Biotechnological potential of the seaweed Cladophora rupestris (Chlorophyta, Cladophorales) lipidic extract.

Recently, with the advent of modern technologies, various marine organisms including algae are being studied as sources of natural substances effective on classical microorganisms and able to also combat the new trend of acquired resistance in microbes. In the present study the antimicrobial activity of the lipidic extract of the green seaweed Cladophora rupestris collected in a Mediterranean area, in two sampling periods (January and April), was assayed. The chemical characterization of the lipidic fractions was performed by gas-chromatography and multinuclear and multidimensional NMR spectroscopy. In the lipidic extract of C. rupestris collected in January an antibacterial activity against Enterococcus sp., Streptococcus agalactiae and Vibrio cholerae non-O1 was recorded; by contrast, bacterial inhibition was measured on several Vibrio species only in April. The fatty acid profile of C. rupestris lipidic extract, analyzed by gas chromatography, resulted mainly composed of palmitic, myristic, oleic, α linolenic, palmitoleic and linoleic acids. Moreover, since α-linolenic acid was the predominant ω3 fatty acid in April, we suggest its involvement in the antibacterial activity observed in this month, taking also into account that pure α-linolenic acid resulted effective towards some vibrios strains. C. rupestris fatty acid profile revealed also an interesting composition in polyunsaturated fatty acids in both the considered periods with the ω6/ω3 ratio lower than 1, leading to conclude that this macroalga may be employed as a natural source of ω3. Finally, the (1)H NMR spectrum in CDCl3 of algal lipid fractions showed the characteristic signals of saturated (SAFAs) and unsaturated fatty acids (UFAs) as well as other metabolites and a marked difference in free fatty acids (FFAs) content for the two examined algal lipid fractions. It is noteworthy that C. rupestris lipidic extracts show, by NMR spectroscopy, the signal pattern of polyhydroxybutyrate, a natural biocompatible and biodegradable polymer. In conclusion, on account of its antimicrobial activity, nutritional value and bioplastic content, C. rupestris lipidic extract can be considered a promising source for future biotechnological applications.

The lipidic extract of the seaweed Gracilariopsis longissima (Rhodophyta, Gracilariales): a potential resource for biotechnological purposes?

In recent years seaweeds increasingly attracted interest in the search for new drugs and have been shown to be a primary source of bioactive natural products including antibiotics. In the present investigation the antimicrobial activity of Gracilariopsis longissima lipidic extract was assayed and its chemical characterization was carried out by means of advanced analytical techniques such as gas-chromatography and multinuclear and multidimensional NMR spectroscopy. G. longissima lipidic extract showed an antibacterial activity against several Vibrio species. These results are interesting considering both the resistance against antibiotics developed by vibrios and the need to control fish and shellfish diseases due to vibriosis. Analysis of fatty acid methyl esters performed by gas-chromatography showed that palmitic acid methyl ester (16:0) was the predominant saturated fatty acid (42%), while, among monounsaturated fatty acids, oleic acid methyl ester (18:1) prevailed (8.5%). Because the palmitic acid represents the main component of fatty acids we hypothesized its involvement in the antibacterial activity observed. However, a pure sample of palmitic acid did not show an antibacterial activity. The fatty acid profile of G. longissima revealed also an interesting composition in polyunsaturated fatty acids and in particular the ratio of ω-3 to ω-6 fatty acids was >1 thus suggesting that this macroalga may be used as a natural source of ω3. Moreover, the (1)H NMR spectrum in CDCl(3) of algal lipid fraction shows the characteristic signals of saturated and unsaturated fatty acids as well as other metabolites. Interestingly, in the lipid extract the presence of polyhydroxybutyrate, a linear biodegradable and biocompatible polyester, was clearly identified by NMR spectroscopy. In conclusion, the lipidic extract of G. longissima on account of its antimicrobial activity, nutritional value and content in biodegradable and biocompatible polyester represents an interesting potential biotechnological resource.

Bacterial accumulation by the Demospongiae Hymeniacidon perlevis: a tool for the bioremediation of polluted seawater.

Sponges can filter large amounts of water, which exerts an important grazing impact on free bacteria, an important component of the diet of sponges. We examined the accumulation of bacteria in the Demospongiae (Hymeniacidon perlevis). Analyses were performed on homogenates from unstarved and starved sponges in seawater from their sampling site (the Ionian Sea). Culturable heterotrophic bacteria (22 degrees C), total culturable bacteria (37 degrees C) and vibrios densities were measured on marine agar 2216, plate count agar and TCBS agar, respectively. Total and fecal coliforms, as well as fecal streptococci, were determined by the most probable number method (MPN). H. perlevis was able to accumulate all of the six microbiological groups. Bacterial groups differed in their resistance to digestion by H. perlevis. Our data suggest that H. perlevis may accumulate, remediate and metabolize bacteria and that they may be employed as a useful bioindicator and bioremediator.

Effect of zinc on lysozyme-like activity of the seastar Marthasterias glacialis (Echinodermata, Asteroidea) mucus.

Lysozyme represents the best characterized enzyme involved in the self-defense from bacteria. In this study we analysed the effects of zinc on the lysozyme-like activity of the seastar Marthasterias glacialis mucus. This activity, detected by measuring the cleared lysis area of dried Micrococcus lysodeikticus cell walls on Petri dishes, was significantly reduced in presence of zinc. The results are discussed in the light of elucidating the possible relationship between environmental contaminants and increased disease susceptibility in seastars due to the decrease of antibacterial protection. The benefits of using the test of lysozyme activity to monitoring environmental pollution are highlighted.

Culturable heterotrophic bacteria in seawater and Mytilus galloprovincialis from a Mediterranean area (Northern Ionian Sea-Italy).

Although Mar Piccolo of Taranto (Ionian Sea, Italy) is one of the most important Mytilus galloprovincialis farming areas, data concerning the natural bacterial microbiota of these mussels and their surrounding environment are still scant. This study was carried out seasonally, throughout a year, to determine culturable heterotrophic bacteria both in the water and mussels samples collected at three sampling sites in the Northern Ionian Sea: S. Vito, Lido Gandoli and Lido Silvana. Culturable heterotrophic bacteria abundance was determined by spread plate on Marine Agar. Heterotrophic bacteria were identified by several morphological, culture and biochemical methods. Bacterial concentrations were higher in the mussel samples compared to the corresponding seawater throughout the year. Among Gram negative heterotrophic bacteria, Aeromonas prevailed both in the water (18%) and mussel samples (40%). Other genera such as Moraxella, Pseudomonas, Alcaligenes, Acinetobacter, Flavobacterium, Chromobacterium, Photobacterium and Flexibacter were present with different percentages of isolation. Bacilli were predominant among Gram positive bacteria. Some genera (Lucibacterium and Vibrio) were present only in mussel samples. The results obtained contribute to improve the knowledge on both the bacterial abundance and diversity in mussels and the surrounding seawater in the Northern Ionian Sea.

Epibiotic Vibrio luminous bacteria isolated from some hydrozoa and bryozoa species.

Luminous bacteria are isolated from both Hydrozoa and Bryozoa with chitinous structures on their surfaces. All the specimens of the examined hydroid species (Aglaophenia kirchenpaueri, Aglaophenia octodonta, Aglaophenia tubiformis, Halopteris diaphana, Plumularia setacea, Ventromma halecioides), observed under blue light excitation, showed a clear fluorescence on the external side of the perisarc (chitinous exoskeleton) around hydrocladia. In the bryozoan Myriapora truncata, luminous bacteria are present on the chitinous opercula. All the isolated luminous bacteria were identified on the basis of both phenotypic and genotypic analysis. The isolates from A. tubiformis and H. diaphana were unambiguously assigned to the species Vibrio fischeri. In contrast, the isolates from the other hydroids, phenotypically assigned to the species Vibrio harveyi, were then split into two distinct species by phylogenetic analysis of 16S rRNA gene sequences and DNA-DNA hybridization experiments. Scanning electron microscopy analysis and results of culture-based and culture-independent approaches enabled us to establish that luminous vibrios represent major constituents of the bacterial community inhabiting the A. octodonta surface suggesting that the interactions between luminous bacteria and the examined hydrozoan and bryozoan species are highly specific. These interactions might have epidemiological as well as ecological implications because of the opportunistic pathogenicity of luminous Vibrio species for marine organisms and the wide-distribution of the hydrozoan and bryozoan functioning as carriers.

Vibrio harveyi associated with Aglaophenia octodonta (Hydrozoa, Cnidaria).

A previously unknown association between a luminous bacterium, Vibrio harveyi, and a benthic hydrozoan, Aglaophenia octodonta, is described. Aglaophenia hydrocladia showed a clear fluorescence in the folds along the hydrocaulus and at the base of the hydrotheca, suggesting the presence of luminous bacteria. This hypothesis was confirmed by isolation of luminous bacteria from Aglaophenia homogenates. Phenotypic characterization of bacterial isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a single species: V. harveyi. The association between V. harveyi and A. octodonta has epidemiological as well as ecological significance. Therefore, A. octodonta may function as habitat "islands" providing a unique set of environmental conditions for luminous bacteria colonization, quite different from those already recorded from the plankton for other Vibrio species.

Viable heterotrophic bacteria in water and sediment in 'Mar Piccolo' of Taranto (Ionian Sea, Italy).

Samples of water and sediment were collected from October 1996 to September 1997 in 'Mar Piccolo' of Taranto (Ionian Sea, Italy). Mar Piccolo is a semi-enclosed basin subject to pollution and receives a considerable amount of sewage and industrial waste. Qualitative and quantitative analyses of the composition of the microbial flora were conducted on samples from six stations. The highest bacterial densities, in water and in sediment samples, were found in summer and the lowest, in autumn. Among Gram-negative bacteria, the predominant genus was Aeromonas; Photobacterium and Pseudomonas were also found. Gram-positive bacilli were abundant at all sampling points. Faecal contamination indicators demonstrated that all the stations examined in Mar Piccolo are influenced by anthropogenic pollution throughout the year.

Antibacterial activity in the coelomocytes of the sea urchin Paracentrotus lividus.

Naturally present antibacterial activity directed against Vibrio alginolyticus was demonstrated in coelomocytes lysate (CL) and cell-free coelomic fluid (CF) of the marine echinoderm Paracentrotus lividus. Kinetic analysis revealed that 5 min of contact was enough to induce significant bactericidal effect. Maximum activity required 30 min of contact. Nonsensitive to the effect of trypsin, the activity was almost completely suppressed by incubation with subtilisin. Purified from CL by three successive steps of chromatography (gel filtration, anion exchange, reverse phase), active antibacterial protein appeared as a single polypeptide chain of approximate molecular weight of 60 kDa.

Antibacterial protection in Marthasterias glacialis eggs: characterization of lysozyme-like activity.

Eggs from Marthastherias glacialis exert antibacterial action on marine bacterial strains and show a lysozyme-like activity. This one depends on pH and ionic strength of sample and reacting medium. This hydrolase, purified by gel filtration and ion-exchange chromatography, could be responsible for the bacterial growth inhibitory activity observed.

Sea urchin coelomic fluid agglutinin mediates coelomocyte adhesion.

The sea urchin Paracentrotus lividus coelomic fluid was found to contain agglutinin which agglutinates animal erythrocytes and promotes adhesion of autologous coelomocytes. Hemagglutinating activity depended upon the presence of calcium ions and was relatively heat-stable. Through a combination of methods including ammonium sulfate precipitation and both size exclusion and ion exchange chromatographies, we purified the anti-rabbit agglutinating factor. The intact agglutinin migrates as a single band with an apparent M(r) of over 200,000. Three distinct protein bands with a calculated M(r) of 174,000, 137,000, and 76,000, respectively were observed under reducing conditions. The purified agglutinin strongly promoted the in vitro adhesion of autologous coelomocytes.