Sławomir Wąsik

Laser interferometric and cultivation methods for measurement of colistin/ampicilin and saponin interactions with smooth and rough of Proteus mirabilis lipopolysaccharides and cells

Laser interferometry is commonly used in permeability studies of soluble substances. In this study a modification that allowed testing partially insoluble mixtures is presented. The modification relies on the measurement of diffusion from 1% agarose gel. As a model for this study, two Proteus mirabilis strains were used that differ in polysaccharide content: smooth P. mirabilis S1959 strain and its Re-type mutant, strain R45. By laser interferometry and precipitation it is shown that R45 lipopolysaccharide is more effective in binding colistin. It has been shown with the laser interferometric method that saponins, which are detergent-like substances of plant origin, partially enhance the interaction of colistin with the S and Re types of P. mirabilis. These results were confirmed with whole cell Proteus studies. The saponin partially inhibited the growth of the S and Re P. mirabilis strains at doses of 31-500 microg/ml. A sub-inhibitory dose--15 microg/ml of saponins alone do not reduced the numbers of P. mirabilis S1959 and R45 cells. However, the presence of colistin or amipicillin and 15 microg/ml of saponins reduced the amount of P. mirabilis S1959 and R45 cells. The saponins enhanced sensitivities of S and R P. mirabilis cells towards colistin and amipicillin. One may proposed that saponins binds to lipid A part of LPS may resulted on an increase in bacterial cell wall outer-membrane permeabilities and by that facilitated antibiotics penetration into the bacterial cells. In conclusion, the laser interferometric method is a useful tool for studies of lipopolysaccharide-antibiotic interactions even if the tested substances are not fully soluble in water.

Morphological changes in Proteus mirabilis O18 biofilm under the influence of a urease inhibitor and a homoserine lactone derivative

Proteus mirabilis is a pathogenic gram-negative bacterium that frequently causes kidney infections, typically established by ascending colonization of the urinary tract. The present study is focused on ureolytic activity and urease inhibition in biofilms generated by P. mirabilis O18 cells. Confocal microscopy revealed morphological alterations in biofilms treated with urea and a urease inhibitor (acetohydroxamic acid, AHA), as some swarmer cells were found to protrude from the biofilm. The presence of a quorum-sensing molecule (N-butanoyl homoserine lactone, BHL) increased biofilm thickness and its ureolytic activity. Laser interferometric determination of diffusion showed that urea easily diffuses through P. mirabilis biofilm, while AHA is blocked. This may suggest that the use of urease inhibitors in CAUTIs may by less effective than in other urease-associated infections. Spectroscopic studies revealed differences between biofilm and planktonic cells indicating that polysaccharides and nucleic acids are involved in extracellular matrix and biofilm formation.

Laser interferometry analysis of ciprofloxacin and ampicillin diffusion from liposomal solutions to water phase

The paper presents experimental investigations of diffusion of antibiotics (ciprofloxacin or ampicillin) into the water phase from mixtures of neutral or negatively charged liposomes, and antibiotic–liposome interactions. Using the laser interferometry technique, the amounts and fluxes of released antibiotics, concentration field evolution, and the velocity of the concentration boundary layer’s “growth” were determined. To avoid the limitations of membranes, a measurement system without the artificial boundary of phases with a free water–solution interface has been proposed. It was found that the diffusion of anionic and neutral liposomes into the water phase was insignificant and mainly the diffusion of antibiotics was measured. Differences in the diffusion kinetics of ciprofloxacin and ampicillin from liposomal solutions to the water phase were observed. Ampicillin diffused more efficiently than ciprofloxacin regardless of the liposomal solution type. Moreover, the amount of ampicillin and ciprofloxacin released from the anionic liposomal phase was higher than that from the neutral one. Our results confirm that ciprofloxacin at neutral pH shows little tendency to bind neutral liposomes. Additionally, it was also observed that ciprofloxacin disrupts negatively charged liposomes as a final effect of antibiotic–lipid interactions.

The use of lysozyme modified with fluorescein for the detection of Gram-positive bacteria.

Lysozyme (1,4-β-N-acetylmuramidase) is commonly applied in the food, medical, and pharmaceutical industries. In this study, we tested a novel application of fluorescein-modified lysozyme (using carboxyfluorescein with a triazine-based coupling reagent) as a new tool for the detection of Gram-positive soil bacteria. The results, obtained by cultivation methods, fluorescence analysis, and laser interferometry, showed that, after optimization, fluorescein-modified lysozyme could be used to evaluate the prevalence of Gram-positive bacteria essential in bioremediation of soils with low pH, such as those degraded by sulfur.

Laser interferometric investigation of solute transport through membrane-concentration boundary layer system

We investigate diffusive transport in a membrane system with a horizontally mounted membrane under concentration polarization conditions performed by a laser interferometry method. The data obtained from two different theoretical models are compared to the experimental results of the substance flux. In the first model, the membrane is considered as infinitely thin, while in the second one as a wall of finite thickness. The theoretical calculations show sufficient correspondence with the experimental results. On the basis of interferometric measurements, the relative permeability coefficient (ζs) for the system, consisting of the membrane and concentration boundary layers, was also obtained. This coefficient reflects the concentration polarization of the membrane system. The obtained results indicate that the coefficient ζs of the membrane-concentration boundary layer system decreases in time and seems to be independent of the initial concentration of the solute.

The effects of nickel(II) complexes with imidazole derivatives on pyocyanin and pyoverdine production by Pseudomonas aeruginosa strains isolated from cystic fibrosis

Pseudomonas aeruginosa infection is problematic in patients with cystic fibrosis (CF). P. aeruginosa secretes a diversity of pigments, such as pyocyanin and pyoverdine. The aim of this study was to evaluate the effects of complexes of nickel(II) ([Ni(iaa)2(H2O)2]·H2O (iaa = imidazole-4-acetate anion), [Ni(1-allim)6](NO3)2 (1-allim = 1-allylimidazole) and NiCl2 on pyocyanin and pyoverdine production by 23 strains of P. aeruginosa isolated from cystic fibrosis under growth conditions specific for the CF respiratory system. The antibacterial effects and biophysical properties of the tested substances were measured by spectrofluorometric techniques, as well as by laser interferometry, confocal and atomic force microscopy. The cytotoxic properties of all compounds were measured by Annexin/IP assay against A549 cells. All tested compounds have no effect on pyocyanin production and decrease the pyoverdine secretion in about 40% of tested P. aeruginosa strains at non-cytotoxic range of concentrations. Imidazole-4-acetate anion and 1-allylimidazole have good diffusion properties in the mature P. aeruginosa PAO1 biofilm. In conclusion, the tested nickel(II) complexes do not have clinical implications in P. aeruginosa eradication in cystic fibrosis. The diffusion properties of 1-allylimidazole and imidazole-4-acetate and their lack of effect on A549 cells suggest that they might be considered for chemical synthesis with other transition metals.

Laser interferometric analysis of glucose and sucrose diffusion in agarose gel.

The paper presents the investigation results of glucose and sucrose diffusion in agarose gel studied with laser interferometry method and the results of fluorescence analysis of the macroscopic gel structure. The diffusion kinetics of these substances released from aqueous solutions of a molar concentration of 0.05 M into the agarose solutions of concentrations of 0.5% and 3% in two gravitational configurations of measuring system was analysed. In the first configuration the solute diffused according, whereas in the second one - opposite to the gravitational force. The diffusion was analysed in the time period between 120 and 2400 s with a time interval of Δt = 120 s. We observed that the convective instabilities were damped well by the agarose gel, which gives the possibility of the interferometric studies of the diffusive transport for other substances in different gravitational configurations of the system. The time characteristics of glucose and sucrose fluxes in both configurations of the system and the gravitational polarisation coefficient values were obtained. The substantial differences in fluxes of glucose and sucrose diffused according and opposite to the gravitational force were observed. Additionally, we observed the differences between the diffusive fluxes of these substances in both configurations in dependence on the gel solution concentration (which is associated with gel porosity dependent on its concentration) and the kind of diffused substance.

Laser Interferometric Determination of Liposomes Diffusion Through Artificial Membranes

Cellulose membranes have been used in the medical field as wound dressing and artificial skin material for the treatment of skin wounds such as burns, ulcers, and grafts and as an adjuvant in dermal abrasions (Cheng et al., 2009; Fontana et al., 1990). Using liposomes as drug-delivery strategies in connection with a cellulose-based wound dressing might have important impact on increasing the efficacy of wound healing. The concentration of liposomes used in this therapy is crucial. The amount of liposomes might permit increasing the efficacy of their diffusion through the cellulose membrane. In the present study we propose a laser interferometric method as a novel technique to investigate liposome diffusion through membranes and cellulose-based wound dressing to optimize drug delivery.

Laser Interferometry Method as a Novel Tool in Endotoxins Research

Optical properties of chemical substances are widely used at present for assays thereof in a variety of scientific disciplines. One of the measurement techniques applied in physical sciences, with a potential for novel applications in biology, is laser interferometry. This method enables to record the diffusion properties of chemical substances. Here we describe the novel application of laser interferometry in chitosan interactions with lipopolysaccharide by detection of colistin diffusion. The proposed model could be used in simple measurements of polymer interactions with endotoxins and/or biological active compounds, like antibiotics.

Testing Sorption Properties of Halloysite by Means of the Laser Interferometry Method

The objective of the present study has been to test the laser interferometry method in terms of its usability for investigating sorption properties of minerals. This method was used to test the absorption capacity of halloysite with reference to glucose, which is often found in industrial wastewater and whose excess can disturb the environmental eco-balance. The sorption capacity of halloysite was thus determined indirectly, basing on the comparison of concentration profiles as well as time characteristics of glucose quantities released from the control solution and from the solution incubated with a halloysite adsorbent. An analysis of glucose diffusion was conducted in a two-chamber membrane system. On the basis of the obtained concentration profiles, the evolution of the concentration field was determined; so were the removal efficiency (%) and the amount of glucose adsorbed at equilibrium (qe, mg/g). The obtained results confirm good sorption properties of halloysite with respect to the investigated substance as well as usability of the method for this kind of investigations. The presented tests suggest that the measurement set-up can be optimised in such as way that visual rendering and testing the kinetics of the adsorbed substance direct release from the studied material become possible.