Genomic insights into the expansion of carbapenem-resistant Klebsiella pneumoniae within Portuguese hospitals.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) are a public health concern, causing infections with a high mortality rate, limited therapeutic options and challenging infection control strategies. In Portugal, the CR-KP rate has increased sharply, but the factors associated with this increase are poorly explored. In order to address this question, phylogenetic and resistome analysis were used to compare the draft genomes of 200 CR-KP isolates collected in 2017-2019 from five hospitals in the Lisbon region, Portugal. Most CR-KP belonged to sequence type (ST) 13 (29%), ST17 (15%), ST348 (13%), ST231 (12%) and ST147 (7%). Carbapenem resistance was conferred mostly by the presence of KPC-3 (74%) or OXA-181 (18%), which were associated with IncF/IncN and IncX plasmids, respectively. Almost all isolates were multi-drug resistant, harbouring resistance determinants to aminoglycosides, beta-lactams, trimethoprim, fosfomycin, quinolones and sulphonamides. In addition, 11% of isolates were resistant to colistin. Colonizing and infecting isolates were highly related, and most colonized patients (89%) reported a previous hospitalization. Moreover, among the 171 events of cross-dissemination identified by core genome multi-locus sequence typing data analysis (fewer than five allelic differences), 41 occurred between different hospitals and 130 occurred within the same hospital. The results suggest that CR-KP dissemination in the Lisbon region results from acquisition of carbapenemases in mobile genetic elements, influx of CR-KP into the hospitals by colonized ambulatory patients, and transmission of CR-KP within and between hospitals. Prudent use of carbapenems, patient screening at hospital entry, and improvement of infection control are needed to decrease the burden of CR-KP infection in Portugal.

Photobiomodulation and salivary glands: a systematic review.

To date, there is no compilation of evidence-based information associating photobiomodulation effect and salivary glands. This systematic review aims to assess photobiomodulation effect of low intensity laser on salivary glands in the presence of systemic diseases. MEDLINE databases were searched in duplicate through December 2018. In vivo studies and clinical trials were included if photobiomodulation was performed in salivary glands of animal (rat or mice) or human in the presence of systemic disease. The methodological quality was assessed in duplicate using the modified Newcastle-Ottawa scale (NOS). Search strategy identified 483 potentially eligible articles, and 449 were included. The Boolean search naturally leads to a high amount of works the majority of which were excluded because the analysis of the title and abstract demonstrated it was not focusing on PBM. Only 34 studies were selected for the full-text analysis, of which 5 were excluded due to non-use of photobiomodulation, 4 due to lack of control group, 2 because they were studies of cell cultures and 1 because they did not have the total of animals used. Thus, 21 papers were included for the critical evaluation of the impact of photobiomodulation on the major salivary glands; the studies used rats (n = 10) and humans (n = 11). Although studies reported an increase in the salivary rate, decrease in pain, and increase in quality of life after the PBM, the lack of standards for the application of light and reporting of the parameters, make it hard to reproduce the results. This topic is still in need for further research.

Influence of UV-B radiation on lead speciation in the presence of natural particles of estuarine waters.

The influence of UV-B irradiation on filtered and non-filtered water samples collected in a non-polluted area of Tagus estuary was evaluated in this study. In the laboratory, both samples were titrated with lead (Pb(+2)) followed by differential pulse anodic stripping voltammetry (DPASV), before and after 1 and 10 days under UV-B irradiation. Metal-ligand complexing parameters were obtained based on a macromolecular heterogeneous ligand described by two distinct sites with a labile behavior, and a third small homogeneous weaker group, which concentration was determined from a potentiometric titration. Under UV-B radiation, the complexing strength decreased with time in both (filtered and non-filtered) irradiated samples, but this effect was more pronounced in the non-filtered water, which might be due to some adsorption of dissolved macromolecular ligands on the particles and/or to further degradation of dissolved organic matter (DOM). Furthermore, the presence of particles favored the break-down of the macromolecular ligand under long UV-B exposure time. These results present ecological implications for the estuarine ecosystems such as bioavailability and toxicity.

Electrochemical methods for speciation of trace elements in marine waters. Dynamic aspects.

The contribution of electrochemical methods to the knowledge of dynamic speciation of toxic trace elements in marine waters is critically reviewed. Due to the importance of dynamic considerations in the interpretation of the electrochemical signal, the principles and recent developments of kinetic features in the interconversion of metal complex species will be presented. As dynamic electrochemical methods, only stripping techniques (anodic stripping voltammetry and stripping chronopotentiometry) will be used because they are the most important for the determination of trace elements. Competitive ligand exchange-adsorptive cathodic stripping voltammetry, which should be considered an equilibrium technique rather than a dynamic method, will be also discussed because the complexing parameters may be affected by some kinetic limitations if equilibrium before analysis is not attained and/or the flux of the adsorbed complex is influenced by the lability of the natural complexes in the water sample. For a correct data interpretation and system characterization the comparison of results obtained from different techniques seems essential in the articulation of a serious discussion of their meaning.

Fractions composition study of the pyrolysis oil obtained from sewage sludge treatment plant.

In this work the parameters of Low Temperature Conversion--LTC were applied in a centrifuged sludge from a sewage treatment plant located in Rio de Janeiro, Brazil. Before the conversion, the sludge was dried and analyzed by TGA to observe its behavior with increasing temperature. The chemical composition of the crude pyrolysis oil was analyzed by FTIR, 1H NMR and GC-MS. The results showed that the oil is a mixture of hydrocarbons, oxygenated and nitrogenated compounds. Using a catalytic treatment it was possible to fractionate the oil where the predominant constituents were hydrocarbons showing that the cracking was effective. An important result was the difference between the calorific value of dry sludge (10 MJ kg(-1)), the pyrolysis oil (36 MJ kg(-1)) and one of the fractions separated by catalytic cracking (40 MJ kg(-1)) when compared with commercial diesel (45 MJ kg(-1)).

Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain.

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 ± 1) × 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.

Matrix effect on paralytic shellfish toxins quantification and toxicity estimation in mussels exposed to Gymnodinium catenatum.

Paralytic shellfish toxins were quantified in whole tissues of the mussel Mytilus galloprovincialis exposed to blooms of the dinoflagellate Gymnodinium catenatum in Portuguese coastal waters. A validated liquid chromatography method with fluorescence detection, involving pre-chromatographic oxidation was used to quantify carbamoyl, N-sulfocarbamoyl and decarbamoyl toxins. In order to test for any matrix effect in the quantification of those toxins, concentrations obtained from solvent and matrix matched calibration curves were compared. A suppression of the fluorescence signal was observed in mussel extract or fraction in comparison to solvent for the compounds dcGTX2 + 3, GTX2 + 3 and GTX1 + 4, while an enhancement was found for C1 + 2, dcSTX, STX, B1, dcNEO and NEO. These results showed that a matrix effect varies among compounds. The difference of concentrations between solvent and matrix matched calibration curves for C1 + 2 (median = 421 ng g⁻¹) exceeded largely the values for the other quantified compounds (0.09-58 ng g⁻¹). Those differences were converted into toxicity differences, using Oshima toxicity equivalence factors. The compounds C1 + 2 and dcNEO were the major contributors to the differences of total toxicity in the mussel samples. The differences of total toxicity were calculated in ten mussel samples collected during a 10-week blooming period in Portuguese coastal lagoon. Values varied between 53 and 218 µg STX equivalents kg⁻¹. The positive differences mean that the estimated toxicity using solvent calibration curves exceed the values taking into account the matrix. For the toxicity interval 200-800 µg STX equivalents kg⁻¹ an increase was found between 44 and 28%.

Improvement in soil and sorghum health following the application of polyacrylate polymers to a Cd-contaminated soil.

Contamination of soils with cadmium (Cd) is a serious global issue due to its high mobility and toxicity. We investigated the application of insoluble polyacrylate polymers to improve soil and plant health. Sorghum was grown in a Cd-contaminated sandy soil. Polyacrylate polymers at 0.2% (w/w) were added to half of the soil. Control soil without plants was also included in the experiment. Growth of sorghum was stimulated in the polymer-amended soil. The concentration of Cd in the shoots, and the activities of catalase and ascorbate peroxidase decreased in plants from polymer-amended soil compared with unamended control. The amount of CaCl(2)-extractable Cd in the polymer-amended soil was 55% of that in the unamended soil. The Cd extracted in sorghum shoots was 0.19 mg per plant grown on soil without polymer and 0.41 mg per plant grown on polymer-amended soil. The total amount of Cd removed from each pot corresponded to 1.5 and more than 6% of soil CaCl(2)-extractable Cd in unamended and polymer-amended soil, respectively. The activities of soil acid phosphatase, beta-glucosidase, urease, protease and cellulase were greatest in polymer-amended soil with sorghum. In conclusion, the application of polyacrylate polymers to reduce the bioavailable Cd pool seems a promising method to enhance productivity and health of plants grown on Cd-contaminated soils.

Competition of copper and/or calcium in nickel-pectin interaction by potentiometric and voltammetric techniques.

The interaction between nickel and pectin extracted from citrus fruit was studied in 0.10 M KNO(3), at pH 5.5 and 25 degrees C. Differential pulse and/or square wave polarography were used to determine free nickel. For a high coverage degree (theta) of the pectin by the metal ion a good fitting was observed between experimental results and the model that includes both complex species, ML and ML(2) (M for the metal ion and L for the ligand). In the ML(2) species, Ni(II) interacts with two carboxylate groups of different chains, resulting in an inter-chain association. For low theta values, the formation of ML(2) is hindered due to the repulsion between the negative charges of carboxylic groups in two independent segments of pectin. The influence of calcium or copper ions on the free nickel concentration, in the presence of pectin, may lead to a decrease in free nickel concentration, contrary to what would be expected from direct competition between Ca(II) or Cu(II) and Ni(II) for the pectin binding sites. This is due to the partial neutralisation of the negative carboxylic charges by the positive charges of the divalent cations, which favours NiL(2) formation through the association of independent chains.

Adaptation of a commercial ion selective fluoride electrode to a tubular configuration for analysis by flow methodologies.

The determination of fluoride ions in water samples is accomplished by using a tubular flow through detector constructed by drilling a channel through a commercially available LaF(3) crystal electrode in such a way that the original contacts of the non-modified unit are maintained. Its performance when incorporated in both FIA and SIA systems was evaluated and the results show that the tubular unit retains the characteristics of the non-modified electrode. In SIA conditions an extended linear range of response and lower detection limit were achieved when compared with the electrode performance in FIA conditions. These aspects together with the additional advantage of low sample and reagent consumptions in SIA when compared to FIA, makes the incorporation of the proposed tubular ISE in a SIA system the preferred approach for on line determination and monitoring of fluoride content in natural water samples.

Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study.

A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 +/- 4 and 42 +/- 5 microM was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 +/- 0.5) x 10(6) and (1.0 +/- 0.5) x 10(6) M(-1) s(-1) for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer.

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E (0)', of 230 +/- 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 +/- 0.2 x 10(5) M(-1) s(-1) was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.

Redox chemistry of low-pH forms of tetrahemic cytochrome c3.

Desulfovibrio vulgaris Hildenborough cytochrome c(3) contains four hemes in a low-spin state with bis-histidinyl coordination. High-spin forms of cytochrome c(3) can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV-visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.

Influence of the sediment on lead speciation in the Tagus estuary.

The aim of this work is to study the influence of the Tagus estuarine sediment on lead speciation in the overlying natural water. The water sample in the presence of the sediment was contaminated three times with Pb(II) in a laboratory experiment. In different periods of time, at 1-7 days after each contamination, small volumes of water were titrated with lead. The titration was followed by anodic stripping voltammetry in differential pulse mode. Before and after contamination systematic analysis of the voltammetric parameters (peak current, peak potential and peak width) were carried out to get a clear picture of Pb(II) complexation in the soluble fraction in contact with the sediment. Two main types of organic ligands, macromolecular ligands and small compounds, were detected before contamination. Both of them form labile complexes (degree of lability within the timescale of some milliseconds). The small compounds, with a diffusion coefficient similar to that of the free metal ion, present a homogeneous behaviour in terms of Pb complexation. On the other hand macromolecular ligands, with a diffusion coefficient of 1.2 x 10(-6) cm2s(-1), can be described by two different binding groups, which might be of phenolic and carboxylic type as presented by humic matter. The sediment eliminated lead contamination (10(-6) moldm(-3) was the maximum concentration added) from 12 dm3 of water (surface of 8 dm2) within 2 days. It was also found that the sediment released organic ligands responsible for both labile and inert Pb complexes "seen" by voltammetry. The release of organic ligands that decreases the bioavailability of Pb(II) was clearly detected 1 week after contamination. Therefore, the sediment acts as a buffer for lead through two mechanisms against lead contamination: removing lead ions from the solution and releasing organic ligands to the water column. In a short period of time the sediment responds as a self-cleaning system for Pb(II) contamination in the estuarine water, which may have a very important influence in environmental pollution.

The finding of Echinostoma (Trematoda: Digenea) and hookworm eggs in coprolites collected from a Brazilian mummified body dated 600-1,200 years before present.

The identification of parasites from ancient cultures expands our list of parasites infective to extant humans. A partially mummified human body from the archeological site of Lapa do Boquete, Minas Gerais State, Brazil, was recently discovered. It was interred between 600 and 1,200 yr ago. Dietary analysis showed that the mummified body was from a society that had a mixed subsistence of agriculture and gathering of wild foods. Coprolites from the body contained numerous helminth eggs. The eggs were identified as those of Echinostoma sp. and hookworm. Hookworm infection in pre-Columbian populations is already established, but this is the first evidence of Echinostoma sp. eggs found in human coprolites. The diagnosis of a true infection, as opposed to false parasitism, is discussed. The possibility of Echinostoma ilocanum infection is discussed, as this is a common species found in humans in the Asiatic region, which could have been introduced in South America in the pre-Columbian period. Alternative possibilities are also considered, including indigenous Brazilian Echinostoma species.

Flow amperometric determination of pharmaceuticals with on-line electrode surface renewal.

In this article, a flow system developed for the amperometric determination of a great variety of pharmaceuticals that are known to lead the rapid poisoning of the working electrode surface is described. The referred system was made up of two parallel flow channels that shared the voltammetric detector of tubular configuration, whose movement in the manifold followed the concept of multi-site location of detector. In this way, after each measurement, the conditioning of the working electrode was possible through the passage by its surface of a regeneration solution without implying the alteration of the carrier that flowed in the analytical channel of the manifold. The methodology proposed was evaluated through the determination of two drugs belonging to two distinct therapeutic groups: an antihypertensive (diltiazem) and a non-steroid anti-inflammatory (nimesulide). The results obtained after evaluation of various pharmaceutical formulations on the Portuguese market were in the case of diltiazem compared with those supplied by the reference US Pharmacopoeia XXIV method, with no statistically significant differences having been observed for a confidence interval of 95%. In the case of nimesulide, since no official reference method exists, a series of recovery experiments were proceeded with and a mean value of 101.1% with a R.S.D. of 0.7% was obtained.

Square-wave voltammetric techniques for determination of psychoactive 1,4-benzodiazepine drugs.

Direct square-wave voltammetry (SWV) and square-wave cathodic stripping voltammetry (SWCSV) at hanging mercury drop electrodes have been developed for determination of the psychoactive 1,4-benzodiazepine compounds clonazepam, bromazepam, midazolam, diazepam, medazepam, and flurazepam over a wide range of concentrations. Analysis was performed with better precision, lower detection limits, and much more rapidly than by previously reported voltammetric techniques. The applicability of both procedures is discussed. The methods were applied to the analysis of commercially available tablets with minimum sample manipulation. The accuracy of the results was accessed by comparison with those obtained by use of the methods described in official pharmacopoeias. A critical comparison with those procedures is also presented.

[Subdural empyema secondary to sinusitis: four pediatric cases]. / Empiema subdural secundario a sinusitis: cuatro casos pediátricos.

INTRODUCTION: Perinasal sinus infections is a common and benign condition in most pediatric cases. Because of the widespread use of antibiotics, intracranial extension of sinusitis is rarely seen today. Nevertheless, the clinician must be aware of the gravity of this condition, because late recognition and delay in treatment can increase mortality and morbidity. The authors made a retrospective study of pediatric patients admitted to Garcia de Orta Hospital between 1996 and 2001 with the diagnosis of subdural empyema and sinusitis. CASE REPORTS: Four patients were identified, with ages between 9 and 13 years. Prodromal manifestations of sinusitis were present in all, followed several days later by headaches, fever, vomiting and neurological abnormalities. Two patients presented in the emergency department with an acute confusional state and a non convulsive status epilepticus. The other two patients had a longer duration of disease, severe deterioration of consciousness and focal neurologic signs. Medical treatment was started in all cases at admission, but none improved significantly before being submitted to surgical intervention, which was repeated several times in two patients. Streptococcus milleri and anaerobic organisms were isolated. There was no mortality and global evolution was favorable, with a median follow up of 32 months. CONCLUSIONS: Clinical presentation of subdural empyema can be relatively inespecific, requiring a high degree of suspicion. Facing a young adolescent with fever of unknown origin associated with any neurological abnormality and previous history of sinusitis, neuroradiological investigation shoul be asked. Early diagnosis and treatment are the mainstays of successful outcome.

Copper-psychoactive drug complexes: a voltammetric approach to complexation by 1,4-benzodiazepines.

Copper complexation by the 1,4-benzodiazepines medazepam, diazepam, flurazepam, nitrazepam, and clonazepam was investigated using differential pulse polarography and cyclic voltammetry at a mercury electrode in 0.10 M KNO3 and pH 7.0 +/- 0.1. Because the 1,4-benzodiazepines are easily reduced at a mercury electrode through the two-electron reduction of the 4,5-azomethine functional group, copper reduction, as well as that of the ligands, was analyzed under varying experimental conditions. In most situations adsorption phenomena occurred and their influence on voltammetric signals had to be carefully analyzed. The voltammetric behavior was then interpreted in terms of complex formation. The results showed that all benzodiazepines can act as ligands toward copper(II) ions, forming 1:1 and 1:2 complexes with similar stabilities. The stoichiometric acidity constants of the benzodiazepines under study were also determined by potentiometric titration in water-ethanol medium and 0.10 M KNO3 and then extrapolated to 0% concentration of ethanol.