Immobilization pattern of morphologically different microorganisms on bacterial cellulose membranes.

The aim of this study was to assess the immobilization pattern of microorganisms characterized by varying cell shapes and sizes (rod-shaped bacteria Lactobacillus delbruecki, spherical-shaped yeast Saccharomyces cerevisiae and hyphae forms of Yarrowia lipolytica) on bacterial cellulose of various material properties. The 'adsorption-incubation' method was used for the purposes of immobilization. The immobilization pattern included adsorption efficiency, ability of the immobilized cells to multiply within the carrier expressed as incubation efficiency and the degree of release of the immobilized cells from the carrier. The efficiency of adsorption and incubation was affected by the morphology of the immobilized cells and increased together with cellulose surface area. For smaller bacterial cells a higher level of loading was obtained on the same surface as compared to larger yeast cells. During incubation, the number of immobilized bacterial and yeast cells increased significantly in comparison to the number of cells adsorbed on the carrier during the adsorption step. Despite the morphological differences between the S. cerevisiae and Y. lipolytica cells, there were no statistically significant differences in the efficiency of adsorption and incubation. It was also revealed that the release ratio values obtained for L. delbruecki and S. cerevisiae increased along with cellulose surface area. Interestingly, Y. lipolytica cells in the pseudohyphae and hyphae forms penetrated deeply into the three-dimensional network of BC nanofibrils which prevented subsequent cell release. It was confirmed that carrier selection must be individually matched to the type of immobilized cells based especially on its porosity-related parameters.

Metabolomics analysis of fungal biofilm development and of arachidonic acid-based quorum sensing mechanism.

The infections caused by filamentous fungi are becoming worldwide problem of healthcare systems due to increasing drug-resistance of this microorganism and increasing number of immunocompromised nosocomial patients. These infections are related with Aspergillus ability to form sessile communities referred to as the biofilms. The small compounds known as quorum sensing (QS) molecules allow this microorganism to coordinate all processes taking place during biofilm formation and maturation. In the study presented, the HRMAS 1 H NMR metabolomic approach was applied to define composition of extra and intracellular metabolites produced by biofilmic and planktonic (aka free-swimming) cultures of this microorganism and to evaluate impact of quorum sensing molecule, arachidonic acid (AA) on biofilm formation. The Scanning Electron Microscopy was used to confirm Aspergillus ability to form biofilm in vitro, while multivariate and univariate data analysis was applied to analyze data obtained. The Aspergillus strain was able to form strong biofilm structures in vitro. The statistical analysis revealed significant changes of metabolite production depending on Aspergillus culture type (biofilm vs. plankton), time and presence of QS molecules. The data obtained, if developed, might be used in future NMR diagnostics as markers of Aspergillus biofilm-related infections and lead to shorten time between pathogen identification and introduction of treatment.

Increased water content in bacterial cellulose synthesized under rotating magnetic fields.

The current study describes properties of bacterial cellulose (BC) obtained from Komagataeibacter xylinus cultures exposed to the rotating magnetic field (RMF) of 50 Hz frequency and magnetic induction of 34 mT for controlled time during 6 days of cultivation. The experiments were carried out in the customized RMF exposure system adapted for biological studies. The obtained BC displayed an altered micro-structure, degree of porosity, and water-related parameters in comparison to the non-treated, control BC samples. The observed effects were correlated to the duration and the time of magnetic exposure during K. xylinus cultivation. The most preferred properties in terms of water-related properties were found for BC obtained in the setting, where RMF generator was switched off for the first 72 h of cultivation and switched on for the next 72 h. The described method of BC synthesis may be of special interest for the production of absorbent, antimicrobial-soaked dressings and carrier supports for the immobilization of microorganisms and proteins.

Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro.

PURPOSE: It is widely thought that inflammation and osteoclastogenesis result in hydroxyapatite (HA) resorption and sequestrum formation during osseous infections, and microbial biofilm pathogens induce the inflammatory destruction of HA. We hypothesized that biofilms associated with infectious bone disease can directly resorb HA in the absence of host inflammation or osteoclastogenesis. Therefore we developed an in vitro model to test this hypothesis. MATERIALS AND METHODS: Customized HA discs were manufactured as a substrate for growing clinically relevant biofilm pathogens. Single-species biofilms of Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans and mixed-species biofilms of C albicans plus S mutans were incubated on HA discs for 72 hours to grow mature biofilms. Three different non-biofilm control groups also were established for testing. HA discs were then evaluated by means of scanning electron microscopy, micro-computed tomography metrotomography, x-ray spectroscopy, and confocal microscopy with planimetric analysis. In addition, quantitative cultures and pH assessment were performed. Analysis of variance was used to test for significance between treatment and control groups. RESULTS: All investigated biofilms were able to cause significant (P < .05) and morphologically characteristic alterations in HA structure as compared with controls. The highest number of alterations observed was caused by mixed biofilms of C albicans plus S mutans. S mutans biofilm incubated in medium with additional sucrose content was the most detrimental to HA surfaces among single-species biofilms. CONCLUSIONS: Our findings suggest that direct microbial resorption of bone is possible in addition to immune-mediated destruction, which has important translational implications for the pathogenesis of chronic bone infections and for targeted antimicrobial therapeutics.

The Application of Impedance Microsensors for Real-Time Analysis of Pseudomonas aeruginosa Biofilm Formation.

Biofilms formed by nosocomial pathogens represent a major threat to patients undergoing invasive procedures. As prophylaxis remains the most efficient anti-biofilm option, it is of paramount importance to develop diagnostic tools able to detect biofilm at the early stage of formation. The present study investigates the ability of impedance microsensors to detect Pseudomonas aeruginosa biofilm presence using the impedance spectroscopy method. The measured data were analyzed using Electrical Equivalent Circuit modelling (EEC). It allowed to recognize conduction and polarization phenomena on the sensors surface and in its environment. The impedance assay results, confirmed by means of electron microscopy and quantitative cultures, indicate that specific EEC parameters may be used for monitoring the development of pseudomonal biofilm.

Efficacy of antiseptics containing povidone-iodine, octenidine dihydrochloride and ethacridine lactate against biofilm formed by Pseudomonas aeruginosa and Staphylococcus aureus measured with the novel biofilm-oriented antiseptics test.

Increasing data suggesting that microorganisms in the biofilm form are among the leading agents of persistent infections of chronic wounds require the development of new approaches to treatment. The aim of this article was to compare the efficacy of three commonly used antiseptics using a biofilm-oriented approach. Biofilm-oriented antiseptics test (BOAT), the innovative method, allows to estimate, in a quick and reliable manner, the in vitro activity of working solutions of antiseptics in real contact times against bacteria in the biofilm form and to use the results in the selection of an appropriate antiseptic to treat local infections in the clinical practice.

Application of bacterial cellulose experimental dressings saturated with gentamycin for management of bone biofilm in vitro and ex vivo.

Bacterial cellulose is one of the most promising polymers of recent years. Herein, we present a possibility of BC application as a carrier of gentamycin antibiotic for the treatment and prevention of bone infections. We have shown that BC saturated with gentamycin significantly reduces the level of biofilm-forming bone pathogens, namely Staphylococcus aureus and Pseudomonas aeruginosa, and displays very low cytotoxicity in vitro against osteoblast cell cultures. Another beneficial feature of our prototype dressing is prolonged release of gentamycin, which provides efficient protection from microbial contamination and subsequent infection. Moreover, it seems that bacterial cellulose (BC) alone without any antimicrobial added, may serve as a barrier by significantly hampering the ability of the pathogen to penetrate to the bone structure. Therefore, a gentamycin-saturated BC dressing may be considered as a possible alternative for gentamycin collagen sponge broadly used in clinical setting. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:30-37, 2020.

Stabilisation and Growth of Metastable Form II of Fluconazole in Amorphous Solid Dispersions.

The crystallisation of metastable drug polymorphs in polymer matrices has been reported as a successful approach to enhance the solubility of poorly water-soluble drug molecules. This can be achieved using different polymers, drug to polymer ratios and formulation techniques enabling the formation of stable nuclei and subsequent growth of new or metastable drug polymorphs. In this work we elucidated the polymorphism behaviour of a model compound fluconazole (FLU) embedded in solid dispersions with amorphous Soluplus® (SOL) obtained using spray drying and fusion methods. The effect of humidity on the stability of FLU in the obtained dispersions was also evaluated. FLU at a drug content below 40 wt. % stayed amorphous in the dispersions prepared using the fusion method and crystallised exclusively into metastable form II at a drug content above 40 wt. % and 70% relative humidity (RH) conditions. In contrast, a mixture of forms I, II and hydrate of FLU was detected in the spray dried formulations after 14 days of storage at 40 °C/40% RH, with preferential growth of thermodynamically stable form I of FLU. This study highlights the importance of preparation techniques and the drug:polymer ratio in the formulation of amorphous solid dispersions and provides further understanding of the complex crystallisation behaviour of amorphous pharmaceuticals encapsulated in the polymer matrixes.

Functionalized Magnetic Bacterial Cellulose Beads as Carrier for Lecitase® Ultra Immobilization.

Bacterial cellulose spheres subjected to amination and inlaid modification with superparamagnetic molecules were analyzed with regard to possibility of their application as an immobilization carrier of Lecitase® Ultra (LU) enzyme. The starting point to obtain the carrier was synthesis of bacterial cellulose spheres performed in shaking cultures of Komagataeibacter xylinus. These spheres were subsequently subjected to a multi-stage modification to increase the efficiency of the immobilization process and to separate product from the reaction medium. Maximal yield of Lecitase® Ultra immobilization equaled 70%. It was also found that immobilization process did not affect the pH and LU temperature optimum. Moreover, immobilized enzyme exhibited similar temperature stability profile as its native form. The immobilization process did not significantly affect the enzyme KM value. The immobilized enzyme retained over 70% of its initial activity after 8 cycles of use. The immobilized enzyme displayed good storage stability and retained 80% of its initial activity after 4 weeks at 4 °C. The potential application of such modified cellulose-based carrier may be correlated with lower costs of process thanks to higher enzyme's reusability in comparison to unbound enzyme. Moreover, data presented in the current study may serve as proof of a concept of cellulose-based carrier utilization for immobilization of enzymes other than LU and of high industrial importance.

In vitro efficacy of gentamicin released from collagen sponge in eradication of bacterial biofilm preformed on hydroxyapatite surface.

Biofilm-related infections of bones pose a significant therapeutic issue. In this article we present in vitro results of the efficacy of gentamicin released from a collagen sponge carrier against Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae biofilms preformed on hydroxyapatite surface. The results indicate that high local concentrations of gentamicin released from a sponge eradicate the biofilm formed not only by gentamicin-sensitive strains but, to some extent, also by those that display a resistance pattern in routine diagnostics. The data presented in this paper is of high clinical translational value and may find application in the treatment of bone infections.