Diversity and characterisation of coagulasenegative staphylococci isolated from healthy individuals in Portugal

In the past few years the interest in coagulase-negative staphylococci (CoNS) has significantly increased in human medicine. CoNS are common commensal colonisers of the human skin, although now also recognised as major nosocomial pathogens. Over the last decades, several studies have been carried out in order to understand the pathogenicity mechanisms of CoNS. The well known determinants in the pathogenesis of CoNS infections are their ability to form biofilms and an exceptional resistance to several antibiotics. Nevertheless, there is a lack of studies regarding the commensal lifestyle of these microorganisms. Additionally, it is now hypothesised that commensal bacteria might be a reservoir of pathogenic determinants. Therefore, the work described throughout this thesis was aimed to perform a phenotypic and genotypic characterisation of different CoNS species isolated from healthy Portuguese individuals. A total of 61 CoNS isolates, comprising 7 different species, were obtained and characterised at the level of biofilm formation and antibiotic susceptibility profiles. According to the results, biofilm formation ability and presence of biofilm-associated genes were commonly found features, highlighting their pivotal role in the colonising lifestyle of CoNS. This study also addressed the correlation between phenotypic and genotypic characteristics of biofilm formation, corroborating and raising questions about the importance of some genes in this process. Moreover, it was observed a great proportion of isolates with decreased susceptibility and multiple resistances to some important antibiotics. A significant association between antibiotic resistance and biofilm formation was also demonstrated, and some hypotheses about the nature of such association were provided. Lastly, the expression patterns of two biofilm-associated genes at two distinct biofilm developmental stages were determined, confirming their importance in the accumulative stage of biofilm formation. Overall, the results presented in this thesis indicate that staphylococcal skin flora might be an important reservoir of potentially pathogenic bacteria and, simultaneously, bring to light new perceptions about the molecular basis of staphylococcal biofilm formation, and the nature of the association between antibiotic resistance and biofilm formation.

Phenotypic impact of deregulated expression of class I histone deacetylases in urothelial cell carcinoma of the bladder

Deregulated expression of histone deacetylases (HDACs) has been implicated in tumorigenesis. Herein, we investigated class I HDACs expression in bladder urothelial cell carcinoma (BUCC), its prognostic value and biological significance. Significantly increased transcript levels of all HDACs were found in BUCC compared to 20 normal mucosas, and these were higher in lower grade and stage tumors. Increased HDAC3 levels were associated with improved patient survival. SiRNA experiments showed decrease cell viability and motility, and increased apoptosis. We concluded that class I HDACs play an important role in bladder carcinogenesis through deregulation of proliferation, migration and apoptosis, constituting putative therapeutic targets

Principal components and generalized linear modeling in the correlation between hospital admissions and air pollution

OBJECTIVE To analyze the association between concentrations of air pollutants and admissions for respiratory causes in children. METHODS Ecological time series study. Daily figures for hospital admissions of children aged < 6, and daily concentrations of air pollutants (PM10, SO2, NO2, O3 and CO) were analyzed in the Região da Grande Vitória, ES, Southeastern Brazil, from January 2005 to December 2010. For statistical analysis, two techniques were combined: Poisson regression with generalized additive models and principal model component analysis. Those analysis techniques complemented each other and provided more significant estimates in the estimation of relative risk. The models were adjusted for temporal trend, seasonality, day of the week, meteorological factors and autocorrelation. In the final adjustment of the model, it was necessary to include models of the Autoregressive Moving Average Models (p, q) type in the residuals in order to eliminate the autocorrelation structures present in the components. RESULTS For every 10:49 μg/m3 increase (interquartile range) in levels of the pollutant PM10 there was a 3.0% increase in the relative risk estimated using the generalized additive model analysis of main components-seasonal autoregressive – while in the usual generalized additive model, the estimate was 2.0%. CONCLUSIONS Compared to the usual generalized additive model, in general, the proposed aspect of generalized additive model − principal component analysis, showed better results in estimating relative risk and quality of fit.

Correlation modeling for a distributed scalable video codec based on the HEVC standard

The growing heterogeneity of networks, devices and consumption conditions asks for flexible and adaptive video coding solutions. The compression power of the HEVC standard and the benefits of the distributed video coding paradigm allow designing novel scalable coding solutions with improved error robustness and low encoding complexity while still achieving competitive compression efficiency. In this context, this paper proposes a novel scalable video coding scheme using a HEVC Intra compliant base layer and a distributed coding approach in the enhancement layers (EL). This design inherits the HEVC compression efficiency while providing low encoding complexity at the enhancement layers. The temporal correlation is exploited at the decoder to create the EL side information (SI) residue, an estimation of the original residue. The EL encoder sends only the data that cannot be inferred at the decoder, thus exploiting the correlation between the original and SI residues; however, this correlation must be characterized with an accurate correlation model to obtain coding efficiency improvements. Therefore, this paper proposes a correlation modeling solution to be used at both encoder and decoder, without requiring a feedback channel. Experiments results confirm that the proposed scalable coding scheme has lower encoding complexity and provides BD-Rate savings up to 3.43% in comparison with the HEVC Intra scalable extension under development. © 2014 IEEE.

Phylogeny and phenotypic characterization of pathogenic cryptococcus species and closely related saprobic taxa in the tremellales

Eukaryotic Cell, Vol.8, Nº3

High resolution stratigraphy and miocene facies correlation in Lisbon and Setúbal Peninsula (Lower Tagus basin, Portugal)

Correlation between facies associations (marine, estuarine and distal fluviatile environments) and disconformities, observed between Foz da Fonte (SW of Setúbal Peninsula) and Santa Iria da Azóia (NE of Lisbon) are presented. The precise definition of the marine-continental facies relationships improved very much the chronology of the depositional sequence boundaries. Tectonic and eustatic controls are discussed on the basis of subsidence rates variation.

High-resolution correlation of coastal cliff sections in the Lagos- Portimao Formation (Lower - Middle Miocene, central Algarve, Portugal)

This paper describes a high-resolution stratigraphic correlation scheme for the early to middle Miocene Lagos-Portimão Formation of central Algarve, southern Portugal. The Lagos Portimão-Formation of central Algarve is a 60 m thick package of horizontally bedded siliciclastics and carbonates. The bryozoan and mollusc dominated biofacies is typical of a shallow marine, warm-temperate climatic environment. We define four stratigraphic marker beds based on biofacies, lithology, and gamma-ray signatures. Marker bed 1 is a reddish shell bed composed predominantly of bivalve shells in various stages of fragmentation. Marker bed 2 is a fossiliferous sandstone / sandy rudstone characterized by bryozoan masses. Marker bed 3 is also a fossiliferous sandstone with abundant larger foraminifers and foliate bryozoans. Marker bed 4 is composed of three distinct layers; two fossiliferous sandstones with an intercalated shell bed. The upper sandstone unit displays thickets of the bryozoan Celleporaria palmate associated with the coral Culizia parasitica. This stratigraphic framework allows to correlate isolated outcrops within the stratigraphic context of the Lagos-Portimão Formation and to establish high resolution chronostratigraphic Sr-isotopic dating.

The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Solubilities of C‑Tetraalkylcalix[4]resorcinarenes in SCCO2: experimental measurements, characterization, and correlation

The solubilities of two C-tetraalkylcalix[4]resorcinarenes, namely C-tetramethylcalix[4]resorcinarene and C-tetrapentylcalix[4]resorcinarene, in supercritical carbon dioxide (SCCO2) were measured in a flow-type apparatus at a temperature range from (313.2 to 333.2) K and at pressures from (12.0 to 35.0) MPa. The C-tetraalkylcalix[4]resorcinarenes were synthesized applying our optimized procedure and fully characterized by means of gel permeation chromatography, infrared and nuclear magnetic resonance spectroscopy. The solubilities of the C-tetraalkylcalix[4]resorcinarenes in SCCO2 were determined by analysis of the extracts obtained by HPLC with ultraviolet (UV) detection methodology adapted by our team. Four semiempirical density-based models, and the SoaveRedlichKwong cubic equation of state (SRK CEoS) with classical mixing rules, were applied to correlate the solubility of the calix[4]resorcinarenes in the SC CO2. The physical properties required for the modeling were estimated and reported.

Off-the-person electrocardiography: performance assessment and clinical correlation

Previous work by our group introduced a novel concept and sensor design for “off-the-person” ECG, for which evidence on how it compares against standard clinical-grade equipment has been largely missing. Our objectives with this work are to characterise the off-the-person approach in light of the current ECG systems landscape, and assess how the signals acquired using this simplified setup compare with clinical-grade recordings. Empirical tests have been performed with real-world data collected from a population of 38 control subjects, to analyze the correlation between both approaches. Results show off-the-person data to be correlated with clinical-grade data, demonstrating the viability of this approach to potentially extend preventive medicine practices by enabling the integration of ECG monitoring into multiple dimensions of people’s everyday lives. © 2015, IUPESM and Springer-Verlag Berlin Heidelberg.

Evaluation of phenotypic markers associated with pathogenicity in the genus Listeria

A total of 130 Listeria strains were tested in order to evaluate lecithinase production and capacity for Congo red adsorption as markers of pathogenicity. The strains were identified according to acid production from sugars and by the CAMP test and the data were correlated with the ability to produce keratoconjunctivitis in guinea pigs. L. monocytogenes cultures presented 51.8% and 88.8% positivity rates for Congo red adsorption and lecithinase production, respectively, whereas 80.8% and 100% for L. innocua cultures were negative for the two test, respectively

Viscosity and self-diffusion coefficients of dialkyl adipates: a correlation scheme with predictive capabilities

A correlation and predictive scheme for the viscosity and self-diffusivity of liquid dialkyl adipates is presented. The scheme is based on the kinetic theory for dense hard-sphere fluids, applied to the van der Waals model of a liquid to predict the transport properties. A "universal" curve for a dimensionless viscosity of dialkyl adipates was obtained using recently published experimental viscosity and density data of compressed liquid dimethyl (DMA), dipropyl (DPA), and dibutyl (DBA) adipates. The experimental data are described by the correlation scheme with a root-mean-square deviation of +/- 0.34 %. The parameters describing the temperature dependence of the characteristic volume, V-0, and the roughness parameter, R-eta, for each adipate are well correlated with one single molecular parameter. Recently published experimental self-diffusion coefficients of the same set of liquid dialkyl adipates at atmospheric pressure were correlated using the characteristic volumes obtained from the viscosity data. The roughness factors, R-D, are well correlated with the same single molecular parameter found for viscosity. The root-mean-square deviation of the data from the correlation is less than 1.07 %. Tests are presented in order to assess the capability of the correlation scheme to estimate the viscosity of compressed liquid diethyl adipate (DEA) in a range of temperatures and pressures by comparison with literature data and of its self-diffusivity at atmospheric pressure in a range of temperatures. It is noteworthy that no data for DEA were used to build the correlation scheme. The deviations encountered between predicted and experimental data for the viscosity and self-diffusivity do not exceed 2.0 % and 2.2 %, respectively, which are commensurate with the estimated experimental measurement uncertainty, in both cases.

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.

Role of ß-lactamase operon on mecA expression in Staphylococcus aureus

RESUMO: Os Staphylococcus aureus resistentes à meticilina (MRSA, do inglês “methicillin-resistant Staphylococcus aureus”) são um dos principais agentes responsáveis por infeções hospitalares. Os MRSA são resistentes a praticamente todos os antibióticos β-lactâmicos devido a dois mecanismos principais: produção de β-lactamase (bla), codificada pelo gene blaZ, e produção de uma proteína de ligação à penicilina (PBP2a, do inglês “penicillin binding protein 2”), codificada pelo gene mecA. Estes dois genes são regulados por sistemas homólogos, constituídos por um sensor-transdutor (BlaR1 e MecR1) e um repressor (BlaI e MecI), de tal modo que ambos os sistemas são capazes de co-regular os genes mecA e blaZ, embora com eficiências de indução muito diferentes. De facto, a indução mediada pelo sistema mecI-mecR1 é tão lenta que se acredita que este sistema não está funcional na maioria das estirpes MRSA. No entanto, dados recentes do nosso laboratório, demonstram a ausência de relação entre a presença do gene mecI e o nível de resistência à meticilina em estirpes MRSA epidémicas, e também que, o fenótipo de resistência da grande maioria das estirpes não é perturbado pela sobre-expressão em trans do repressor mecI. Curiosamente, as duas estirpes em que a expressão da resistência foi afetada pela sobre-expressão do mecI são negativas para o locus da β-lactamase, o que sugere que este locus pode interferir diretamente com a repressão do gene mecA mediada pelo MecI. Nesta tese de mestrado esta hipótese foi explorada usando estratégias de biologia molecular e ensaios fenotípicos da resistência aos -lactâmicos. Os resultados obtidos demonstram que a presença do plasmídeo nativo da β-lactamase não só anula a repressão mediada pelo MecI, como também aumenta o nível de resistência das estirpes parentais. Várias hipóteses foram então formuladas para explicar estas observações. Dados preliminares, em conjunto com evidências experimentais publicadas, sugerem que o BlaI forma hetero-dímeros com o MecI que, após a indução, são inativados eficientemente pelo BlaR1. Em conclusão, estes resultados apresentam novas perspetivas para o mecanismo de regulação do mecA e para uma nova importante função do operão da β-lactamase para o fenótipo das estirpes MRSA.-------------------ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen and is also emerging in the community. MRSA is cross-resistant to virtually all β-lactam antibiotics and has acquired two main resistance mechanisms: production of β-lactamase (bla), coded by blaZ, and production of penicillin binding protein 2a (PBP2a), coded by mecA. Both genes are regulated by homologous sensor-transducers (BlaR1 and MecR1) and repressors (BlaI and MecI), and coregulation of mecA and blaZ by both systems has been demonstrated, although with remarkable different efficiencies. In fact, induction of mecA by mecI-mecR1 is so slow that it is believed it is not functional in most MRSA strains. However, recent data from our laboratory has unexpectedly demonstrated that not only there is no correlation between the presence of mecI gene and the resistance level in epidemic MRSA strains, but also that for most strains there were no significant changes on the resistance phenotype upon the mecI overexpression in trans. Interestingly, the two strains in which mecI overexpression affected the resistance expression were negative for the bla locus, suggesting that this locus may interfere directly with the MecI-mediated repression of mecA and account for those puzzling observations. In this master thesis we have explored this hypothesis using molecular biology strategies and phenotypic analysis of -lactam resistance. The data obtained demonstrate that the presence of a wild-type plasmid containing the bla locus not only disrupts the MecImediated repression, but also significantly enhances the expression of resistance. Several preliminary hypotheses were formulated to explain these observations and preliminary data, together with published evidence, support the working model that BlaI forms functional hetero-dimers with MecI, which upon induction are readily inactivated by BlaR1. These results provide new insights into the regulatory mechanism(s) of mecA and open new perspectives for the role of β-lactamase operon in the MRSA phenotype.

Phenotypic and molecular characterization of Salmonella Enteritidis strains isolated in São Paulo, Brazil

In São Paulo State, Brazil, the epidemic increase in isolation of Salmonella Enteritidis has been observed since 1994. A total of 105 S. Enteritidis strains (72 from human and 33 from non-human sources) isolated during the period 1975-1995, previously characterized by phage typing, was analyzed by antimicrobial susceptibility, plasmid profile, and ribotyping. Over 70% of the strains were susceptible to all antimicrobial agents tested, however, multiple resistance to antimicrobials was observed among the studied strains, mainly those from hospitalized patients. Phage type 8 (PT-8) was predominant among the strains isolated during the period of 1975-1992, but in the following years, PT-4 was the most frequent phage type identified. Seven different plasmid profiles were detected and 96% of the isolates harbored a plasmid of approximately 36 MDa. Ribotyping discriminated fourteen ribotypes (R1 to R14) among the strains examined. By analysis of dendrogram the strains were included in three groups with similarity level of 60%. The obtained results indicate that, a single ribotype (R11), determined for PT-4 strains isolated from 1993, characterizes the epidemic clone of S. Enteritidis in our region.