Dissemination of NDM-1 carbapenemase-producer Providencia stuartii strains in Romanian hospitals: a multicentre study.

Several Romanian hospitals have noted increasing isolation of Providencia stuartii strains in recent years, with an alarming rate of carbapenem resistance. In order to provide molecular epidemiological data regarding their dissemination, 77 P. stuartii strains collected from five hospitals located in different regions of Romania were analysed. All strains harboured IncA/C plasmid, and 67 carried the blaNDM-1 gene. Six clonal clusters were differentiated by pulsed-field gel electrophoresis. The predominant subtype was found in all five hospitals. Our study highlights the need for efficient infection-control measures, the optimization of antibiotic use and the targeted surveillance for carbapenemase-producing P. stuartii.

Illumina short-read and MinION long-read WGS to characterize the molecular epidemiology of an NDM-1 Serratia marcescens outbreak in Romania.

Background and Objectives: Serratia marcescens is an emerging nosocomial pathogen, and the carbapenemase blaNDM has been reported in several surveys in Romania. We aimed to investigate the molecular epidemiology of S. marcescens in two Romanian hospitals over 2010-15, including a neonatal NDM-1 S. marcescens outbreak. Methods: Isolates were sequenced using Illumina technology together with carbapenem-non-susceptible NDM-1-positive and NDM-1-negative Klebsiella pneumoniae and Enterobacter cloacae to provide genomic context. A subset was sequenced with MinION to fully resolve NDM-1 plasmid structures. Resistance genes, plasmid replicons and ISs were identified in silico for all isolates; an annotated phylogeny was reconstructed for S. marcescens. Fully resolved study NDM-1 plasmid sequences were compared with the most closely related publicly available NDM-1 plasmid reference. Results: 44/45 isolates were successfully sequenced (S. marcescens, n = 33; K. pneumoniae, n = 7; E. cloacae, n = 4); 10 with MinION. The S. marcescens phylogeny demonstrated several discrete clusters of NDM-1-positive and -negative isolates. All NDM-1-positive isolates across species harboured a pKOX_NDM1-like plasmid; more detailed comparisons of the plasmid structures demonstrated a number of differences, but highlighted the largely conserved plasmid backbones across species and hospital sites. Conclusions: The molecular epidemiology is most consistent with the importation of a pKOX_NDM1-like plasmid into Romania and its dissemination amongst K. pneumoniae/E. cloacae and subsequently S. marcescens across hospitals. The data suggested multiple acquisitions of this plasmid by S. marcescens in the two hospitals studied; transmission events within centres, including a large outbreak on the Targu Mures neonatal unit; and sharing of the pKOX_NDM1-like plasmid between species within outbreaks.

Seroprevalence of Bartonella species, Coxiella burnetii and Toxoplasma gondii among patients with hematological malignancies: A pilot study in Romania.

Patients receiving immunosuppressive cancer treatments in settings where there is a high degree of human-animal interaction may be at increased risk for opportunistic zoonotic infections or reactivation of latent infections. We sought to determine the seroprevalence of selected zoonotic pathogens among patients diagnosed with haematologic malignancies and undergoing chemotherapeutic treatments in Romania, where much of the general population lives and/or works in contact with livestock. A convenience sample of 51 patients with haematologic cancer undergoing chemotherapy at a referral clinic in Cluj-Napoca, Romania, was surveyed regarding animal exposures. Blood samples were obtained and tested for evidence of infection with Bartonella species, Coxiella burnetii and Toxoplasma gondii, which are important opportunistic zoonotic agents in immunocompromised individuals. 58.8% of participants reported living or working on a farm, and living or working on a farm was associated with contact with livestock and other animals. 37.5% of participants were IgG seroreactive against one or more of five Bartonella antigens, and seroreactivity was statistically associated with living on farms. Farm dwellers were 3.6 times more likely to test IgG seroreactive to Bartonella antibodies than non-farm dwellers. 47.1% of the participants tested T. gondii IgG positive and 13.7% tested C. burnetii IgG positive, indicating past or latent infection. C. burnetii IgM antibodies were detected in four participants (7.8%), indicating possible recent infection. These results indicate that a large proportion of patients with haematologic cancer in Romania may be at risk for zoonotic infections or for reactivation of latent zoonotic infections, particularly with respect to Bartonella species. Special attention should be paid to cancer patients' exposure to livestock and companion animals in areas where much of the population lives in rural settings.

Species distribution and susceptibility profile to fluconazole, voriconazole and MXP-4509 of 551 clinical yeast isolates from a Romanian multi-centre study.

This is the first multi-centre study regarding yeast infections in Romania. The aim was to determine the aetiological spectrum and susceptibility pattern to fluconazole, voriconazole and the novel compound MXP-4509. The 551 isolates were identified using routine laboratory methods, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and DNA sequence analysis. Susceptibility testing was performed using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) method and breakpoints. The yeasts originated from superficial infections (SUP, 51.5 %), bloodstream infections (BSI, 31.6 %) and deep-seated infections (DEEP, 16.9 %), from patients of all ages. Nine genera and 30 species were identified. The 20 Candida species accounted for 94.6 % of all isolates. C. albicans was the overall leading pathogen (50.5 %). Lodderomyces elongisporus is reported for the first time as a fungaemia cause in Europe. C. glabrata and Saccharomyces cerevisiae, as well as the non-Candida spp. and non-albicans Candida spp. groups, showed decreased fluconazole susceptibility (<75 %). The overall fluconazole resistance was 10.2 %. C. krusei accounted for 27 of the 56 fluconazole-resistant isolates. The overall voriconazole resistance was 2.5 % and was due mainly to C. glabrata and C. tropicalis isolates. Fluconazole resistance rates for the three categories of infection were similar to the overall value; voriconazole resistance rates differed: 4 % for BSI, 3.2 % for DEEP and 1.4 % for SUP. The antifungal activity of MXP-4509 was superior to voriconazole against C. glabrata and many fluconazole-resistant isolates. There was a large percentage of non-albicans Candida isolates. A large part of the high fluconazole resistance was not acquired but intrinsic, resulting from the high percentage of C. krusei.

Nebivolol induces a hyperpolarizing effect on smooth muscle cells in the mouse renal artery by activation of beta-2-adrenoceptors.

Nebivolol is a highly selective beta(1)-adrenoceptor antagonist with vasodilator properties involving the vascular endothelium, but its effect on the smooth muscle cells (SMC) is still unclear. In this paper, we tested the effect of nebivolol on renal artery smooth muscle cells and investigated the cellular mechanism involved. To this purpose, the denuded renal arteries isolated from mice were studied in vitro using the myograph and the nitric oxide (NO) sensor techniques, while the SMC in culture were analyzed by the patch-clamp technique. The myograph technique was used to assay the vasodilator effect of nebivolol on the arterial muscular layer, and to establish the optimal dose of the drug to be tested on single SMC by the patch-clamp technique. Using both the myograph and the patch-clamp techniques, we examined the potential contribution of beta(2)-adrenoceptors and Ca(2+)-activated K(+) channels to the nebivolol-induced effects, by exposing the denuded arteries and SMC cultures to specific inhibitors such as butoxamine (100 micromol/l), tetraethylammonium (TEA, 1 mmol/l), and iberiotoxin (100 nmol/l). The direct measurement of NO using the NO sensor enabled us to evaluate if nebivolol induces/or not the release of NO in denuded renal arteries. The results of this study show that nebivolol exerts vasodilator effects on the SMC in the denuded renal arteries and the maximal response is achieved at a concentration of 50 micromol/l. Nebivolol effects involve binding to the beta(2)-adrenoceptors and the subsequent activation of Ca(2+)-activated K(+) channels in SMC, with no contribution of NO. Taken together, the study brings new insights into the mechanism underlying the nebivolol-induced arterial vasodilation.

Physiology. Cold current in thermoreceptive neurons.

We sense the temperature of our skin and surroundings using specific thermoreceptors, which are sensitive to cold and warmth, but little is known about how these receptors transduce temperature into electrical activity. We have discovered an inward ionic current that is activated by moderate cooling in a small number of rat sensory neurons. This current has features that are found in intact cold receptors, including sensitization by menthol, adaptation upon sustained cooling, and modulation by calcium, and is likely to be important in cold sensing.

A system for applying rapid warming or cooling stimuli to cells during patch clamp recording or ion imaging.

We describe a system for superfusing small groups of cells at a precisely controlled and rapidly adjustable local temperature. Before being applied to the cell or cells under study, solutions are heated or cooled in a chamber of small volume ( approximately 150 microl) and large surface area, sandwiched between four small Peltier elements. The current through the Peltier elements is controlled by a microprocessor using a PID (proportional-integral-derivative) feedback algorithm. The chamber can be heated to at least 60 degrees C and cooled to 0 degrees C, changing its temperature at a maximum rate of about 7 degrees C per second; temperature ramps can be followed under feedback control at up to 4 degrees C per second. Temperature commands can be applied from the digital-to-analogue converter of any laboratory interface or generated digitally by the microprocessor. The peak-to-peak noise contributed by the system does not exceed that contributed by a patch pipette, holder and headstage, making it suitable for single channel as well as whole cell recordings.

Cold transduction by inhibition of a background potassium conductance in rat primary sensory neurones.

Transduction in cutaneous cold receptors is poorly understood at present. We have studied this question using dorsal root ganglion (DRG) neurones in primary culture as a model of the otherwise inaccessible receptor terminal. Whole-cell recordings during cooling from 32 to 20 degrees C revealed a large depolarization (>8mV) in 22 of 88 DRG neurones (25%), sometimes accompanied by action potentials. In cold-sensitive neurones cooling inhibited a time-independent background K+ current (Icold) which was resistant to tetraethylammonium and 4-aminopyridine. Ouabain elicited a substantially smaller depolarization than cooling, and no action potentials. We conclude that excitation by cooling in this model is primarily due to inhibition of Icold and that the previously suggested role of the Na+/K+ adenosine triphosphatase is secondary. We suggest that Icold may underlie cold transduction in cutaneous thermoreceptors.

Slowed inactivation at positive potentials in a rat axonal K+ channel is not due to preferential closed-state inactivation.

We have investigated slow inactivation in a rat axonal K+ channel, the I channel. Using voltage steps to potentials between -70 mV and +80 mV, from a holding potential of -100 mV, we observed a marked slowing of inactivation at positive potentials: the time constant was 4.5+/-0.4 s at -40 mV (mean +/- S.E.M.), increasing to 14.7+/-2.0 s at +40 mV. Slowed inactivation at positive potentials is not consistent with published descriptions of C-type inactivation, but can be explained by models in which inactivation is preferentially from closed states (which have been developed for Kv2.1 and some Ca2+ channels). We tested two predictions of preferential closed-state models: inactivation should be more rapid during a train of brief pulses than during a long pulse to the same potential, and the cumulative inactivation measured with paired pulses should be greater than the inactivation at the same time during a continuous pulse. The I channel does not behave according to these predictions, indicating that preferential closed-state inactivation does not explain the slowing of inactivation we observe at positive potentials. Inactivation of the I channel therefore differs both from C-type inactivation, as presently understood, and from the inactivation of Kv2.1.

Hydrodynamic effects on the solute transport across endothelial pores and hepatocyte membranes.

In this short note we propose a simple and rapid procedure to calculate the net quantity of metabolites absorbed by hepatocytes from blood plasma. The blood movement through sinusoids determines an opposed circulation of plasma through the space of Disse. Hydrodynamic considerations lead to the conclusion that hepatocytes absorb for their own synthesis processes a quantity of metabolites in a volume flow of the order of 10(-12) nl s(-1) through a sieve plate surface with an area of 1 microm2. At pathological temperature (40 degrees C), the excess of the net absorbed volume flow for the entire sinusoidal surface of the mammalian liver may be as high as 1.9 nl s(-1). Some observations on the effect of red and white blood cells on the chylomicron traffic through endothelial pores are made.

Interaction of the antioxidant flavonoid quercetin with planar lipid bilayers.

Our capacitance and conductance measurements on reconstituted planar lipid bilayers (BLM) suggest an insertion of the flavonoid quercetin (QCT) in the membranes, which is concentration- and pH-dependent. Interaction of the flavonoid with the membrane has no impact on either structure or integrity of the lipid bilayer. The QCT molecules penetrate the lipid bilayer by intercalating between the flexible acyl chains of the phospholipids, the deepest insertion occuring in acidic medium, when QCT is neutral and completely liposoluble. Results indicated that aggregation of QCT within the hydrophobic core is accompanied by an increase of the transmembrane conductance following an alteration of the hydrophobic barrier for small electrolytes. By contrast, within alkaline media where QCT is deprotonated, the reaction site of the flavonoid is restricted to the hydrophilic domain of the membrane. This significantly changes the double layer capacitance as the negatively charged QCT molecules become sandwiched between polar headgroups at the bilayer surface. At highest alkaline pH, the transmembrane conductance was not affected, since QCT did not perturb the molecular packing of the hydrocarbonic acyl chains of the phospholipids. Results also demonstrated that changes in physical properties of the lipid bilayers following interstitial QCT embedding within either the hydrophobic domain or the polar headgroup domain may be related to both its lipophilic nature and interactions with the electric dipole moments of the polar headgroups of phospholipids. Data also demonstrated that translocation of QCT in the polar part of the lipid bilayer, at physiological pH and salt conditions, may be correlated with its optimized radical scavenging activity. This paper discusses the significance of the free radical scavenging capacity and antioxidant efficiency of QCT.

Cu2+ reveals different binding sites of amiloride and CDPC on the apical Na channel of frog skin.

The effect of Cu2+ ions, present in the mucosal bathing solution, on the transepithelial short-circuit current (Isc) and conductance (Gt) and on the blocker-induced noise of apical Na channels, was studied on the isolated ventral skin of the frog Rana temporaria. Cu2+ effects were concentration-dependent, the full effect being reached at 50 micromol/l. Cu2+ increased Isc and Gt; this effect was eliminated by high concentrations of amiloride (30 micromol/l) and of CDPC (150 micromol/l). Cu2+ markedly reduced the corner frequency (fc) of the Na channel noise, while having virtually no effect on the fc of CDPC-induced noise. Cu2+ reduces the association rate constant of amiloride to the Na channel to one third; this effect is interpreted as indicating competition between Cu2+ and amiloride for the same (negatively charged) binding site on the channel, while CDPC appears to bind on a different site.

Characteristics of ionic transport processes in fish intestinal epithelial cells.

A general mathematical version of the cell model of a leaky epithelium for the NaCl absorption is presented, analysed and integrated numerically. The model consists in the adequate differential equations that describe the rate of change of the intracellular ion concentrations and are expressed in strict accordance with the law of mass conservation. The model includes many state variables representing ion concentrations, the cell volume, and membrane potentials. Ion movements are described by the Michaelis-Menten kinetics or by the constant field flux equation (Goldman-Hodgkin-Katz). In this paper, we model the intracellular ion concentrations, change in the cell volume, the transmembrane flux and membrane potentials of intestinal epithelium of both fresh water and sea water fish, and generate several simulations (in both the steady state and the transient state analysis) that appear to accord with prior experimental data in this area. For the ion movements of the sea water fish intestine, there were included a Na+/K+ pump, a K(+)-Cl- symport system, the K+ and Cl- channels in the basolateral membrane, whereas a Na(+)-K(+)-2Cl- cotransporter for NaCl absorption and K+ channels are located in the apical membrane. In the fresh water fish intestinal cells, the NaCl absorption is performed by two coupled antiporters Na+/H+ and Cl-/HCO3- presumably responsible for the intracellular pH regulation. In this type of cells, Na+ and K+ channels are located within the apical membrane, whereas Cl- channels are located within the basolateral membrane. The osmotically induced water transport across the apical and basolateral membranes has been taken into account as well. The simulations plot the steady state values for membrane potential difference, short-circuit current and intracellular ionic concentrations using the magnitude of the transmembrane flux through the Na+/K+ pump and Na(+)-K(+)-2Cl- cotransporter, or the basolateral Cl- permeability as dependent variables. The model behaves appropriately with regard to several experimental studies regarding the hyperpolarization (sea water fish intestine) and depolarization (fresh water fish intestine) of the apical membrane potential and inhibition of the short-circuit flux with reduced NaCl absorption. The model is also used to make several analytical predictions regarding the response of the membrane potential and ionic concentrations to variations in the basolateral Cl- flux. Furthermore, maintaining conservation of both mass and electroneutrality and taking into account the osmolar forces is an important advantage, because it allows a rigorous analysis of the relationship between membrane potential difference, volume and flux. The model can be used in the analysis and planning of the experiments and is capable of predicting the instantaneous values of ionic fluxes and intracellular concentrations and of cell volume.

Fourier analysis of potential oscillations of a liquid membrane for the discrimination of taste substances.

Electrical potential oscillations were obtained across a liquid membrane composed of nitrobenzene/picric acid placed between two aqueous phases in the presence of various taste (i.e. salty, sweet and bitter) substances. The influence of these compounds on electrical oscillations was studied using Fourier analysis to establish a "fingerprint" of the substance that can be correlated with its taste index. Various concentrations of each substance were tested to obtain a Fourier spectrum with discrete peaks which can be further processed. The electrical oscillations consisted of a number of weak damped oscillators, and the Fourier spectra of these signals were found to have a number of discrete peaks of decreasing amplitude at low frequencies (0-0.5 Hz). A correlation of the frequency of the first peak of the Fourier spectrum with the taste index was found for bitter substances, whereas for salty substances the amplitude of the first two peaks of the spectrum was correlated with the taste index.

The action of Cu2+ on the frog epithelium.

The mechanism of action of Cu2+ when applied to the external side of the frog skin preparation was investigated. Cu2+ acts most probably on the external barrier of this preparation, since it increases the transport pool of Na proportionally to the increase in the short-circuit current (I(sc)). Cu2+ does not open new routes for the Na+ entry since the stimulated I(sc) is still completely abolished by amiloride. The I(sc) dependence of Na concentration in the external medium is modified by cooper, since the Ksm value increases in addition to changes in I(sc).

Evaluation of the stimulatory capacity of procaine on Na transport through frog skin.

Procaine has different effects on various ionic conductive pathways through the frog skin. We investigated the season and temperature dependence of the stimulation by mucosal procaine, of the Na-conductive pathway. For this stimulation, we found higher half-maximal saturation constants (KNa) in winter animals (6.38 +/- 0.8 mmol/l), than in summer ones (4.03 +/- 0.7 mmol/l). Summer frogs kept for 2 weeks at 4 degrees C, reacted like winter frogs (6.24 +/- 0.8 mmol/l). However, the maximal sodium currents (INa max) did not depend on temperature adaptation. Procaine-induced increased of KNa is associated with an increase of INa. The effects of procaine associated with BIG (benzoylimidazole-2-guanidine) were non-additive, while with vasopressin they were additive. A biphasic, dose-dependent response was recorded after procaine application to the inner surface. Vasopressin counteracted the serosal procaine-induced inhibition of the Na-transport.

Procaine effects on sodium and chloride transport in frog skin.

Procaine, a tertiary amine, has previously been shown to stimulate reversibly transepithelial Na transport across frog skin after application from the epithelial side. In the present study with intracellular recording from principal, i.e. amiloride-sensitive cells, we demonstrate that the stimulation results from increase in apical membrane Na permeability. A second effect of procaine (10-25 mmol/l) in the outside perfusion solution is a reversible increase of transepithelial conductance which drastically exceeds the predicted response of the transcellular Na pathway. It requires presence of chloride on the epithelial side and depends on the non-ionized molecule of procaine. Abolition of apical membrane Na uptake by amiloride or Na-free mucosal incubation decreases the magnitude but does not prevent the stimulatory effect of procaine. The origin of this gain in conductance from stimulation of a Cl-specific pathway is demonstrated by a highly significant correlation between the increases in electrically determined tissue conductance and partial Cl conductance, obtained from measurements of influx and efflux of Cl-36. Measurements with microelectrodes indicate that the stimulated Cl-specific pathway is distinct from the principal cells. Since procaine activates a conductive pathway with similar response pattern as spontaneously existing Cl conductance, it might be a valuable tool for investigating mode and way of Cl movement across epithelial tissues.

Procaine has opposite effects on passive Na and K permeabilities in frog skin.

Procaine has opposite effects on the active transport of Na+ when applied on the mucosal side of the frog skin [where it produces a stimulation of the short-circuit current (Isc)] or when added on the serosal side (where it produces an inhibition of Isc). In an attempt to reveal and localize the primary effect of procaine on either the apical or latero-basal membranes of the epithelial cells, we have tried to "chemically dissect" both membrane functions with inhibitors and ionophores. When applied on the apical side of the latero-basally depolarized epithelium, 25 mmol/l procaine increases Isc and Voc (transepithelial open-circuit potential), while decreasing the transepithelial resistance. The E1-E2 linearity domain of the I-V curves is narrowed. On the serosal side of the depolarized epithelium, the same concentration of procaine does not affect Isc and Voc (which are already inhibited) but it produces an increase in the transepithelial resistance (Rt). Procaine influence on the passive K+ permeability was studied by using the ionophore nystatin, which is assumed to form channels permeable to K+, when applied on the amiloride blocked apical membrane. In nystatin-treated epithelia, 25 mmol/l procaine on the apical side decreased Isc, Voc and Rt. In parallel experiments during Cl- substitution by SO2-(4), the procaine effects on Isc and Voc are no longer maintained, but transient.(ABSTRACT TRUNCATED AT 250 WORDS)

Procaine effects on the sodium transport in frog skin.

A study on the influence of procaine on the sodium transport properties in frog skin was carried out. The application of procaine hydrochloride on either the mucosal or the serosal sides of the isolated frog skin has opposite effects. When added to the mucosal compartment, the procaine (as well as two procaine based drugs: Gerovital H3 and Aslavital) biphasically increase the short-circuit current (Isc) with a noticeable "recline" phenomenon, and decrease the slope resistance, as given by the I-V curves. When applied in the serosal compartment, Isc is decreased and the slope resistance of the epithelium is increased. The procaine effect on the apical membranes shows a pronounced dependence on the external sodium concentration. The shift of the E2 inflection point (which indicates the critical intensity of the electric field at which the epithelial conductance changes), with respect to the transepithelial open-circuit potential, shows a rapid and quasi-exponential increase following the application of 25 mM procaine in addition to the different mucosal Na concentrations.