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 1The Iberian Pyrite Belt extends from Portugal to Spain and is one of the most important pyrite regions in the world. Its aquatic reservoirs display extreme conditions characterized by low pH and high concentrations of heavy metals. In this study, the diversity of microeukaryotes was analysed at the abandoned mines of Sa˜o Domingos (Portugal) and at Rio Tinto (Spain). DNA was extracted from water samples and a set of eukaryotic universal primers directed to the small subunit rRNA genes (rDNA) was used. The amplicons were analysed by molecular cloning and temperature gradient gel electrophoresis (TGGE). In addition, a fungi-specific primer set was also used in TGGE experiments. The fungi-specific primers contributed to a substantial increase in the number of fungal taxa found due, probably, to the relative low density of fungal structures. Several microorganisms, belonging (or closely related) to the ascomycetous yeast Pichia acaciae, the basidiomycetous yeasts Cryptococcus humicola and Cystofilobasidium bisporidii, the green algae Chlamydomonas noctigama and Chlorella protothecoides var. acidicola and some uncultured microeukaryotes were present at both localities, which suggests that specific microorganisms are adapted to the peculiar conditions of the Iberian Pyrite Belt extreme environments. However, in spite of the similarities, a higher algal richness was observed at S. Domingos, whereas for R. Tinto the richness of fungi was more prominent

Assessment of Metronidazole Susceptibility in Helicobacter pylori: Statistical Validation and Error Rate Analysis of Breakpoints Determined by the Disk Diffusion Test

Metronidazole susceptibility of 100 Helicobacter pylori strains was assessed by determining the inhibition zone diameters by disk diffusion test and the MICs by agar dilution and PDM Epsilometer test (E test). Linear regression analysis was performed, allowing the definition of significant linear relations, and revealed correlations of disk diffusion results with both E-test and agar dilution results (r(2) = 0.88 and 0.81, respectively). No significant differences (P = 0.84) were found between MICs defined by E test and those defined by agar dilution, taken as a standard. Reproducibility comparison between E-test and disk diffusion tests showed that they are equivalent and with good precision. Two interpretative susceptibility schemes (with or without an intermediate class) were compared by an interpretative error rate analysis method. The susceptibility classification scheme that included the intermediate category was retained, and breakpoints were assessed for diffusion assay with 5-μg metronidazole disks. Strains with inhibition zone diameters less than 16 mm were defined as resistant (MIC > 8 μg/ml), those with zone diameters equal to or greater than 16 mm but less than 21 mm were considered intermediate (4 μg/ml < MIC ≤ 8 μg/ml), and those with zone diameters of 21 mm or greater were regarded as susceptible (MIC ≤ 4 μg/ml). Error rate analysis applied to this classification scheme showed occurrence frequencies of 1% for major errors and 7% for minor errors, when the results were compared to those obtained by agar dilution. No very major errors were detected, suggesting that disk diffusion might be a good alternative for determining the metronidazole sensitivity of H. pylori strains

Photoprotective Bioactivity Present in a Unique Marine Bacteria Collection from Portuguese Deep Sea Hydrothermal Vents

Abstract: Interesting biological activities have been found for numerous marine compounds. In fact, screening of phylogenetically diverse marine microorganisms from extreme environments revealed to be a rational approach for the discovery of novel molecules with relevant bioactivities for industries such as pharmaceutical and cosmeceutical. Nevertheless, marine sources deliverables are still far from the expectations and new extreme sources of microbes should be explored. In this work, a marine prokaryotic collection from four Mid-Atlantic Ridge (MAR) deep sea hydrothermal vents near the Azores Islands, Portugal, was created, characterized and tested for its photoprotective capacity. Within 246 isolates, a polyphasic approach, using chemotaxonomic and molecular typing methods, identified 23-related clusters of phenetically similar isolates with high indexes of diversity. Interestingly, 16S rRNA gene sequencing suggested the presence of 43 % new prokaryotic species. A sub-set of 139 isolates of the prokaryotic collection was selected for biotechnological exploitation with 484 bacterial extracts prepared in a sustainable upscalling manner. 22 % of the extracts showed an industriall

Yamadazyma barbieri f.a. sp nov., an ascomycetous anamorphic yeast isolated from a Mid-Atlantic Ridge hydrothermal site (-2300m) and marine coastal waters

Two yeast strains that are members of the same species were isolated from different marine habitats, i.e. one from Mid-Atlantic Ridge ocean water samples located in the direct vicinity of black smokers near the Rainbow deep-sea hydrothermal vent and one from Brazilian marine water samples off the Ipanema beach. Strains CLIB 1964(T) and CLIB 1965 are anamorphic ascomycetous yeasts affiliated to the Yamadazyma clade of Saccharomycetales. Interestingly, these strains were phylogenetically and distinctly positioned into a group of species comprising all species of the genus Yamadazyma isolated from marine habitats including deep-sea hydrothermal vents, i.e. Candida atmosphaerica, C. spencermartinsiae, C. atlantica, C. oceani and C. taylorii. These strains differed significantly in their D1/D2 domain sequences of the LSU rRNA gene from the closely related species mentioned above, by 2.6, 3.0, 3.4, 3.8 and 6.0 %, respectively. Internal transcribed spacer region sequence divergence was also significant and corresponded to 4.6, 4.7, 4.7, 12.0 and 24.7% with C. atlantica, C. atmosphaerica, C. spencermartinsiae, C. oceani and C. taylorii, respectively. Phenotypically, strains CLIB 1964(T) and CLIB 1965 could be distinguished from closely related species by their inability to assimilate L-sorbose. CLIB 1964(T) (= CBS 14301(T) = UBOCC-A-214001(T)) is the designated type strain for Yamadazyma barbieri sp. nov. The MycoBank number is MB 815884