The hemolytic ability, the presence of cyl genes, and the diagnostic accuracy of cytolysin molecular detection were investigated in the genus Enterococcus by using 164 strains from 20 different species (26 reference strains, 42 clinical isolates from human and veterinary origin, and 96 isolates from ewe cheese and milk). Hemolysis was assayed with sheep and horse erythrocytes and under aerobic or anaerobic conditions. Screening of cytolysin genes ( cylL _L , cylL _S , cylM , cylB , and cylA ) was performed with new specific primers and the anaerobic assay of beta-hemolysis was used as the “gold standard” for the evaluation of cyl gene-based PCRs. Since beta-hemolysis and cyl genes were found in 10 and 14 species, respectively, the hemolytic ability seems to be spread throughout the genus Enterococcus . Beta-hemolysis was observed in 6 of 26 (23%) reference strains, 14 of 42 (33%) clinical isolates, and 6 of 96 (6%) food isolates. The presence of cyl genes was detected in 15 of 26 (58%) reference strains, 37 of 42 (88%) clinical isolates, and 67 of 96 (70%) food isolates. These data indicate a virulence potential in food isolates, reinforcing the need of their safety assessment. Analysis of phenotypic-genotypic congruence suggests a divergent sequence evolution of cyl genes and the effect of environmental factors in the regulation of cytolysin expression. Evaluation of the diagnostic accuracy of cytolysin molecular detection points to cylL _L -based PCR and cylL _L L _S MBA -based PCR as the most reliable approaches. Nevertheless, the low sensitivity (46%) and gene variability indicated by our study strongly recommend the phenotypic assay for the assessment of hemolytic ability in enterococci, followed by the molecular screening of cyl genes in nonhemolytic strains to evaluate their virulence potential.

The biosynthesis of Ag and Au nanoparticles (NPs) was investigated using an extremophilic yeast strain isolated from acid mine drainage in Portugal. Three distinct studies were performed, namely, the growth of yeast strain in presence of metal ions, the use of yeast biomass for the metal nanoparticles synthesis, and of the supernatant obtained after 24-hour incubation of yeast biomass in water. The extremophilic strain under study was able to grow up to an Ag ion concentration of 1.5 mM whereas an increase of Au ion concentration over 0.09 mM caused a strong inhibitory effect. A successful route for the metal NPs synthesis was obtained using the yeast biomass. When the washed yeast cells were in contact with Ag or Au solutions, AgNPs smaller than 20 nm were produced, as for the AuNPs diameter ranged from 30 to 100 nm, as determined through transmission electron microscopy and confirmed by energy-dispersive X-ray spectra. The supernatant-based strategy provided evidence that proteins were released to the medium by the yeasts, which could be responsible for the formation and stabilisation of the Ag NPs, although the involvement of the cell wall seems fundamental for AuNPs synthesis.

In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene andrpoC1phylogenies. A highly congruent association was observed between STTR- and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon–Wiener (J′) indices a high diversity was observed within all species, withPlanktothrix agardhiishowing the lowest diversity values (D=0.83;J′=0.88) andAphanizomenon flos-aquaethe highest ones (D=J′=0.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.