Chitosan-based matrix as a carrier for bacteriophages.

Wound healing is a dynamic and complex process where infection prevention is essential. Chitosan, thanks to its bactericidal activity against gram-positive and gram-negative bacteria, as well as anti-inflammatory and hemostatic properties, is an excellent candidate to design dressings for difficult-to-heal wound treatment. The great advantage of this biopolymer is its capacity to be chemically modified, which allows for the production of various functional forms, depending on the needs and subsequent use. Moreover, chitosan can be an excellent polymer matrix for bacteriophage (phage) packing as a novel alternative/supportive antibacterial therapy approach. This study is focused on the preparation and characteristics of chitosan-based material in the form of a film with the addition of Pseudomonas lytic phages (KTN4, KT28, and LUZ19), which would exhibit antibacterial activity as a potential dressing that accelerates the wound healing. We investigated the method of producing a polymer based on microcrystalline chitosan (MKCh) to serve as the matrix for phage deposition. We described some important parameters such as average molar mass, swelling capacity, surface morphology, phage release profile, and antibacterial activity tested in the Pseudomonas aeruginosa bacterial model. The chitosan polysaccharide turned out to interact with phage particles immobilizing them within a material matrix. Nevertheless, with the high hydrophilicity and swelling features of the prepared material, the external solution of bacterial culture was absorbed and phages went in direct contact with bacteria causing their lysis in the polymer matrix. KEY POINTS: • A novel chitosan-based matrix with the addition of active phages was prepared • Phage interactions with the chitosan matrix were determined as electrostatic • Phages in the matrix work through direct contact with the bacterial cells.

The Antibacterial Effect of PEGylated Carbosilane Dendrimers on P. aeruginosa Alone and in Combination with Phage-Derived Endolysin.

The search for new microbicide compounds is of an urgent need, especially against difficult-to-eradicate biofilm-forming bacteria. One attractive option is the application of cationic multivalent dendrimers as antibacterials and also as carriers of active molecules. These compounds require an adequate hydrophilic/hydrophobic structural balance to maximize the effect. Herein, we evaluated the antimicrobial activity of cationic carbosilane (CBS) dendrimers unmodified or modified with polyethylene glycol (PEG) units, against planktonic and biofilm-forming P. aeruginosa culture. Our study revealed that the presence of PEG destabilized the hydrophilic/hydrophobic balance but reduced the antibacterial activity measured by microbiological cultivation methods, laser interferometry and fluorescence microscopy. On the other hand, the activity can be improved by the combination of the CBS dendrimers with endolysin, a bacteriophage-encoded peptidoglycan hydrolase. This enzyme applied in the absence of the cationic CBS dendrimers is ineffective against Gram-negative bacteria because of the protective outer membrane shield. However, the endolysin-CBS dendrimer mixture enables the penetration through the membrane and then deterioration of the peptidoglycan layer, providing a synergic antimicrobial effect.

New Approach to Antifungal Activity of Fluconazole Incorporated into the Porous 6-Anhydro-α-l-Galacto-ß-d-Galactan Structures Modified with Nanohydroxyapatite for Chronic-Wound Treatments-In Vitro Evaluation.

New fluconazole-loaded, 6-Anhydro-α-l-Galacto-ß-d-Galactan hydrogels incorporated with nanohydroxyapatite were prepared and their physicochemical features (XRD, X-ray Diffraction; SEM-EDS, Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy; ATR-FTIR, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy), fluconazole release profiles and enzymatic degradation were determined. Antifungal activity of pure fluconazole was tested using Candida species (C. albicans, C. tropicalis, C. glabarata), Cryptococcus species (C. neoformans, C. gatti) and Rhodotorula species (R. mucilaginosa, R. rubra) reference strains and clinical isolates. Standard microdilution method was applied, and fluconazole concentrations of 2-250 µg/mL were tested. Moreover, biofilm production ability of tested isolates was tested on the polystyrene surface at 28 and 37 ± 0.5 °C and measured after crystal violet staining. Strains with the highest biofilm production ability were chosen for further analysis. Confocal microscopy photographs were taken after live/dead staining of fungal suspensions incubated with tested hydrogels (with and without fluconazole). Performed analyses confirmed that polymeric hydrogels are excellent drug carriers and, when fluconazole-loaded, they may be applied as the prevention of chronic wounds fungal infection.

Experimental and Theoretical Analysis of Metal Complex Diffusion through Cell Monolayer.

The study of drugs diffusion through different biological membranes constitutes an essential step in the development of new pharmaceuticals. In this study, the method based on the monolayer cell culture of CHO-K1 cells has been developed in order to emulate the epithelial cells barrier in permeability studies by laser interferometry. Laser interferometry was employed for the experimental analysis of nickel(II) and cobalt(II) complexes with 1-allylimidazole or their chlorides' diffusion through eukaryotic cell monolayers. The amount (mol) of nickel(II) and cobalt(II) chlorides transported through the monolayer was greater than that of metals complexed with 1-allylimidazole by 4.34-fold and 1.45-fold, respectively, after 60 min. Thus, laser interferometry can be used for the quantitative analysis of the transport of compounds through eukaryotic cell monolayers, and the resulting parameters can be used to formulate a mathematical description of this process.

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 (ζ(s)) 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 ζ(s) of the membrane-concentration boundary layer system decreases in time and seems to be independent of the initial concentration of the solute.

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 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 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.

Subdiffusion equation with Caputo fractional derivative with respect to another function in modeling diffusion in a complex system consisting of a matrix and channels.

Anomalous diffusion of an antibiotic (colistin) in a system consisting of packed gel (alginate) beads immersed in water is studied experimentally and theoretically. The experimental studies are performed using the interferometric method of measuring concentration profiles of a diffusing substance. We use the g-subdiffusion equation with the fractional Caputo time derivative with respect to another function g to describe the process. The function g and relevant parameters define anomalous diffusion. We show that experimentally measured time evolution of the amount of antibiotic released from the system determines the function g. The process can be interpreted as subdiffusion in which the subdiffusion parameter (exponent) α decreases over time. The g-subdiffusion equation, which is more general than the "ordinary" fractional subdiffusion equation, can be widely used in various fields of science to model diffusion in a system in which parameters, and even a type of diffusion, evolve over time. We postulate that diffusion in a system composed of channels and a matrix can be described by the g-subdiffusion equation, just like diffusion in a system of packed gel beads placed in water.

[Analysis of antibiotic diffusion from agarose gel by spectrophotometry and laser interferometry methods]. / Analiza dyfuzji antybiotyków z zelu agarozowego metodami spektrofotometrii i interferometrii laserowej.

The aim of this study was to analysis of antibiotics (ampicilin, streptomycin, ciprofloxacin or colistin) release from agarose gel by spectrophotmetry and laser interferometry methods. The interferometric system consisted of a Mach-Zehnder interferometer with a He-Ne laser, TV-CCD camera, computerised data acquisition system and a gel system. The gel system under study consists of two cuvettes. We filled the lower cuvette with an aqueous 1% agarose solution with the antibiotics at initial concentration of antibiotics in the range of 0.12-2 mg/ml for spectrophotmetry analysis or 0.05-0.5 mg/ml for laser interferometry methods, while in the upper cuvette there was pure water. The diffusion was analysed from 120 to 2400 s with a time interval of deltat = 120 s by both methods. We observed that 0.25-1 mg/ml and 0,05 mg/ml are minimal initial concentrations detected by spectrophotometric and laser interferometry methods, respectively. Additionally, we observed differences in kinetic of antibiotic diffusion from gel measured by both methods. In conclusion, the laser interferometric method is a useful tool for studies of antibiotic release from agarose gel, especially for substances are not fully soluble in water, for example: colistin.

Pseudomonas aeruginosa PA5oct Jumbo Phage Impacts Planktonic and Biofilm Population and Reduces Its Host Virulence.

The emergence of phage-resistant mutants is a key aspect of lytic phages-bacteria interaction and the main driver for the co-evolution between both organisms. Here, we analyze the impact of PA5oct jumbo phage treatment on planktonic/cell line associated and sessile P. aeruginosa population. Besides its broad-spectrum activity and efficient bacteria reduction in both airway surface liquid (ASL) model, and biofilm matrix degradation, PA5oct appears to persist in most of phage-resistant clones. Indeed, a high percentage of resistance (20/30 clones) to PA5oct is accompanied by the presence of phage DNA within bacterial culture. Moreover, the maintenance of this phage in the bacterial population correlates with reduced P. aeruginosa virulence, coupled with a sensitization to innate immune mechanisms, and a significantly reduced growth rate. We observed rather unusual consequences of PA5oct infection causing an increased inflammatory response of monocytes to P. aeruginosa. This phenomenon, combined with the loss or modification of the phage receptor, makes most of the phage-resistant clones significantly less pathogenic in in vivo model. These findings provide new insights into the general knowledge of giant phages biology and the impact of their application in phage therapy.

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.

How to determine a boundary condition for diffusion at a thin membrane from experimental data.

We present a method of deriving a boundary condition for diffusion at a thin membrane from experimental data. Based on experimental results obtained for normal diffusion of ethanol in water, we show that the derived boundary condition at a membrane contains a term with a Riemann-Liouville fractional time derivative of order 1/2. Such a form of the boundary condition shows that a transfer of particles through a thin membrane is a "long-memory process." The presented method is an example that an important part of the mathematical model of physical processes may be derived directly from experimental data.

The O-specific polysaccharide lyase from the phage LKA1 tailspike reduces Pseudomonas virulence.

Pseudomonas phage LKA1 of the subfamily Autographivirinae encodes a tailspike protein (LKA1gp49) which binds and cleaves B-band LPS (O-specific antigen, OSA) of Pseudomonas aeruginosa PAO1. The crystal structure of LKA1gp49 catalytic domain consists of a beta-helix, an insertion domain and a C-terminal discoidin-like domain. The putative substrate binding and processing site is located on the face of the beta-helix whereas the C-terminal domain is likely involved in carbohydrates binding. NMR spectroscopy and mass spectrometry analyses of degraded LPS (OSA) fragments show an O5 serotype-specific polysaccharide lyase specificity. LKA1gp49 reduces virulence in an in vivo Galleria mellonella infection model and sensitizes P. aeruginosa to serum complement activity. This enzyme causes biofilm degradation and does not affect the activity of ciprofloxacin and gentamicin. This is the first comprehensive report on LPS-degrading lyase derived from a Pseudomonas phage. Biological properties reveal a potential towards its applications in antimicrobial design and as a microbiological or biotechnological tool.

A proposed integrated approach for the preclinical evaluation of phage therapy in Pseudomonas infections.

Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications.

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.

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

Lysozyme (1,4-ß-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.

Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm.

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

Analysis of ciprofloxacin and gentamicin diffusion in Proteus mirabilis O18 biofilm by laser interferometry method.

Laser interferometry is a measurement technique used in physical sciences, with a potential for new applications in microbiology. Our previously studies, focused on the quantitative analysis of antibiotics diffusion through membranes or their releasing from gel structure, indicate that this method might be useful in analysis of substances diffusion across the bacterial biofilms. As antibiotic - biofilm interaction model, we tested above method for determination of ciprofloxacin or gentamicin diffusion through Proteus mirabilis O18 biofilm. Laser interferometry analysis of antibiotics diffusion showed that the amount of ciprofloxacin transported through mature biofilm is 1.9 times higher than gentamicin. It was correlated with lower level of gentamicin in compare to the level of ciprofloxacin in biofilm, which amounts were predicted in biofilm during diffusion process by laser interferometry method. We suggest that the analysis of antibiotic diffusion in biofilm might by helpful in evaluation of effectiveness of antibacterial agents.

The presence of anti-LPS antibodies and human serum activity against Proteus mirabilis S/R forms in correlation with TLR4 (Thr399Ile) gene polymorphism in rheumatoid arthritis.

OBJECTIVES: Proteus mirabilis strains are human pathogens responsible for urinary tract infections, which may also be involved in rheumatoid arthritis (RA). DESIGN AND METHODS: We determined whether the binding site of anti-LPS antibodies on the O-polysaccharide part of P. mirabilis LPS correlates with the level of TLR4 (Thr399Ile) gene polymorphism in the sera of RA patients. We investigated the deposition of C3d and C5b complement components on the P. mirabilis LPS. The ELISA method used in this study was optimized with LAL test and laser interferometry. RESULTS: Depending on LPS P. mirabilis used in these studies, the amount of antibodies in RA patients sera varied. We did not observe a correlation between anti-LPS antibodies binding and the level of TLR4 (Thr399Ile) gene polymorphism. We found that the lower complement components deposition by O49 in contrast to O9 LPS correlates with its reduced sensitivities to human complement-mediated killing. CONCLUSION: The immunological response against P. mirabilis LPS might play a role in rheumatoid arthritis.