, salinity and total phytoplankton biomass) was studied along longitudinal and latitudinal transects. Hydrolysis rates of aminopeptidase (LAP), beta-glucosidase (GLU) and alkaline phosphatase (AP) and -for the first time in this area- the corrispective free enzymes (dissolved in waters) were determined. Results showed decreasing trends in the TNB from coastal to off-shore stations and from surface to depth for LAP, that were related positively to temperature (T) and negatively to salinity (S) and depth (p

The abundance, biomass and size-structure of planktonic populations, and the microbial metabolic processes were studied in the Sicily Channel, one of the most important spawning areas in the Mediterranean for anchovy (Engraulis encrasicolus), a pelagic species of commercial interest. Results showed that prokaryotes contribute for the 83% of total carbon biomass. Microphytoplankton abundances and biomasses were dominated by autotrophic nanoflagellates and dinoflagellates (36 identified species) and contribute 11% of total biomass. The microzooplanktonic biomass showed its maximum at the surface or subsurface and its contribution was low (4%). In agreement with the general oligotrophy of the investigated area, the study highlights the prevalence of pico-sized fractions within the whole phytoplankton biomass expressed as chlorophyll content, suggesting the importance of picophytoplankton in sustaining the microbial food web. At the same time, the levels of microbial hydrolytic activities are related to productive processes recycling the organic matter and releasing nutrients (P and N), indicating also an active functioning of ecosystem at low trophic levels. Autotrophic production exceeded oxidation by respiration; at the same time, the co-variation of prokaryotic activities and eggs distribution with temperature in summer was observed. The results obtained confirmed that the area acted as a nursery for small fish and both autotrophic and heterotrophic processes supported by microorganisms were in synergy.

In the Ross Sea, warm Circumpolar Deep Water (CDW) intrudes onto the continental shelf preferably on the western sector and flows into the ice shelf cavities, affecting the ice shelf melting rates. In fact, the intrusion of the warm CDW onto the Antarctic shelf area is one of the recognized drivers of ice shelf melting. Moreover, this water mass is the primary source of heat, salt, nutrients and iron (Fe) onto the Antarctic continental shelves playing an important role on the shelf physical and biological processes. An oceanographic cruise was performed in the framework of the CELEBeR (CDW effects on glacial melting and on bulk of Fe in the Western Ross Sea) project (funded by Italian National Research Programme in Antarctica) in January - February 2017 aboard of R/V Italica, addressed to study microbial community level physiological profiles (by Biolog ecoplate) and extracellular enzymatic activities (EEA) of leucine-aminopeptidase, beta-glucosidase and alkaline phosphatase in the intruding CDW of the Western Ross Sea. The investigated areas covered: a) the Antarctic continental shelf break area near Cape Adare (North Victoria Land); b) the coastal area near the edge of the Mariner and Aviator glaciers; c) the Drygalski Ice Tongue and the nearby Terra Nova Bay polynya. The functional diversity (catabolic potential) was assessed using the Average Well Color Development (AWCD). Moreover, Richness (R) and Shannon-Weaver index (H), analysis of variance (ANOVA) and principal component analysis (PCA) were used to depict the differences of the microbial functional diversity in the studied areas. Differences in potential metabolic pathways were detected in the different sampling areas. The measured enzymatic activity rates suggested the potential ability of the microbial community to decompose proteins, carbohydrates and organic phosphates. Different levels of EEA were measured as a function of the CDW proximity - or not - to the glaciers and the trophism of the investigated area.

Studies in coastal areas of Terra Nova Bay (TNB) highlighted the sudden variability of planktonic biomass with ecological implications on the heterotrophic microbial community and the fate of the organic substance on a seasonal scale. With the aim to monitoring the evolution of the prokaryotic and viral abundance and the hydrolysis of organic matter, in the frame of the CEFA project (Italian PNRA) two fixed stations (Faranda Field - FF; Michaud Pier - MP), were repeatedly sampled. In FF, a sea ice covered station, the prokaryotic and viral abundances were monitored from the 9th of November 2015 to the 19th of January 2016 with Image Analysis (IA) and Cytofluorimetry (FC). Starting from the 4th of January 2016 the potential enzymatic activities on proteins, carbohydrates and organic phosphates were determined. Moreover, the MP station was monitored from the 12th of January to the 8th of February 2016. The prokaryotic abundance in FF station varied in the ranges 0.73-3.38 and 0.26-1.85cellsx105ml-1 by IA and FC, respectively. Cells with high nucleic acid content (HNA) accounted from 32 to 84% of the total FC cells. Virus like particles by FC ranged between 8.08 and 22.3VPLx105ml-1 and V1 group accounted for the 64-94% of the total. The main activity in all samples taken from FF station (under ice) was the phosphatase with the highest values at the beginning of the sampling and showing a decreasing trend when the station was defrosted. This enzyme can be produced in surface water by both bacteria and phytoplankton. The glucosidase activity, showed lower values than phosphatase with a similar trend. The peptidase activities instead increased in the sampling at the end of January in correspondence of the ice absence. In the MP station, the prokaryotic abundance varied in the ranges 1.00-12.3 and 1.03-4.07cellsx105ml-1 by IA and FC, respectively. Cells with high nucleic acid content (HNA) accounted from 47 to 84% of the total FC cells. Virus like particles by FC resulted to be below the detection limits. High variability of enzymatic activities was observed with time in this station. Generally phosphatase prevailed on other activities with the maximum values on the 13rd and 22nd of January; thereafter it decreased with the ice melting. ?-glucosidase, showed peaks on January 16th and from 24th to 29th of January (bay covered by ice), then it decreased. The amino-peptidase showed variable values with peaks on the 12th and 17th of January.

The response of both microbial activities and prokaryotic abundances to environmental variability was studied in a transitional Mediterranean system (Oliveri–Tindari, Italy) during two yearly surveys (1997–'98 and 2005–'06). The total enzymatic (leucine aminopeptidase, β-glucosidase, alkaline phosphatase) and respiratory activity rates as well as of the abundances of total prokaryotes, culturable heterotrophic bacteria, faecal coliforms and enterococci were measured in surface waters of four brackish ponds, together with temperature, salinity, dissolved oxygen, pH, inorganic nutrients, chlorophyll- a and particulate organic carbon and particulate nitrogen determinations. The seasonal and interannual patterns of microbial parameters were investigated in relation to environmental variations. Both the microbial activities and the abundances of culturable heterotrophic bacteria, faecal coliforms and enterococci varied significantly among the ponds, reflecting their haline and trophic variability. Significant seasonal and interannual variations were found in the patterns of microbial activity, but not in total prokaryotic abundance, in response to temperature, dissolved oxygen and trophic changes. Microbial parameters showed a different response to environmental variability in the two examined periods: during 1997–'98, increases in the microbial activity rates and culturable bacterial counts were positively related with the trophic changes, while during 2005–'06 the patterns of leucine aminopeptidase, alkaline phosphatase and respiratory activities and of total prokaryotic and culturable bacteria abundances were uncoupled with those of temperature, salinity, dissolved oxygen, particulate organic carbon and chlorophyll- a . Changes in the relative importance of environmental and trophic variables between the two considered periods explain this discrepancy.

This study examines the effects of temporal changes on microbial parameters in a brackish aquatic ecosystem. To this aim, the abundances of prokaryotes and vibrios together with the rates of enzymatic hydrolysis of proteins by leucine aminopeptidase (LAP), polysaccharides by ß-glucosidase (GLU) and organic phosphates by alkaline phosphatase (AP), heterotrophic prokaryotic production (HPP), respiration (R), were seasonally investigated, during a two years period in the coastal area of Cape Peloro (Messina, Italy), constituted by two brackish lakes (Faro and Ganzirri). In addition, physical and chemical parameters (temperature, salinity, nutrients) and particulate organic carbon and nitrogen (POC, PN) were measured. The influence of multiple factors on prokaryotic abundances and activities was analysed. The results showed that Cape Peloro area is characterised by high seasonal variability of the microbial parameters that is higher than the spatial one. Combined changes in particulate matter and Temperature (T), could explain the variability in vibrios abundance, GLU and R activities in both lakes, indicating a direct stimulation of the warm season on the heterotrophic prokaryotic metabolism. Positive correlations between T (from 13.3 to 29.6°C) and HPP, LAP, AP, POC, PN are also observed in Ganzirri Lake. Moreover the trophic status index and most of the microbial parameters show significant seasonal differences. This study demonstrates that vibrios abundance and microbial activities are responsive to the spatial and seasonal changes of examined area. The combined effect of temperature and trophic conditions on the microbial parameters lead us to suggest their use as potential indicators of the prokaryotic response to climate changes in temperate brackish areas.

Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 ?g dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.

Monitoring of bacterial pathogens is important for marine environmental protection, because the presence of these microorganisms can be a serious risk for human health. For this reason, a portable sensor implemented as an electronic embedded system featuring disposable measurement cells was used to evaluate the ability and sensitivity of detection of Escherichia coli (E. coli) as an indicator of fecal pollution in transitional environments and a water sample added with E. coli (102 CFU/mL) was assayed. The first result obtained from the laboratory experiment seems promising for the determination of E. coli in environmental samples, though further improvements will be needed for the field application of this sensor in marine and brackish waters.

We report on investigations of prokaryotic abundance, biomass, extracellular enzymatic activity, heterotrophic production and respiration in the full water column (~ 1200 m) of a deep convection site (the Southern Adriatic Pit), carried out on six cruises in 2006-2008. Vertical distribution of prokaryotic abundance varied vertically and temporally and ranged from 1.2 to 20.4 x 105 cell ml-1. Cell volumes, generally increased with depth and in the deep layers the lowest and highest mean cell volumes were observed in a period of no convective process (Feb-07) and a period of stratification (Jun-08) following the convection process occurred in Feb-08. Prokaryotic biomass decreased with depth and was related with both the seasonal cycles of organic matter and the hydrological processes. The picophytoplankton ranged in the upper layer (UL) from 0.089 to 10.71 x 104 cell ml-1 and cells were also recorded till 500 m depth in Feb-08. The variations of enzymatic activities in UL as well as leucine- aminopeptidase/ß-glucosidase ratio showed a seasonal trend probably linked with the productive processes of the photic layer. An inverse relation between Alkaline phosphatase (AP) and phosphate concentrations was found (AP = 0.0003 PO4 -1.7714, R2 = 0.333, P < 0.05) indicating that the presence of inorganic phosphorus in deep waters induced a reduction of total AP; such evidence may be related to deep water formation.. Generally cell-specific enzymatic activities increased with depth as did cell-specific carbon dioxide production rates, while cell-specific prokaryotic heterotrophic production had an opposite trend. High values of prokaryotic growth efficiency registered in the deep layers in Nov-06 reflected a supply of preformed C transported within the deep water masses. Overall, in 2007 in which no convective phenomenon was observed, the variability of prokaryotes metabolism was governed by the seasonal cycle of the organic matter, while in Nov-06 and Jun-08 the dynamics of deep water ventilation influenced the trend along the water column of many microbial parameters. The yearly trophic balance of the study site appeared to move towards autotrophy only in UL, whilst in the whole water column, the prokaryotic carbon demand exceeded POC availability rained down from euphotic zone. This mismatch was balanced by the DOC entrapped in the "younger waters" of new formation that alters the normal flux of the biological pump and fuels the deep marine biota in this area of deep water convection.

The distribution of extracellular enzymatic activities (EEA) [leucine aminopeptidase (LAP), s-glucosidase (GLU), alkaline phosphatase (AP)], as well as that of prokaryotic abundance (PA) and biomass (PB), dissolved organic carbon (DOC), particulate organic carbon and particulate total nitrogen (POC, PTN), was determined in the epi-, meso-, and bathypelagic waters of the Mediterranean Sea along a West–East transect and at one Atlantic station located outside the Strait of Gibraltar. This study represents a synoptical evaluation of the microbial metabolism during early summer. Decreasing trends with depth were observed for most of the parameters (PA, PB, AP, DOC, POC, PTN). Significant differences between the western and eastern basins of the Mediterranean Sea were found, displaying higher rates of LAP and GLU and lower C/N ratios more in the eastern than in the western areas. Conversely, in the epipelagic layer, PA and PB were found to be higher in the western than in the eastern basins. PB was significantly related to DOC concentration (all data, n = 145, r = 0.53, P

The Ionian Sea represents a suitable basin for studying the biogeochemical processes mediated by microbial activities. Due to its characteristics of a crossing region between the Western and Eastern Mediterranean Sea, it is one of the most affected sites by changes in water mass composition and dynamics, caused by the Eastern Mediterranean Transient (EMT). To date, relatively few data exist on microbial activities in pelagic areas of the Ionian Sea. From 1998 to 2004 during different research cruises, prokaryotic parameters (abundance, extracellular enzyme activities -leucine aminopeptidase, ß-glucosidase, alkaline phosphatase-, bacterial production and respiration) were measured together with the culturable bacteria and the main physical, chemical and trophic parameters (temperature, salinity, particulated organic matter). The aim of the study was to describe the spatial and temporal variability of microbial activities involved in the carbon and phosphorus cycles, in different layers. Results showed that organic matter transformation mediated by the microbial community displayed a significant increase in Autumn, pointing out the occurrence of significant changes also at meso- and bathy- pelagic depths. Unlike the dark ocean, the bacterial growth efficiency in the Ionian Sea, which increased with depth, seemed to vary its role from a source to a sink of carbon in the epi- and in meso-and bathypelagic layers, respectively. The mechanism of phosphatase regulation showed a weak inverse correlation between specific phosphatase and inorganic P in all seasons except Autumn. It is worth mentioning that the reported results constitute, to the best of our knowledge, one of the available datasets, giving information about microbial activities in the Ionian Sea.

In the framework of the VECTOR DIVCOST Project, a 2-year investigation was started in 2006, with the aim of testing the sensibility of microbial parameters to environmental changes and of assessing whether they can provide information about functional changes in the carbon cycle. The investigation was performed in the surface waters of two small brackish ponds (Ganzirri and Faro), located in the Cape Peloro transitional area (Sicily, Italy). The seasonal changes in both the microbial compartment [bacterioplankton, vibrios, exoenzymatic hydrolysis of proteins and polysaccharides, bacterial secondary production (HBP) and community respiration] and the trophic state of suspended matter [total suspended matter (TSM), particulate organic carbon (POC), particulate organic nitrogen (PON), C ⁄N] were analysed in relation to the hydrological characteristics [temperature, salinity, oxygen, fluorescence, NH4 ,N O 2, NO3 ,P O4]. Despite marked differences in the nutritional input and the diversification in both carbon budget and trophic level, the two ponds show similar trends in many of the investigated factors, hardly influenced by seasonal variations. Temporally coupled trends were observed for some parameters (enzyme activities, vibrios abundances, respiratory activity), whereas others (POC, PON, heterotrophic bacterial production, bacterioplankton) showed a seasonal shift between the two lakes. The different behaviour found for the some biotic parameters suggests that their response to environmental conditions may be modulated differently between the two lakes, which, despite their spatial proximity and reciprocal connection, do not always show contemporaneous functional processes.

In this study is reported the first episode of a Shell Disease Syndrome (SDS) in wild specimens of Carcinus aestuarii from the polluted Volturno River estuary. Collected crabs showed erosive and ulcerative “black spot” lesions on the exoskeleton; histologically the lesions ranged from a mild to extensive and severe damages and intense haemocyte infiltration. Bacterial isolation confirmed the presence of some marine bacterial species with chitinolytic activity. The authors suggest that the detection of sds in wild crabs living in polluted waters could be used to monitor the health status of this environment.

La comunità microbica costituisce l'anello di sutura tra componenti biotiche e abiotiche dell'ecosistema marino e contribuisce ai processi di base della pompa biologica del carbonio. Infatti, mediante i suoi comparti etero ed autotrofici, essa è strettamente collegata sia con le catene trofiche. Il link tra ecologia microbica e risorse ittiche nasce dalla consapevolezza che i fattori biofisici (ambientali) e le dinamiche trofodinamiche (interazioni tra specie) - insieme ai processi di sfruttamento delle risorse biologiche (pesca) - sono le principali forzanti che influenzano la produttività ittica. Di conseguenza, una corretta politica di gestione ecocompatibile del patrimonio naturale non può prescindere dal monitoraggio delle condizioni ambientali e delle dinamiche trofiche che regolano la funzionalità degli ecosistemi. Urge inoltre approfondire le conoscenze sulle relazioni tra il cambiamento globale e risorse marine, attraverso lo studio delle relazioni tra le reti trofiche e la biogeochimica negli oceani. La problematica diviene particolarmente importante nelle aree costiere e di transizione che subiscono contemporaneamente l'impatto di pressioni antropiche e naturali. Nel Mar Mediterraneo, diversi documenti hanno sottolineato i rapporti tra i parametri microbici e le condizioni chimico fisiche delle acque, così come tra le masse d'acqua. E' anche noto che tenui cambiamenti nelle variabili ambientali possono drasticamente modificare l'abbondanza, la distribuzione e la disponibilità delle popolazioni ittiche e dei loro prodotti di riproduzione. Inoltre, nelle zone di produzione ittica, i processi di trasferimento del carbonio organico attraverso le catene trofiche microbiche hanno un'importanza fondamentale e nei mari oligotrofici, quale è il Canale di Sicilia, la scarsa funzionalità nei primi livelli trofici si potrebbe tradurre in un disequilibrio ecosistemico con implicazioni sui consumatori secondari e terziari, in termini di produttività e biodiversità. In tale ottica, gli obiettivi di ricerca di SP2-WP1-AZ2-UO05 sono stati quelli di approfondire le conoscenze sui popolamenti microbici in un'area di spawning e di verificare l'esistenza o meno di interrelazioni tra la rete trofica microbica e lo sviluppo di uova e larve di Engraulis encrasicolus - specie ittica pelagica di interesse commerciale. Gli scopi della ricerca sono stati quindi molto articolati ed hanno affrontato tre principali tematiche: 1) lo studio quantitativo delle biomasse (nelle frazioni dimensionali: pico-, nano- e micro-plantoniche); 2) lo studio delle popolazioni produttive e degradative della sostanza organica svolto dalle popolazioni auto- ed eterotrofiche; 3) il flusso biogeochimico del carbonio nell'area di studio. E' stato inoltre valutato - per la prima volta in un'area oligotrofica del Mediterraneo - il contributo dell'attività enzimatica libera (non aggregata a cellule microbiche).

An integrated study was carried out to follow the temporal evolution of microbiological parameters during a 48 h period, in relation to the deep chlorophyll maximum (DCM) at a coastal station. The micro-organisms showed an active role in the environment and a different distribution, without a clear diel cycle. The phytoplankton community, responsible for the DCM, consisted mainly of diatoms. Their distribution in relation to pycnocline showed an opposite trend with respect to picophytoplankton. Total bacterioplankton contributed to enzymatic degradation of particulated organic carbon (by producing β-glucosidase and aminopeptidase), with peaks related to changes in the main water current. We estimated that about 25% of particulate organic carbon per day may be hydrolysed by bacteria. The living bacterioplankton represented 20% of the total. The picophytoplankton fraction contributed significantly to the high values of alkaline phosphatase, suggesting a fast P regeneration. Respiration showed significant co...

The carbon transfer through the microbial community in two areas of the Northern Adriatic Sea was estimated by proteolytic and respiratory activities during four oceanographic surveys carried out in June, 1996, 1997 and February, 1997, 1998. In front of the Po Delta (area A), the mean rates of proteolytic activity range from 4.9 to 9.9 r µg r C r h r l; near Ancona (area B), they range from 3.1 to 7.6 r µg r C r h r l. Respiratory rates vary between 0.19 and 2.29 and between 0.24 and 1.40 r µg r C r h r l in areas A and B, respectively. In general, high rates occur in the surface layers, within the first 10 r m of depth. In area A, proteolytic and respiratory rates undergo seasonal course, with high activity in warm periods. In area B, respiration and bacterioplankton abundance increase from the first to the second year, whilst proteolytic activity decreases. The sequence of metabolic steps in the carbon transfer within the bacteria, from the biotic vs . the abiotic compartment, was drawn in order to defi...

The EU Marine Strategy Framework Directive 2008/56/EC (MSFD) defines a framework for Community actions in the field of marine environmental policy in order to achieve and/or maintain the Good Environmental Status (GES) of the European seas by 2020. Microbial assemblages (from viruses to microbial-sized metazoa) provide a major contribution to global biodiversity and play a crucial role in the functioning of marine ecosystems, but are largely ignored by the MSFD. Prokaryotes are only seen as "microbial pathogens", without defining their role in GES indicators. However, structural or functional prokaryotic variables (abundance, biodiversity and metabolism) can be easily incorporated into several MSFD Descriptors (i.e. D1. Biodiversity, D4. Food webs, D5. Eutrophication, D8. Contaminants and D9. Contaminants in seafood) with beneficial effects. The present review provides a critical analysis of the current MSFD descriptors and illustrates the reliability and advantages of the potential incorporation of some prokaryotic variables within the set of indicators of marine environmental quality. Following a cost/benefit analysis against scientific and economic criteria, we conclude that marine microbial components, and particularly prokaryotes, are highly effective for detecting the effects of anthropogenic pressures on marine environments and for assessing changes in the environmental health status. We thus recommend the inclusion of these components in future implementations of the MSFD.

The present research reports the first description of Shell Disease Syndrome in European spiny lobsters Palinurus elephas (Fabricius 1787), which occurred in an experimental aquaculture facility in Sicily (Italy). Both bacterial characterization and histopathological examination of the exoskeleton at site of lesions was carried out. Infected specimens showed tail fan erosions, and in one case uropod ulceration and complete loss of periods. Identified species included: Listonella anguillarum 50.5%, Vibrio parahaemolyticus 27.5% and Vibrio alginolyticus 22%. Microscopic evaluation of lesions indicate the presence of inflammatory responses, which include melanization and pseudomembrane formation, similar to those described for other crustaceans affected by SDS.

Aim: Leucine aminopeptidase (LAP), an enzyme involved in the decomposition of-natural peptides, was measured in: different marine and brackish ecosystems, together with some environmental and microbiological parameters. Methods and Results: The fluorogenic compound L-leucine-7-amido-4-methyl coumarin was specifically used for the determination of this in situ activity. The enzyme data obtained from this comparative study highlighted the strong spatial and temporal variability of the distribution of LAP in aquatic ecosystems, which was sometimes related to the course of environmental variables such as salinity and organic carbon content. Conclusions: LAP assay has proved to be a rapid method providing useful information on the microbial metabolic processes involved in the mineralization of organic matter. Significance and Impact of the Study: The determination of the potential rates of extracellular enzyme activity is of great ecological importance to extend knowledge on the role played by bacteria in aquatic biogeochemical cycles.

The determination of Escherichia coli in marine waters through a rapid method, the microscopic indirect immunofluorescent technique, is evaluated in comparison with the conventional count on m-FC agar medium. The data obtained in seawater samples, collected monthly along the Messina coastline, show good sensitivity of the analysis and agreement between the microscopic and culture technique, with a detection limit of 10(2) cells 100 ml(-1) for immunofluorescence.

I microorganismi giocano un ruolo primario nella modulazione dei flussi della materia biogenica e nella sua trasformazione tra comparti biotici e abiotici e, negli ecosistemi marini, la rete trofica microplanctonica gioca un ruolo primario per il sostentamento e lo sviluppo dei livelli trofici superiori. In tale contesto, l'analisi ed il monitoraggio degli stock ittici non può prescindere dalla conoscenza delle strette interrelazioni esistenti tra le suddette reti trofiche. Tale approccio ecosistemico alla gestione della pesca nel Canale di Sicilia consente la valutazione delle dinamiche tra comunità di differenti taglie dimensionali, ed amplia le conoscenze sulle interazioni tra i diversi comparti biologici. Tali dinamiche infatti evolvono e coevolvono all'interno della matrice acquatica. Obiettivi della ricerca sono: ofornire un approccio ecosistemico alla gestione delle risorse ittiche implementando i dati sperimentali delle campagne di pesca attraverso il monitoraggio delle biomasse microbiche nelle diverse categorie dimensionali planctoniche (aspetto legato alle reti trofiche) odeterminare i flussi di elementi biogeochimici veicolati attraverso il comparto microbico (misure di produzione, degradazione e mineralizzazione della sostanza organica -pompa biologica) e loro ruolo funzionale nel supportare i livelli trofici superiori.

Tra le malattie batteriche, le infezioni da cocchi Gram positivi stanno assumendo sempre piu? importanza. Lactococcus garvieae (sin. Enterococcus seriolicida) e? l'agente patogeno che colpisce ricciole di piccola taglia. Nel presente lavoro, viene riportato il primo caso di infezione oculare (Pop-eye) in un esemplare adulto di ricciola, Seriola dumerilii. Nei primi giorni del mese di Settembre 2008 un esemplare femmina di circa 18 anni di eta? e del peso di circa 35 Kg, stabulato fin dallo stadio giovanile (40 g circa) presso l'Impianto Sperimentale di Acquacoltura dell'IAMC - CNR di Messina, mostrava atassia, letargia, anoressia, esoftalmia unilaterale destra con una grave emorragia accompagnata da essudato biancastro. Sono stati effettuati dei tamponi dall'occhio con lesione, seminati poi su terreni di coltura. Le colonie sospette sono state isolate e identificate mediante indagini colturali e biochimiche (API 20 STREP).

Poly(vinyl chloride) (PVC) composites containing increasing amounts (2-20%, w/w) of silver zeolite (SZ) were prepared by melt mixing and characterized by thermal, mechanical and rheological analyses. The addition of large amount of SZ did not influence the processability and the formability of the composites, if compared to neat plasticized PVC. The antibacterial activity of PVC SZ composites was tested on Escherichia coli and Staphylococcus epidermidis and resulted promising both in culture broth and on agar plate and also in sterile urine seeded with these strains, for simulation purposes. In sterile urine, composites induced a significant reduction (4-6 log units) of viability of both strains already at 24 h, inhibiting E. coli growth up to 20 days, whereas their antimicrobial action against S. epidermidis vanished within 5 days. The silver release in sterile urine was determined up to 20 days. It was found that the highest amount of silver ions was released during the first day (0.365 ppm), whilst from days 6 to 20 the silver release decreased, reaching a steady daily mean value of 0.02 ppm.

The antibiotic resistance patterns of aetiological agents responsible for vibriosis and pasteurellosis were studied to contribute to control the spread of these two bacterial diseases in Mediterranean fish farming. Strains of Photobacterium damsela ssp. piscicida, Vibrio fluvialis, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio metschnikovii, isolated from Italian aquaculture (fish, shellfish and crustaceans) sites, were assayed for their susceptibility to some antibacterial agents currently used in farming practices. Kirby Bauer and Minimum Inhibitory Concentration (M.I.C.) tests were performed. The bacterial strains showed resistance to ampicillin, carbenicillin, kanamycin, cefalothin, while they were sensitive to chloramphenicol, nitrofurantoin and tobramycin; the sulfadiazine-trimethoprim association was completely ineffective. Conversely, flumequine showed the lowest M.I.C. value (0.97 �g mL-1), suggesting its marked antibiotic effect. Considering that quinolone resistance can be transmitted only by selection of mutations and not by other genetic mechanisms, this study stresses the importance of a more responsible use of this antibacterial drug in aquaculture.

A synthesis of field data carried out in the Mediterranean Sea are presented, aimed at contributing to the knowledge of three prokaryotic-mediated processes and their implications on the Carbon cycle. The distribution of exoenzymatic activities, secondary production and respiration rates was studied together with the prokaryotic abundances. Particular attention was paid to the meso- and bathypelagic layers which play an important role in the Mediterranean carbon cycle. The study is noteworthy because of its large spatial scale spanning the entire Mediterranean Sea over 4 years. In addition, two Atlantic stations in front of the Gibraltar Strait were investigated. The longitudinal distribution of prokaryotic activities and abundance along the MED showed different trends along the depthlayers. In particular, higher exoenzymatic rates were detected in the Eastern basin compared to the Western one; carbon respiration rate showed patterns variable with the sampling periods in the epipelagic and bathypelagic layers, while a consistent Westwards decreasing trend at the mesopelagic layers occurred. Specific enzyme activities per cell showed high values in the deepest layers for leucine aminopeptidase. Comparison with Carbon respiration rate data collected before the 2000s showed changing patterns of microbial heterotrophic processes in the Mediterranean Sea.

A two-year ecological study in the Oliveri-Tindari (Messina) brackish water ponds evaluated the quantitative and qualitative distribution of Vibrio species in relation to the physicochemical conditions of the ponds. The evaluation of 204 samples shows a general relationship between the halophilic vibrios and water temperature. In particular this relationship is stronger for the Vibrio fraction able to grow at 37°C, i. e., those species related to human infections. On the other hand, the halophilic vibrios show no relation with the index of fecal contamination. The ecology of V. parahaemolyticus is discussed in relation to the salinity of Marinello pond. The distribution of V. cholerae non O1, isolated from all ponds, shows that this microorganism can be considered as autochthonous in coastal environments.

A multiplex polymerase chain reaction protocol for the detection of Photobacterium damselae and subspecies piscicida and damselae discrimination, with internal amplification control, was developed. Assay specificity was assessed by testing 19 target and 25 non-target pure cultures. The detection limit was 500 fg, corresponding to 100 genome equivalents. The optimized protocol was also prevalidated with spleen, kidney and blood samples from infected and uninfected sea bass, without any culture step, and it can be proposed as a valid alternative to culture standard methods for the rapid and specific diagnosis of photobacteriosis in fish.

The greater amberjack, Seriola dumerilii (Risso 1810) is a semi-pelagic fish and a world wide species; it is considered a promising candidate for the diversification of Mediterranean aquaculture. In this paper an experimental injection with Listonella (Vibrio) anguillarum was performed to study the immune response of S. dumerilii. Antibody titres to L. anguillarum O1 were determined with indirect ELISA at different times over a period of 42 days. Results showed that the antibody levels against L. anguillarum were significantly higher in the challenged fish compared to the control. They started developing since the 5th day reaching the highest peak on day 20 after injection, indicating a fast response of the immune system. The observed antibody titre was very high versus L. anguillarum if compared to other fish species.

Nel periodo compreso tra ottobre 2007 - ottobre 2008, l'Istituto CNR-IAMC Sezione di Messina ha svolto, con il contributo del CeSPOM, uno studio applicativo finalizzato alla valorizzazione dei prodotti della molluschicoltura del lago di Faro (area lagunare di Capo Peloro), ed all'analisi della loro sicurezza alimentare. L'obiettivo principale del progetto è consistito nel monitoraggio dell'accrescimento di esemplari di Mytilus galloprovincialis allevati nel Lago di Faro, nella valutazione delle loro condizioni igieniche, mediante la ricerca dei più diffusi batteri patogeni di origine antropica, di alcuni batteri autoctoni patogeni opportunisti e dei principali parassiti dei molluschi, quali Giardia lamblia e Cryptosporidium sp.

Three microbial extracellular enzymes, leucine aminopeptidase (LAP), β-glucosidase (β-glu) and alkaline phosphatase (AP), were studied in six small Mediterranean littoral ponds, to evaluate the diversity of microbial activities relative to prevailing environmental conditions. The marked diversification of the trophic states, ranging from oligotrophy to eutrophy, in the ponds was reflected in a range of enzyme patterns at different spatial and temporal scales. There were higher levels and greater variability of microbial activity in the oldest and most ‘confined’ ponds (ranges: 0.55–4360.00 nm h−1, 0.15–76.44 nm h−1, 1.29–1600.00 nm h−1 for LAP, β-glu and AP respectively) compared with the youngest and most seaward ponds (ranges: 22.64–612.0 nm h−1, 0.06–48.89 nm h−1, 0.32–744.0 nm h−1 for LAP, β-glu and AP respectively). The close relationship of the degradative potential with chlorophyll-a and particulate organic carbon could be a consequence of the stimulating effect of phytoplankton-released polymeric compounds (organic matter) and/or a response of the microbial community to warm temperatures, which were recorded from July to September. Within an area less than 1 km2, different aquatic ecosystems coexist and maintain their distinctive properties in terms of microbial biogeochemical processes.

The mussel and fish-farming impact was investigated both in pelagic and sediment environments of Mar Piccolo in Taranto. We studied the yearly distribution of microbiological indicators (heterotrophic bacteria and coliforms), in relation to physical and chemical parameters of surface water at three different sites, characterized by mussel farms, fish cages and Citro springs. The heterotrophic bacteria and Clostridium perfrigens distribution were also examined in relation to organic content and granulometry of sediments. The urban sewages affected the quality of waters of the mussel site, by increasing faecal coliforms in the entire basin during summer. In the benthonic environment the aquaculture activity increased the bio-deposition of the sediment, causing changes in the abundance and the composition (heterotrophic bacteria/clostridia ratio) of microflora. A significant increase of heterotrophic bacteria in the warm season was observed. The clostridia proved to be a good indicator of impact of organic matter because they were significantly higher in the sites than the control stations.

This paper concerns the current debate as to whether oceans are heterotrophic or autotrophic environments. Microbes are responsible for the assimilation and remineralization of CO2 in the sea and microbial processes are involved in the Earth climatic change. The variability of microbial biomass and activities were studied in the Mediterranean Sea which represents a suitable basin for mapping the trophic regimes by the study of biogeochemical processes. The surveys were carried out in the epi-, meso- and bathy-pelagic zones of the Central Mediterranean Sea from 1996 to 2002. Bacterial abundance, leucine aminopeptidase (LAP) and h-glucosidase (h-GLU) activities, bacterial carbon production (BCP), community respiration (R) and primary production (PP) were the main parameters investigated. Biomass and activities seasonally varied in epipelagic and, to some extent, in meso- and bathy-pelagic layers too and several trophic ratios were determined, showing different scenarios in the different areas and seasons. In the euphotic layer, bacterial growth efficiency (BGE) widely ranged between 4% and 50% showing a decreasing trend from estuaries towards pelagic areas in alignment with thetrophic gradient from theNorthernAdriatic to the IonianSea. However, BGE did not correlate to PP and temperature. The Central Mediterranean Sea showed different trophic regimes in the examined areas and seasons. In the Northern Adriatic Sea in winter, the trophic balance appeared to move towards autotrophy and a positive C budget resulted. An opposite scenario happened in summer when the trophic balance moved to heterotrophy and a negative C budget occurred. In the Ionian Sea, the overall balance turned always towards remineralization. However, on annual time scale, the Central Mediterranean Sea seems to show a tendency towards a heterotrophic system. D 2005 Elsevier B.V. All rights reserved.

The purpose of this study was the quantification, through the comparison of different methods, of viable and metabolically active bacteria in marine environments. To quantify the living and active bacterioplankton fractions, we compared the total cell count (TC using DAPI staining), plate count on marine agar (CFU), and three viability-staining methods: nucleoid-containing cell count (NuCC), Live/Dead staining procedure (L/D) and direct viable count (DVC). With respect to TC (mean value 3.0+2.3 105 cell ml21) CFU represented less than 0.1% and DVC cells 1%, both showing significant differences. NuCC and L/D cells were 18.0% and 15.9% of TC, respectively, showing no significant differences and higher percentages in the Ionian Sea than the Adriatic Sea. Moreover, NuCC and L/D were two orders of magnitude greater than the culturable fraction, while active cells (DVC) exceeded CFU by one order of magnitude. The comparison of different staining methods allowed us to confirm the simultaneous presence of different physiological states within the bacterial population in natural marine environments. The NuCC and L/D methods gave comparable values to those of other authors, while the DVC procedure gave lower values than previously reported. This research provides information on the fraction of living and/or metabolically active bacteria in aquatic ecosystems. Since each method has its own detection limits, the study highlights the need to simultaneously compare the different methods to validate their results.