Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strains

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.

Ocorrência e diversidade de leveduras no filoplano de plantas seleccionadas do Parque Natural da Serra da Arrábida

Dissertação apresentada para obtenção do Grau de Doutor em Biologia (especialidade Microbiologia), pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Microeukaryoticdiversity in the extreme environments of the Iberian PyriteBelt: a comparison between universal and fungi-specific primer sets, temperature gradient gel electrophoresis and cloning

FEMS Microbiology Ecology, Vol. 57, nº 1

Primary bioreceptivity of limestones from the mediterranean basin to phototrophic microorganisms

Dissertação apresentada para a obtenção do grau de Doutor em Conservação e Restauro pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Dioszegia antarctica sp. nov. and Dioszegia cryoxerica sp. nov., psychrophilic basidiomycetous yeasts from polar desert soils in Antarctica

International Journal of Systematic and Evolutionary Microbiology, 60

Farysizyma gen. nov., an anamorphic genus in the Ustilaginales to accommodate three novel epiphytic basidiomycetous yeast species from America, Europe and Asia

FEMS Yeast Research, Vol. 8, Nº 3

Growth of phototrophic biofilms from limestone monuments under laboratory conditions

International Biodeterioration & Biodegradation,xxx (2009) 1–8

New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

Meningococcal Disease Caused by Neisseria meningitidis Serogroup B Serotype 4 in São Paulo, Brazil, 1990 to 1996

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988.21 A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: A polyphasic study

Cyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes). The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments. The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N2-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.

Genetic relationships among serogroup B: serotype 4 Neisseria meningitidis strains

We compared the results obtained by serotyping of PorB epitopes using an expanded panel of monoclonal antibodies (mAb) including mAb 7 and mAb 10, with results obtained by RFLP of rRNA genes (ribotyping). The purpose of this study was to assess the correlation between phenotypic- and genotypic- methods for typing N. meningitidis. The ribotypes obtained using ClaI or EcoRV endonucleases grouped the strains in seven and two different patterns, respectively. This additional characterization of PorB epitopes improved the correlation between these two methods of typing N. meningitidis.

Neurocryptococcosis: diagnosis by PCR method

Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

Polymerase Chain Reaction Screening for Fungemia and/or Invasive Fungal Infections in Patients with Hematologic Malignancies

INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore, our results confirm the potential usefulness of PCR serial screening and the clinical applicability in everyday routine. PCR screening offers a noninvasive repeatable aid to the diagnosis of IFI.

Occurrence of Proteus mirabilis associated with two species of venezuelan oysters

The fecal contamination of raw seafood by indicators and opportunistic pathogenic microorganisms represents a public health concern. The objective of this study was to investigate the presence of enteric bacteria colonizing oysters collected from a Venezuelan touristic area. Oyster samples were collected at the northwestern coast of Venezuela and local salinity, pH, temperature, and dissolved oxygen of seawater were recorded. Total and fecal coliforms were measured for the assessment of the microbiological quality of water and oysters, using the Multiple Tube Fermentation technique. Analyses were made using cultures and 16S rRNA gene sequencing. Diverse enrichment and selective culture methods were used to isolate enteric bacteria. We obtained pure cultures of Gram-negative straight rods with fimbriae from Isognomon alatus and Crassostrea rhizophorae. Our results show that P. mirabilis was predominant under our culture conditions. We confirmed the identity of the cultures by biochemical tests, 16S rRNA gene sequencing, and data analysis. Other enterobacteria such as Escherichia coli, Morganella morganii and Klebsiella pneumoniae were also isolated from seawater and oysters. The presence of pathogenic bacteria in oysters could have serious epidemiological implications and a potential human health risk associated with consumption of raw seafood.

Molecular characterization of Cryptosporidium spp. from HIV infected patients from an urban area of Brazil

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.