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Braga T.M.,New University of Lisbon | Marujo P.E.,IBET | Pomba C.,The Interdisciplinary Center | Lopes M.F.S.,New University of Lisbon
Journal of Antimicrobial Chemotherapy | Year: 2011

Objectives: To investigate the role of a putative small multidrug resistance transporter, annotated in Enterococcus faecalis V583 genome as EFA0010 (we will refer to this gene as qacZ), in decreased susceptibility to biocides. Methods: A derivative strain of V583, susceptible to erythromycin (V583ErmS), was complemented with pORI23 carrying the qacZ gene (strain EF-SAVE1). MICs of benzalkonium chloride, chlorhexidine and ethidium bromide were determined for the complemented strain and wild-type. RT-PCR and ethidium bromide efflux assays were performed in order to fully understand the role and specificity of the qacZ gene. The presence of qacZ in 73 enterococcal strains from different origins was investigated by PCR, and MICs of benzalkonium chloride and chlorhexidine were determined for the same strains. Results: The complemented strain, EF-SAVE1, presented a higher MIC of benzalkonium chloride (8 mg/L) than V583ErmS (4 mg/L); the MICs of chlorhexidine and ethidium bromide were the same for both strains, 4 mg/L and 16 mg/L, respectively. Expression of qacZ was found to be higher in EF-SAVE1 and constitutive, i.e. not inducible by any of the three tested biocides. Overexpression of qacZ was not responsible for changes in ethidium bromide efflux. This gene was present in 52% of the enterococcal isolates studied and the MICs of benzalkonium chloride and chlorhexidine ranged between 2 and 8 mg/L. Conclusions: We demonstrate the involvement of the qacZ gene in tolerance to the quaternary ammonium compound benzalkonium chloride, but not ethidium bromide. This work constitutes the first report of a biocide resistance mechanism in E. faecalis, and reveals its dissemination amongst the genus Enterococcus. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. Source


Ribeiro T.,New University of Lisbon | Santos S.,IBET | Marques M.I.M.,Instituto Gulbenkian Of Ciencia | Gilmore M.,Massachusetts Eye and Ear Infirmary | De Fatima Silva Lopes M.,New University of Lisbon
International Journal of Antimicrobial Agents | Year: 2011

Subinhibitory concentrations of vancomycin are known to induce a cell-wall stimulon in some Gram-positive pathogens, but this has never been studied in the genus Enterococcus. In this study, Enterococcus faecalis V583 strain was submitted to a subinhibitory concentration of vancomycin. DNA microarray technology was used to analyse the transcriptomic changes induced by this antibiotic. EF2292, annotated as a hypothetical protein in the E. faecalis V583 genome, was highly induced in response to vancomycin exposure, to similar levels as the vanB operon genes. We investigated further and provide evidence for co-transcription of ef2292 with vanY BWH BBX B genes. It was also demonstrated that expression of ef2292 is under the control of vanR BS B and it is proposed to name it vanV. This gene was found not to be required for vancomycin resistance under the conditions tested, thus coding for another accessory protein in the vanB operon. vanV was detected in some, but not all, E. faecalis carrying the vanB operon, suggesting that this operon can have different composition amongst E. faecalis isolates. © 2011 Elsevier B.V. and the International Society of Chemotherapy. Source


Vicente T.,IBET | Peixoto C.,IBET | Alves P.M.,IBET | Carrondo M.J.T.,IBET | Carrondo M.J.T.,New University of Lisbon
Journal of Chromatography A | Year: 2010

Product-related impurities constitute a major burden in the production of recombinant viral vectors for gene therapy and vaccination; it impairs not only the biological efficacy of the preparation but the process yield/productivity. Recombinant baculovirus was used as an enveloped virus model to address this issue. Given that ion-exchange chromatography is a process of choice for purification of viral vectors, the analysis of the electrostatic behavior can be instrumental for the improvement of impurity removal. The main species, product (infective virus particle) and product-derived impurities (dsDNA-, glycoprotein-, and envelope-deprived baculovirus particles), were isolated and correspondent ζ potentials were analyzed through dynamic light scattering. A model of the virus based on the viral components critical for biological function is proposed. The contribution of these viral components to the overall particle electrostatic interaction energy profile (calculated between the particle and a putative ion-exchange surface) was assessed as a function of ionic strength and pH. This resulted in a deterministic tool capable of distinguishing the electrostatic properties of the infective virus particle from the major virus-related impurities. Within an ion-exchange bind-elute process, this knowledge helps narrow the optimization space in early stage process development for viral vectors by predicting the best selectivity conditions. © 2010 Elsevier B.V. Source


Pereira V.J.,IBET | Pereira V.J.,New University of Lisbon | Marques R.,IBET | Marques M.,IBET | And 3 more authors.
Water Research | Year: 2013

The effectiveness of free chlorine for the inactivation of fungi present in settled surface water was tested. In addition, free chlorine inactivation rate constants of Cladosporium tenuissimum, Cladosporium cladosporioides, Phoma glomerata, Aspergillus terreus, Aspergillus fumigatus, Penicillium griseofulvum, and Penicillium citrinum that were found to occur in different source waters were determined in different water matrices (laboratory grade water and settled water). The effect of using different disinfectant concentrations (1 and 3 mg/l), temperatures (21 and 4 °C), and pH levels (6 and 7) was addressed. The sensitivity degree of different fungi isolates to chlorine disinfection varied among different genera with some species showing a higher resistance to disinfection and others expected to be more prone to protection from inactivation by the water matrix components. When the disinfection efficiency measured in terms of the chlorine concentration and contact time (Ct) values needed to achieve 99% inactivation were compared with the Ct values reported as being able to achieve the same degree of inactivation of other microorganisms, fungi were found to be more resistant to chlorine inactivation than bacteria and viruses and less resistant than Cryptosporidium oocysts. © 2012 Elsevier Ltd. Source


Carvalho-Santos Z.,Instituto Gulbenkian Of Ciencia | Machado P.,Instituto Gulbenkian Of Ciencia | Alvarez-Martins I.,Instituto Gulbenkian Of Ciencia | Gouveia S.,Instituto Gulbenkian Of Ciencia | And 11 more authors.
Developmental Cell | Year: 2012

Cilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled by a central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. We characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. We show that assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and that the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135@s role in assembling highly stable MT-based structures, such as motile axonemes and centrioles. Source

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