Addition time plays a major role in the inhibitory effect of chitosan on the production of Pseudomonas aeruginosa virulence factors.

Pseudomonas aeruginosa is a gram-negative bacterium capable of forming persistent biofilms that are extremely difficult to eradicate. The species is most infamously known due to complications in cystic fibrosis patients. The high mortality of cystic fibrosis is caused by P. aeruginosa biofilms occurring in pathologically overly mucous lungs, which are the major cause facilitating the organ failure. Due to Pseudomonas biofilm-associated infections, remarkably high doses of antibiotics must be administered, eventually contributing to the development of antibiotic resistance. Nowadays, multidrug resistant P. aeruginosa is one of the most terrible threats in medicine, and the search for novel antimicrobial drugs is of the utmost importance. We have studied the effect of low molecular weight chitosan (LMWCH) on various stages of P. aeruginosa ATCC 10145 biofilm formation and eradication, as well as on production of other virulence factors. LMWCH is a well-known naturally occurring agent with a vast antimicrobial spectrum, which has already found application in various fields of medicine and industry. LMWCH at a concentration of 40 mg/L was able to completely prevent biofilm formation. At a concentration of 60 mg/L, this agent was capable to eradicate already formed biofilm in most studied times of addition (2-12 h of cultivation). LMWCH (50 mg/L) was also able to suppress pyocyanin production when added 2 and 4 h after cultivation. The treatment resulted in reduced formation of cell clusters. LMWCH was proved to be an effective antibiofilm agent worth further clinical research with the potential to become a novel drug for the treatment of P. aeruginosa infections.

Antibiofilm agent pterostilbene is able to enhance antibiotics action against Staphylococcus epidermidis.

Pterostilbene (PTE) is a naturally occurring compound originally isolated from Pterocarpus spp. It has been widely used in traditional Indian medicine and later discovered to have various beneficial pharmacological effects such as antioxidant properties, hypoglycaemic or antitumor, and antimicrobial activity. This work is focused on demonstrating PTE synergistic effect with erythromycin and tetracycline to reduce their needed effective concentration for suppression of Staphylococcus epidermidis planktonic cells growth and biofilm formation. The secondary aim is to find these combinations effect on the production of its virulence factors. PTE was found to be effective in inhibition of its planktonic cells with MIC80 values 25-37.5 mg l-1. Simultaneously, it decreased the metabolic activity of biofilm cells and was especially effective on a clinical isolate (MBIC80 = 35 mg l-1) in contrast to the conventional antibiotics. In combination, PTE helped the antibiotics to overcome the tolerance of S. epidermidis biofilm cells (5 mg l-1 of each antibiotic with 49 mg l-1 PTE caused more than 85% inhibition of metabolic activity). It permeabilized cytoplasmic membrane of S. epidermidis cells and altered their surface hydrophobicity. Therefore, PTE has a great potential to enhance antibiotics action in the treatment of infections caused by this pathogen.

Natural antioxidant pterostilbene as an effective antibiofilm agent, particularly for gram-positive cocci.

Pterostilbene (PTE), a dimethylated analogue of resveratrol, mostly contained in Vitis vinifera leaves or in other plant sources is well-known for its antioxidant activity. Due to its bioavailability, low hydrophilicity and thus ability to penetrate hydrophobic biological membranes it was found to be an antimicrobial agent. These properties of PTE offer the possibility of its use in the treatment of microbial infections. The emergence of antibiotic resistance of microorganisms is often caused by their ability to form biofilm; new substances with antibiofilm activity are therefore sought. The representatives of opportunistic pathogenic gram-positive and gram-negative bacteria as well as fungi were used for the determination of minimum inhibitory concentrations (MIC50 and MIC80), minimum biofilm inhibitory concentrations (MBIC50 and MBIC80) and minimum biofilm eradication concentrations (MBEC50 and MBEC80) of PTE and commonly used antibiotics erythromycin, polymyxin B or antimycotic amphotericin B. Total biofilm biomass was investigated by crystal violet staining, and the results were confirmed using microscopic techniques. The most significant antibiofilm action was proved for gram-positive cocci, e.g., MBEC50 of PTE for all strains of Staphylococcus epidermidis tested was 25 mg/L. By contrast, the antibiotic ERM did not exhibit antibiofilm activity in most cases. The permeabilization of cell membranes of gram-positive cocci biofilm by MBIC50 and MBEC50 of PTE was confirmed by LIVE/DEAD staining using spinning disc confocal microscopy. PTE significantly influenced the ability of gram-positive cocci to form biofilm and it effectively eradicated pre-formed biofilm in vitro; its potential for the treatment of biofilm-associated infections of Staphylococcus spp. or Enterococcus faecalis is thus apparent.

Mild hydrolysis of nitriles by Fusarium solani strain O1.

High levels of an aromatic nitrilase (about 37 microkat/L culture) were induced in Fusarium solani O1 after transfer of the mycelium from a rich medium into a medium with 20 mmol/L picolinonitrile. The mycelium was entrapped in lense-shaped particles consisting of a polyvinyl alcohol/polyethylene glycol copolymer (LentiKats). The cell-free extract was immobilized by hydrophobic binding onto a Butyl Sepharose column. The enzyme was useful for the mild hydrolysis of nicotinonitrile, isonicotinonitrile and benzonitrile.

Colonization of surfaces by phenolic compounds utilizing microorganisms.

The aim of the present study is to determine optimal adhesive interaction of phenolic compounds utilizing Candida maltosa and Rhodococcus erythropolis when adhering to kaolin, silicone, synthetic foil (Steriking R40) and fluorinated silicones, comparing cell and support surface hydrophobicity. In parallel, the interfering effect of detergents was investigated. Data obtained show that the less hydrophobic supports display high initial cell adhesion when contacted with the cell type with a lower surface hydrophobicity (yeast cell) but most stable yeast biofilms are those formed on highly hydrophobic fluorinated silicones. On the other hand, support hydrophobicity has no effect on bacterial cell detachment; however, bacterial biofilms are denser when growing on more hydrophobic supports. Both detergents interfere (independently on the cell type) with the early and late phases of biofilm development.

The influence of heavy metals on the production of extracellular polymer substances in the processes of heavy metal ions elimination.

Wastewaters from a chemical industry polluted by heavy metal ions represent a hazard for all living organisms. It can mean danger for ecosystems and human health. New methods are sought alternative to traditional chemical and physical processes. Active elimination process of heavy metals ions provided by living cells, their components and extracellular products represents a potential way of separating toxic heavy metals from industrial wastewaters. While the abilities of bacteria to remove metal ions in solution are extensively used, fungi have been recognized as a promising kind of low-cost adsorbents for removal of heavy-metal ions from aqueous waste sources. Yeasts and fungi differ from each other in their constitution and in their abilities to produce variety of extracellular polymeric substances (EPS) with different mechanisms of metal interactions. The accumulation of Cd(2+), Cr(6+), Pb(2+), Ni(2+) and Zn(2+) by yeasts and their EPS was screened at twelve different yeast species in microcultivation system Bioscreen C and in the shaking Erlenmayer's flasks. This results were compared with the production of yeast EPS and the composition of yeast cell walls. The EPS production was measured during the yeast growth and cell wall composition was studied during the cultivations in the shaking flasks. At the end of the process extracellular polymers and their chemical composition were isolated and amount of bound heavy metals was characterized. The variable composition and the amount of the EPS were found at various yeast strains. It was influenced by various compositions of growth medium and also by various concentrations of heavy metals. It is evident, that the amount of bound heavy metals was different. The work reviews the possibilities of usage of various yeast EPS and components of cell walls in the elimination processes of heavy metal ions. Further the structure and properties of yeasts cell wall and EPS were discussed. The finding of mechanisms mentioned above is necessary to identify the functional groups entered in the metals elimination processes.

Host-vector system for phenol-degrading Rhodococcus erythropolis based on Corynebacterium plasmids.

The strain Rhodococcus erythropolis CCM2595, which was shown to degrade phenol, was chosen for genetic studies. To facilitate strain improvement using the methods of gene manipulation, the technique of genetic transfer was introduced and cloning vectors were constructed. Using the plasmid pFAJ2574, an electrotransformation procedure yielding up to 7x10(4) transformants/microg DNA was optimized. Escherichia coli- R. erythropolis shuttle vectors were constructed using the replicons pSR1 and pGA1 from Corynebacterium glutamicum. The small vector pSRK21 (5.8 kb) provides six unique cloning sites and selection of recombinant clones using alpha-complementation of beta-galactosidase in E. coli. This vector, exhibiting high segregational stability under non-selective conditions in R. erythropolis CCM2595, was applied to cloning and efficient expression of the gene coding for green fluorescent protein (gfpuv).

Comparison of yeast (Candida maltosa) and bacterial (Rhodococcus erythropolis) phenol hydroxylase activity and its properties in the phenolic compounds biodegradation.

Aromatic contaminants of the environment, to which belongs phenol and its derivatives, are toxic and in most of the cases hard to degrade. Removal of these pollutants by biological, gentle and effective way, depends on specific environmental conditions in the locality and on the biodegradation potential of the used microbial population. Closer characterization of the biodegradation and enzyme mechanisms is therefore an essential assumption of the successful implementation of microbes. This paper is focused on comparison of the biodegradation activity between the soil yeast Candida maltosa and bacteria Rhodococcus erythropolis towards various aromatics connected with determination of the first enzyme of the phenol biodegradation pathway: phenol hydroxylase (PH). The effect of substrate type, substrate concentration, growth phase of the microorganisms and presence of humic acids in the cultivation medium, on phenol biodegradation and PH activity are discussed.

The enhancement of reproduction and biodegradation activity of eukaryiotic cells by humic acids.

Fourteen samples of humic acids (HA) were screened for ability to influence reproduction and biodegradation activity of eukaryotic cells in the presence of chosen toxic pollutants. Microorganisms Candida maltosa and Rhodotorula mucilaginosa (soil isolates) were used for all tests. It was observed during our experiments that some samples of humic acids served as a protection against the high concentration of toxic pollutants (phenol, naphtalene etc). This effect can be widely used in many bioremediation technologies.

Significance of physical attachment of fungi for bio-treatment of water.

The inhibitory effect of xenobiotics known to damage cell surface structures was studied. The sensitivity of suspended cells of the two fungi Candida maltosa and Fusarium proliferatum was compared with that of artificial or natural biofilms of these fungi. The results obtained indicate that the resistance of attached cell populations to model xenobiotics is increased compared with suspended cells. Only the attached fungal cells had the capacity to degrade acetone and phenol and to adapt to increasing concentrations of these substances, so they seem ideally suited for bioremediation of waste water.

Yeast cell attachment: a tool modulating wall composition and resistance to 5-bromo-6-azauracil.

The attachment of Candida utilis, Kluyveromyces lactis, and Saccharomyces cerevisiae cells stimulates an increase in the content of cell wall polysaccharides and mannoproteins, accompanied by increased resistance to the inhibitory effect of 5-bromo-6-azauracil. The covalent attachment of viable yeasts was accomplished (via dialdehyde-amino spacers) by reaction of aldehyde groups of the carrier with reactive amino groups in accessible cell surface proteins. The employed technique enables the optimization of yeast sources of beta-1,3-, beta-1,6- glucans, mannan, and mannoprotein. The modulatory effect of the cell attachment is discussed.

Use of mineral nutrients and surface-active substances in a biodegradation process modulation.

The capability of a bacterial population to degrade oil hydrocarbons and naphthalene was found to be markedly enhanced by an optimized P:N ratio as well as by proper application of a surface-active compound. The importance of this optimization procedure was shown by both laboratory and technologically performed experiments.