The agar microdilution method - a new method for antimicrobial susceptibility testing for essential oils and plant extracts.

AIMS: To develop a new agar microdilution technique suitable for the assessment of the antimicrobial activity of natural plant products such as essential oils or plant extracts as well as to evaluate the antimicrobial effect of several essential oils and plant extracts. METHODS AND RESULTS: The proposed agar microdilution method was evolved on the basis of the CLSI agar dilution method, approved for conventional antimicrobials. However, this new method combines convenience and time/cost effectiveness typical for microtitre methods with the advantages of the agar dilution of hydrophobic or coloured substances. Different concentrations of the tested agents were added to Eppendorf tubes with molten Mueller-Hinton agar, vortexed and dispensed into the 96-well microplate in a small volume of 100 µl per well which allows for rapid, easy and economical preparation of samples as well as providing a uniform and stable dispersion without separation of the oil-water phases which occurs in methods with liquid medium. Next, the agar microdilution plates were inoculated with four reference bacterial strains. The results of our study demonstrated that the minimal inhibitory concentrations (MICs) were successfully determined using the agar microdilution method even with hydrophobic essential oils or strongly coloured plant extracts. CONCLUSIONS: The new agar microdilution method avoids the problems associated with testing of water insoluble, oily or strongly coloured plant natural products. Moreover, it enables the reliable, cheap and easy MIC determination of such agents. SIGNIFICANCE AND IMPACT OF THE STUDY: In the era of increasing antibiotic resistance high hopes are associated with new drugs of plant origin. However, the lack of standardized and reliable testing methods for assessing antibacterial activity of plant natural products causes impediment to research into this area. This study demonstrated that the agar microdilution method can be used successfully for testing oily and coloured substances.

TiO2 nanotube composite layers as delivery system for ZnO and Ag nanoparticles - an unexpected overdose effect decreasing their antibacterial efficacy.

Enhancement of biocompatibility and antibacterial properties of implant materials is potentially beneficial for their practical value. Therefore, the use of metallic and metallic oxide nanoparticles as antimicrobial coatings components which induce minimized antibacterial resistance receives currently particular attention. In this work, TiO2 nanotubes layers loaded with ZnO and Ag nanoparticles were designed for biomedical coatings and delivery systems and evaluated for antimicrobial activity. TiO2 nanotubes themselves exhibited considerable and diameter-dependent antibacterial activity against planktonic Staphylococcus epidermidis cells but favored bacterial adhesion. Loading of nanotubes with moderate amount of ZnO nanoparticles significantly diminished S. epidermidis cell adhesion and viability just after 1.5h contact with modified surfaces. However, an increase of loaded ZnO amount unexpectedly altered the structure of nanoparticle-nanolayer, caused partial closure of nanotube interior and significantly reduced ZnO solubility and antibacterial efficacy. Co-deposition of Ag nanoparticles enhanced the antibacterial properties of synthesized coatings. However, the increase of ZnO quantity on Ag nanoparticles co-deposited surfaces favored the adhesion of bacterial cells. Thus, ZnO/Ag/TiO2 nanotube composite layers may be promising delivery systems for combating post-operative infections in hard tissue replacement procedures. However, the amount of loaded antibacterial agents must be carefully balanced to avoid the overdose and reduced efficacy.

Enhanced differentiation of osteoblastic cells on novel chitosan/ß-1,3-glucan/bioceramic scaffolds for bone tissue regeneration.

Bone scaffolds for regenerative medicine applications should have the ability to promote adhesion, proliferation and differentiation of osteoblast cells. Osteoconductive, osteoinductive and osteopromotive properties of the material are essential for rapid bone regeneration and new bone formation. In this study, the osteogenic potential of two novel tri-component scaffolds composed of krill chitosan, bacterial ß-1,3-glucan and bioceramics (HAp or a mix of HAp/ß-TCP granules) was investigated. The typical markers of the first (type I collagen), second (bone alkaline phosphatase) and third stages (osteocalcin) of the osteoblast differentiation process were evaluated during in vitro experimentation. The study was carried out using three various osteoblastic cell lines (normal human fetal osteoblast cells hFOB 1.19, human osteoblast-like cells derived from osteosarcoma Saos-2 and mouse calvarial preosteoblast cells MC3T3-E1 Subclone 4). The bone alkaline phosphatase (bALP) and osteocalcin (OC) were determined quantitatively using enzyme-linked immunosorbent assays, and type I collagen (Col I) was evaluated qualitatively using the direct immunofluorescence (DIF) method. The data obtained clearly prove that novel scaffolds have the ability to increase bALP activity, to enhance extracellular matrix synthesis (Col I and OC) and to induce mineralized nodule formation during osteogenic differentiation. In conclusion, novel tri-component materials have osteoconductive and osteopromotive properties, and thus are promising materials in bone tissue engineering applications to accelerate the bone regeneration process.

Enzymatic electrodes nanostructured with functionalized carbon nanotubes for biofuel cell applications.

Nanostructured bioelectrodes were designed and assembled into a biofuel cell with no separating membrane. The glassy carbon electrodes were modified with mediator-functionalized carbon nanotubes. Ferrocene (Fc) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) bound chemically to the carbon nanotubes were found useful as mediators of the enzyme catalyzed electrode processes. Glucose oxidase from Aspergillus niger AM-11 and laccase from Cerrena unicolor C-139 were incorporated in a liquid-crystalline matrix-monoolein cubic phase. The carbon nanotubes-nanostructured electrode surface was covered with the cubic phase film containing the enzyme and acted as the catalytic surface for the oxidation of glucose and reduction of oxygen. Thanks to the mediating role of derivatized nanotubes the catalysis was almost ten times more efficient than on the GCE electrodes: catalytic current of glucose oxidation was 1 mA cm(-2) and oxygen reduction current exceeded 0.6 mA cm(-2). The open circuit voltage of the biofuel cell was 0.43 V. Application of carbon nanotubes increased the maximum power output of the constructed biofuel cell to 100 µW cm(-2) without stirring of the solution which was ca. 100 times more efficient than using the same bioelectrodes without nanotubes on the electrode surface.

Vascular prostheses with covalently bound gentamicin and amikacin reveal superior antibacterial properties than silver-impregnated ones--an in vitro study.

OBJECTIVE: This study aims to compare the antibacterial activities of vascular prostheses: silver-impregnated and modified with covalently immobilised antibiotics. MATERIALS AND METHODS: Six types of protein-sealed vascular prostheses were modified with amikacin and gentamicin according to the method described in the Polish Patent Office. Their antimicrobial properties were estimated against 14 reference and clinical strains and compared with those of InterGard Silver grafts. Cytotoxicity of the tested grafts was estimated against human skin fibroblasts. RESULTS: Prostheses modified with antibiotics in a stable covalent mode were found to be much more effective against bacterial growth and biofilm formation, as well as in case of methicillin-resistant Staphylococcus aureus (MRSA), than InterGard Silver. They inhibited the bacterial growth for at least 30 days, without losing higher than 10% of the initial amount of its drug content. They were also good, non-toxic matrices for growth of human skin fibroblasts. CONCLUSIONS: Prostheses modified with covalently immobilised antibiotic according to our technique are much more effective than InterGard Silver at protection against bacterial growth. They are also compatible with human skin fibroblasts.

Microbial conversion of daunomycin wastes in unsteril soil inoculated with Bjerkandera adusta R59.

Effect of cooperation between native soil microorganisms and white-rot fungus Bjerkandera adusta R59 on degradation of daunomycin post-production wastes in soil was described. Pure cultures of B. adusta R59 strain were capable to decolorize and decompose that cytostatic xenobiotic in liquid media. Presence of R59 strain in studied daunomycin waste/soil systems increased the rate of the antibiotic conversion. The markers of that process were changes of waste biomass or daunomycin concentration (in pulp) and phenolics level and peroxidase activity (in effluent). It was shown that daunomycin in the wastes may be metabolized even up to 20% of its initial concentration. This effect was conjugated with the propagation of native soil microorganisms (microfungi and bacteria) more significant than in parallel system without R59 strain.

Cultivation conditions and properties of extracellular crude lipase from the psychrotrophic fungus Penicillium chrysogenum 9'.

Among 97 fungal strains isolated from soil collected in the arctic tundra (Spitsbergen), Penicillium chrysogenum 9' was found to be the best lipase producer. The maximum lipase activity was 68 units mL(-1) culture medium on the fifth day of incubation at pH 6.0 and 20 degrees C. Therefore, P. chrysogenum 9' was classified as a psychrotrophic microorganism. The non-specific extracellular lipase showed a maximum activity at 30 degrees C and pH 5.0 for natural oils or at pH 7.0 for synthetic substrates. Tributyrin was found to be the best substrate for lipase, among those tested. The Km and Vmax were calculated to be 2.33 mM and 22.1 units mL(-1), respectively, with tributyrin as substrate. The enzyme was inhibited more by EDTA than by phenylmethylsulfonyl fluoride and was reactivated by Ca2+. The P. chrysogenum 9' lipase was very stable in the presence of hexane and 1,4-dioxane at a concentration of 50%, whereas it was unstable in presence of xylene.

Antibacterial activity of gentamicin-bonded gelatin-sealed polyethylene terephthalate vascular prostheses.

OBJECTIVES: To create an antibiotic-modified vascular prosthesis with a prolonged bactericidal activity, susceptible to endothelialisation. METHODS: We used a covalent method of gentamicin sulphate immobilisation to polyethylene terephthalate prosthesis sealed with gelatin. Antibacterial activity was assayed in Luria-Bertani medium against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains. Prosthesis endothelialisation was performed using bovine aorta endothelial cells (BAEC). RESULTS: Gentamicin was bound to vascular prostheses in the amount of 12g per kg of prosthesis. Ninety-seven percent of antibiotic bound in covalent way and remained on the biomaterial for at least 30 days during shaking in PBS solution. Gentamicin-modified prostheses exerted bactericidal or bacteriostatic effect on growth of clinical and reference bacterial strains, prevented biofilm formation and were highly susceptible to endothelialisation. BAEC viability exceeded 90%, which indicated that gentamicin-vascular prostheses were not toxic for these cells. CONCLUSIONS: Covalent gentamicin immobilisation resulted in effective antibacterial protection of vascular prostheses against clinical and reference strains of S. aureus, E. coli and P. aeruginosa and allowed for a strong adherence of endothelial cells to antibiotic-modified prostheses.

Extracellular enzyme activities of Bjerkandera adusta R59 soil strain, capable of daunomycin and humic acids degradation.

Geotrichum-like strain R59, the anamorphic form of the white-rot fungus, Bjerkandera adusta, was isolated from soil. It was found to completely decolorize and degrade 10% daunomycin post-production effluent during 10 days of incubation at 26 degrees C. Strain R59 produced only low levels of ligninolytic enzymes when grown on wheat straw- or beech sawdust-containing media, but in the presence of humic acids derived from brown coal it synthesized significant amounts of laccase and lipase. This phenomenon was coupled with the fungus entering the idiophase and the appearance of aerial mycelium. B. adusta strain R59 was found to completely decolorize 0.03% humic acids from brown coal and lessive soil and to partially decolorize humic acids isolated from a chernozem during 14 days of growth. This ability of strain R59 could be useful in constructing a new generation of biologically active filters for the purification of humic acids-contaminated drinking waters.

A thermostable lipase produced by a newly isolated Geotrichum-like strain, R59.

Growth and production of lipase by a new Geotrichum-like strain, R59, were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, substances probably stimulating the lipase biosynthesis, the temperature, and time of growth were optimized. Sucrose and triolein were the most effective carbon sources for lipase production. Maximum lipase activity (146 U/ml(-1)) was obtained with urea as the nitrogen source. Growth at 30 degrees C, an initial pH of 6.0 and incubation time of 48 h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like strain R59. The enzyme was thermostable and exhibited very high activity after 1 h incubation at 60 degrees C.

The novel non-glycosylated invertase from Candida utilis (the properties and the conditions of production and purification).

The Candida utilis yeast, which is cultivated in liquid media enriched with saccharose, synthesizes the well-known invertase of 300 kDa (EC 3.2.1.26). This enzyme is present both intracellularly in the periplasmic space and extracellularly in the culture broth. However, it was determined that the same C. utilis strain cultured in certain conditions is simultaneously capable of producing another, still unknown form of invertase with a molecular mass of 60 kDa. The presence of the latter enzymatic form was detected in cells as well as in the liquid culture medium. Both invertase forms were purified using a three-step process (ion-exchange chromatography, affinity chromatography, and preparative column electrophoresis) and named, due to their different migration ratio in polyacrylamide gel electrophoresis, F-form (Fast; 60 kDa) and S-form (Slow; 300 kDa). The F-form of invertase was found to be nonglycosylated as opposed to the well-known S-form of invertase from the same source. The physicochemical properties of the F-form of invertase (isoelectric point, substrate specificity, pH, and temperature optima) were determined and compared with those of the S-form of the enzyme.

Selection of amylolytically active Aspergillus niger mutants to 2-deoxy-D-glucose.

As a result of mutagenization and passaging on 2-deoxy-D-glucose containing medium, 10 Aspergillus niger strains resistant to this agent were obtained. These showed (with one exception) an increase in the activity of glucoamylse, the level of which ranged widely in individual cases from several to over 200% in comparison with the parent strain. A weaker rate of glucose accumulation in derepressed strains may account for the fact that the mechanism of their resistance to deoxyglucose is connected with disturbance of the system of glucose transport. However, it is possible that a high activity of acid phosphatase, which the obtained deoxyglucose-resistant cultures showed, may be involved here. Apart from the biochemical character of the catabolic derepression, it seems that it can already be successfully utilized to increase the productivity of industrial mould cultures.

New matrices for the purification of pectinases by affinity chromatography.

Polygalacturonic acid was used as a ligand in the affinity technique for pectinases purification from the filtrate of Aspergillus niger 71 culture. For this purpose four matrices were examined, namely, alkylamine controlled porous glass (CPG), alkylamine silica gel as well as keratin or polyamide coated silica gel. Good results of pectinase purification was obtained on silanized CPG or keratin coated silica gel supports.

Cellulolytic activity of moulds. I. Characteristics of the cellulases complex and xylanase of the strain Aspergillus terreus F-413.

Among 79 strains of moulds belonging to 17 different species, the strain Aspergillus terreus F-413 which showed the highest cellulolytic activity was isolated for further studies. Some properties of the complex of cellulases formed by this strain as well as the dynamics of their synthesis under optimal submerged culture conditions were characterized.