Guilherme Muricy

Isolation, characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil.

Bacteria associated with marine sponges represent a rich source of bioactive metabolites. The aim of this study was to isolate and characterize bacteria with antimicrobial activities from Brazilian sponges. A total of 158 colony-forming units were isolated from nine sponge species. Among these, 12 isolates presented antimicrobial activities against pathogenic bacteria. Based on comparative sequence analysis of their 16S rRNA genes, the sponge-associated bacterial strains could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes (genera Bacillus and Virgibacillus), three with alpha-Proteobacteria (Pseudovibrio sp.) and four with gamma-Proteobacteria (genera Pseudomonas and Stenotrophomonas). The sponge-associated bacterial strains Pseudomonas fluorescens H40 and H41 and Pseudomonas aeruginosa H51 exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria, including strains such as vancomycin-resistant Enterococcus faecium and multiresistant Klebsiella pneumoniae. Bacillus pumilus Pc31 and Pc32, Pseudovibrio ascidiaceicola Pm31 and Ca31 and Pseudovibrio denitrificans Mm37 strains were more effective against Gram-positive bacteria. These findings suggest that the identified strains may contribute to the search for new sources of antimicrobial substances, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

Environmental Shaping of Sponge Associated Archaeal Communities

Background Archaea are ubiquitous symbionts of marine sponges but their ecological roles and the influence of environmental factors on these associations are still poorly understood. Methodology/Principal Findings We compared the diversity and composition of archaea associated with seawater and with the sponges Hymeniacidon heliophila, Paraleucilla magna and Petromica citrina in two distinct environments: Guanabara Bay, a highly impacted estuary in Rio de Janeiro, Brazil, and the nearby Cagarras Archipelago. For this we used metagenomic analyses of 16S rRNA and ammonia monooxygenase (amoA) gene libraries. Hymeniacidon heliophila was more abundant inside the bay, while P. magna was more abundant outside and P. citrina was only recorded at the Cagarras Archipelago. Principal Component Analysis plots (PCA) generated using pairwise unweighted UniFrac distances showed that the archaeal community structure of inner bay seawater and sponges was different from that of coastal Cagarras Archipelago. Rarefaction analyses showed that inner bay archaeaoplankton were more diverse than those from the Cagarras Archipelago. Only members of Crenarchaeota were found in sponge libraries, while in seawater both Crenarchaeota and Euryarchaeota were observed. Although most amoA archaeal genes detected in this study seem to be novel, some clones were affiliated to known ammonia oxidizers such as Nitrosopumilus maritimus and Cenarchaeum symbiosum. Conclusion/Significance The composition and diversity of archaeal communities associated with pollution-tolerant sponge species can change in a range of few kilometers, probably influenced by eutrophication. The presence of archaeal amoA genes in Porifera suggests that Archaea are involved in the nitrogen cycle within the sponge holobiont, possibly increasing its resistance to anthropogenic impacts. The higher diversity of Crenarchaeota in the polluted area suggests that some marine sponges are able to change the composition of their associated archaeal communities, thereby improving their fitness in impacted environments.

Sponges without skeleton: A new Mediterranean genus of Homoscleromorpha (Porifera, Demospongiae)

Abstract A new genus of aspiculate homoscleromorph, Pseudocorticium, is described along with the new species P. jarrei found in a littoral cave located in the western Mediterranean Sea (France). The anatomy and cell composition are described and compared to that of species of the other homoscleromorph genus without skeleton, Oscarella, and to genera with skeleton, Corticium and Plakina. The new sponge differs from Oscarella in its morphology, anatomy, and cytology. An emended definition of the genus Oscarella is proposed. Anatomical and cytological characters carry a high informative content and should be described for a greater number of sponge species.

Antibiotic, cytotoxic and enzyme inhibitory activity of crude extracts from Brazilian marine invertebrates

Herein we present the results of a screening with 349 crude extracts of Brazilian marine sponges, ascidians, bryozoans and octocorals, against 16 strains of susceptible and antibiotic-resistant bacteria, one yeast (Candida albicans), Mycobacterium tuberculosis H37Rv, three cancer cell lines MCF-7 (breast), B16 (murine melanoma ) and HCT8 (colon), and Leishmania tarentolae adenine phosphoribosyl transferase (L-APRT) enzyme. Less than 15% of marine sponge crude extracts displayed antibacterial activity, both against susceptible and antibiotic-resistant bacteria. Up to 40% of marine sponge crude extracts displayed antimycobacterial activity against M. tuberculosis H37Rv. Cytotoxicity was observed for 18% of marine sponge crude extracts. Finally, less than 3% of sponge extracts inhibited L-APRT. Less than 10% of ascidian crude extracts displayed antibacterial activity. More than 25% of ascidian crude extracts were active against M. tuberculosis and the three cancer cell lines. Only two crude extracts from the ascidian Polysyncraton sp. collected in different seasons (1995 and 1997) displayed activity against L-APRT. Less than 2% of bryozoan and octocoral crude extracts presented antibacterial activity, but a high percentage of crude extracts from bryozoan and octororal displayed cytotoxic (11% and 30%, respectively) and antimycobacterial (60%) activities. The extract of only one species of bryozoan, Bugula sp., presented inhibitory activity against L-APRT. Overall, the crude extracts of marine invertebrates herein investigated presented a high level of cytotoxic and antimycobacterial activities, a lower level of antibacterial activity and only a small number of crude extracts inhibited L-APRT. Taxonomic analysis of some of the more potently active crude extracts showed the occurrence of biological activity in taxa that have been previously chemically investigated. These include marine sponges belonging to genera Aaptos, Aplysina, Callyspongia, Haliclona, Niphates, Cliona, Darwinella, Dysidea, Ircinia, Monanchora and Mycale, ascidians of the genera Didemnum, Aplidium, Botrylloides, Clavelina, Polysyncraton and Symplegma, the bryozoan Bugula sp. and octocorals of the genera Carijoa and Lophogorgia. The subsequent chemical investigation of some of the active extracts led to the isolation of several new biologically active secondary metabolites. Our results are in agreement with previous screening programs carried out abroad, that showed a high percentage of bioactive extracts from Porifera, Ascidiacea, Cnidaria and Bryozoa.

Marine sponges of Pernambuco State, NE Brazil

The Brazilian coastline is still poorly studied ftom a faunistic viewpoint. Brazilian sponges are among the less studied in the world, and most of the knowledge about them comes ftom dredging by foreign expeditions such as the Challenger (e.g., Ridley & Dendy, 1887) and the Calypso (BouryEsnault, 1973). Brazilian shallow-water sponges, easily accessed by divers, still remain 1argely unknown (Hajdu et aJ., 1996). In this study, we describe the composition and distribution of the shallow water marine sponge fauna ftom the National Marine Park of Fernando de Noronha and ftom Tamandare, both in Pemambuco State, Northeastem Brazil. Such basic taxonomic data can help the development of management plans for these protected areas.

Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria

Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3) isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

Anatomy, cytology and symbiotic bacteria of four Mediterranean species of Plakina Schulze, 1880 (Demospongiae, Homosclerophorida)

The internal anatomy, cytology and symbiotic bacteria of four Mediterranean species of the sponge genus Plakina Schulze, namely P. trilopha Schulze, P. jani Muricy et al., P. crypta Muricy et al., and P. endoumensis Muricy et al., are investigated under light and transmission electron microscopy in a search for new, taxonomically useful characters. Anatomical traits such as ectosome thickness, ectosomal cavities, size of basal cavities, and mesohyl chambers ratio can be useful to distinguish groups of species within the genus, but give no clear indication of relationships of Plakina with other plakinid genera. The most useful cytological traits at the species level within Mediterranean Plakina are the presence/absence of vacuolar cells and of pseudopodia in pinacocytes, and the number of microvilli in choanocytes. There are no cells with inclusions in the mesohyl, and the simple cytology of Plakina agrees more closely with that of Corticium candelabrum Schmidt, Oscarella lobularis Schmidt and O. tubercula...

Order Homosclerophorida Dendy, 1905, Family Plakinidae Schulze, 1880

Homosclerophorida Dendy (Demospongiae, Homoscleromorpha) contains a single family Plakinidae Schulze (including Oscarellidae Lendenfeld and Corticiidae Vosmaer), with seven valid genera and about 60 valid species worldwide. Species live mainly in shallow waters but a few have been recorded from abyssal depths (up to 2460m). Species are often encrusting, lobate, but massive species are common in some genera (Plakortis, Plakinastrella); surface is usually smooth or microhispid and consistency varies from soft to cartilaginous. All genera possess flagellated exo- and endopinacocytes, a basement membrane lining both choanoderm and pinacoderm, oval to spherical choanocyte chambers with a sylleibid-like or leuconoid organization, and a unique incubated cinctoblastula-type larvae; spicules, when present, are peculiar tetractines (calthrops) and derivatives. Genera are distinguished mainly by four morphological characters: presence of a siliceous skeleton; presence of a cortex associated with a leuconoid aquiferous system and well-developed mesohyl or a sylleibid aquiferous system with poorly developed mesohyl and ectosome; number of spicule size classes; and presence and type of ramifications in the actines of calthrops (tetractinal spicules), with three distinct general morphologies recognized.

THE SESTERTERPENE VARIABILIN AS A FISH-PREDATION DETERRENT IN THE WESTERN ATLANTIC SPONGE Ircinia strobilina

The furanosesterterpene variabilin was identified in an unpalatable crude extract and proved to be a feeding deterrent when offered at 0.23% of artificial diet dry weight to reef fishes in field assays. The icthyodeterrent property of variabilin was expressed when food pellets for the palatability assays were made with calcium alginate, but not carrageenan. Variabilin probably failed to show deterrent activity in the carageenan matrix because of decomposition during the necessary heat treatment.

Investigation of biotechnological potential of sponge‐associated bacteria collected in Brazilian coast

Marine bacteria are a rich source of structurally unique natural compounds, several of which have shown a wide variety of biological activities. In this study, the metabolites present in the culture supernatants of the eight sponge‐associated bacteria were extracted using ethyl acetate, and all extracts showed activity against Staphylococcus aureus. Subsequently, the extracts of the Pseudomonas fluorescens H40 and H41, and Pseudomonas aeruginosa H51 were subjected to solvent partitioning, and the active fractions were submitted to chromatographic separation. Three different active fractions were obtained, one of which was identified as diketopiperazine cyclo‐(L‐Leu‐L‐Pro). This substance was bactericidal for Staph. aureus and Ps. aeruginosa and showed cytotoxic activity against HEp‐2 tumour cells. Putative gene fragments coding for the type I polyketide synthase (PKS‐I) and nonribosomal peptide synthetase (NRPS) domains were PCR‐amplified from five and three strains, respectively. The results suggest that sponge‐associated bacteria analysed in this study may represent a potential source for production of antimicrobial substances against bacterial pathogens of medical importance.