Staphylococcus aureus-A Known Opponent against Host Defense Mechanisms and Vaccine Development-Do We Still Have a Chance to Win?

The main purpose of this review is to present justification for the urgent need to implement specific prophylaxis of invasive Staphylococcus aureus infections. We emphasize the difficulties in achieving this goal due to numerous S. aureus virulence factors important for the process of infection and the remarkable ability of these bacteria to avoid host defense mechanisms. We precede these considerations with a brief overview of the global necessitiy to intensify the use of vaccines against other pathogens as well, particularly in light of an impasse in antibiotic therapy. Finally, we point out global trends in research into modern technologies used in the field of molecular microbiology to develop new vaccines. We focus on the vaccines designed to fight the infections caused by S. aureus, which are often resistant to the majority of available therapeutic options.

Phenolic and Non-Polar Fractions of the Extracts from Fruits, Leaves, and Twigs of Elaeagnus rhamnoides (L.) A. Nelson-The Implications for Human Barrier Cells.

Biological potential of plant extracts are widely described. Because their oral or topical administration is usually recommended, intestinal mucous and skin are the first surfaces exposed to such preparations. Therefore, we asked the question whether phenolic and non-polar fractions of the extracts from fruits, twigs, and leaves of sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) would be able to modulate the functions of human physiological barrier. The study was carried on caucasian colon epithelial-like Caco-2 cells and human foreskin fibroblasts HFF-1 line. Cell secretory activity (ELISA), the expression of cell surface molecules (flow cytometry), cell migration during wound healing in vitro (scratch assay) were assessed. It was demonstrated for the first time, that sea buckthorn extracts can improve intestinal and skin barrier by increasing of ICAM-1 expression on colon epithelial cells and intensification of IL-8 production by fibroblasts. On the other hand, an inhibition of fibroblasts migration in the presence of those preparations was noted. Therefore, greater attention should be paid on precise description of plant extracts effect depended on target cells and their role to give adequate recommendations for such preparations use.

Molecular Mechanisms of Leonurus Cardiaca L. Extract Activity in Prevention of Staphylococcal Endocarditis-Study on in Vitro and ex Vivo Models.

Better understanding the mechanisms of Leonurus cardiaca L. extract (LCE) activity is necessary to prepare recommendations for the use of LCE-based herbal products for preventive/supportive purposes in case of infective endocarditis (IE) and other staphylococcal invasive infections. The aim of the study was to analyze molecular mechanisms of LCE effect on Staphylococcus aureus and blood platelets in the context of their interactions playing a pivotal role in such disorders. Using atomic force microscopy, we demonstrated that adhesion forces of S. aureus were markedly reduced after exposure to LCE at subinhibitory concentrations. The effect resulted from the impact of LCE on S. aureus cell morphology and the composition of phospholipids and fatty acids in bacterial membranes (assessed by HPLC), which modulated their stabilization, hydrophobicity, and charge. Moreover, using FACS we showed also that LCE significantly reduced GP IIb/IIIa expression on blood platelets, thus the disruption of platelet-fibrinogen interactions seems to explain antiplatelet effect of LCE. The obtained results prove the usefulness of LCE in the prevention of S. aureus adhesion, platelet activation, and vegetations development, however, also pointed out the necessity of excluding the cationic antibiotics from the treatment of S. aureus-associated IE and other invasive diseases, when motherwort herb is used simultaneously as an addition to the daily diet.

The Pros and Cons of Cystic Fibrosis (CF) Patient Use of Herbal Supplements Containing Pulmonaria officinalis L. Extract: the Evidence from an In Vitro Study on Staphylococcus aureus CF Clinical Isolates.

The justification for the use of herbal supplements with Pulmonaria officinalis L. extract (POE) in the case of staphylococcal lung colonization/infections characteristic for cystic fibrosis (CF), was examined in vitro. The impact of POE phenolic-rich fraction on the virulence attributes of CF-associated Staphylococcus aureus (S. aureus) clinical strains has been assessed, including pathogen adhesion, biofilm formation on native and protein-conditioned surfaces (mucin, elastin), mature biofilm eradication, staphylococcal protein A expression, α-toxin release, and S. a. adhesion to A549 cells. Cytotoxicity of the extract to lung epithelial cells was also investigated. It was found that POE has bacteriostatic effects at MIC 1⁻2 mg/mL, recognized as of limited efficacy, but at MIC/subMICs it targeted virulence not viability. It usually decreased S. aureus adhesion and less frequently inhibited biofilm formation on native and protein-conditioned surfaces. Observed effect seems to be related to significant reduction by POE of sortase A activity. However, in some cases POE favored the creation of biofilm by staphylococci and S. aureus adhesion to the lung epithelium was not limited. On the other side POE caused significant decrease of S. a. α-toxin synthesis and slightly weakened the expression of SpA. When used at supraMICs POE eradicated mature biofilm, but in some cases with unsatisfying outcomes. Promisingly, POE has been recognized as a safe product, with no cytotoxicity up to 4 mg/mL. These results reflect the positive, negative or neutral anti-staphylococcal properties of POE. It seems that POE may be beneficial as a prophylactic, but not as a therapeutic or supportive agent in the area of CF-integrative medicine. However, introduction the official recommendations needs further in vivo studies.

Phenolic and Nonpolar Fractions of Elaeagnus rhamnoides (L.) A. Nelson Extracts as Virulence Modulators-In Vitro Study on Bacteria, Fungi, and Epithelial Cells.

Butanol extracts from leaves, twigs, and fruits of Elaeagnus rhamnoides (L.) A. Nelson (sea buckthorn, SBT) were fractionated into phenolic and nonpolar lipid components, the chemical composition of which was analyzed. Assuming that an effect on natural microbiota and host epithelial cells needs to be assessed, regardless of the purpose of using SBT formulations in vivo, the minimal inhibitory/biocidal/fungicidal concentrations (MICs/MBCs/MFCs) of the fractions and reference phytocompounds were screened, involving 17 species of Gram-positive and Gram-negative bacteria and Candida species. The MICs of SBT extracts were in the range of 0.25⁻2.0 mg∙mL−1. Since direct antimicrobial activity of the extracts was quite low and variable, the impact of subMIC on the important in vivo persistence properties of model microorganisms S. aureus and C. albicans was evaluated. Tests for adhesion and biofilm formation on an abiotic surface and on surfaces conditioned with fibrinogen, collagen, plasma, or artificial saliva showed the inhibitory activity of the fractions. The effects on fluorescein isothiocyanate (FITC)-labeled staphylococci adhesion to fibroblasts (HFF-1) and epithelial cells (Caco-2), and on fungal morphogenesis, indicated that SBT extracts have high antivirulence potential. Cytotoxicity tests (MTT reduction) on the standard fibroblast cell line showed variable biological safety of the fractions depending on their composition and concentration. The new information afforded by this study, additional to that already known, is of potential practical value in the application of SBT-derived preparations as antivirulence agents.

Novel properties of Hippophae rhamnoides L. twig and leaf extracts - anti-virulence action and synergy with antifungals studied in vitro on Candida spp. model.

Original, chemically characterized Sea buckthorn (SBT) twig and leaf extracts were in vitro studied in terms of anti-Candida activity. Minimum inhibitory concentrations (MICs) of the extracts against C. albicans ATCC 10231 ranged: 250 µg/ml (twig), 31.5 µg/ml (leaf), and against C. glabrata G1 (clinical isolate) - 15.6 µg/ml (twig), 3.9 µg/ml (leaf). Next the extracts have been used at their subMIC. Both extracts significantly enhanced activity of fluconazole (FLC) and caspofungin (CAS) against C. albicans and increased their efficacy against C. glabrata, measured by an agar dilution assay combined with the E-test. The extracts inhibited C. albicans morphogenesis such as germ tube and hyphae formation as well as invasion to the "Spider" Agar. Antiadhesive and anti-biofilm activities of the extracts were evaluated by Alamar Blue reduction assay. It showed not significant reduction in the degree of cell adhesion (by 10-15%) but noticeable decrease of biofilm formation (by 80% in the case of SBT-twig extract). In conclusion, this study provided the evidence that SBT extracts, used at non-cytotoxic concentrations for the fibroblasts (IC50 from 664.8 µg/ml to 1060.4 µg/ml), targeted some of Candida spp. virulence factors essential for the establishment of the infection. SBT twigs, previously regarded as waste material, were shown to be also a valuable source of the substances with promising antimicrobial activity.

Candida albicans/Staphylococcus aureus Dual-Species Biofilm as a Target for the Combination of Essential Oils and Fluconazole or Mupirocin.

The effectiveness of essential oils (EOs), fluconazole (FLU) and mupirocin (MUP) used alone or in combination against mono-species and mixed Candida albicans/Staphylococcus aureus biofilms was examined. An experimentally established dual-species biofilm model, verified by fluorescence microscopy and viable cell counting, was used. Selected commercial EOs were tested: geranium, citronella and clove oils, which have been chemically characterized and found to differ in the content of the main components (qualitative and quantitative). As expected, C. albicans and S. aureus biofilms were less susceptible to fluconazole and mupirocin action, respectively, compared to the planktonic counterparts. However, the drug effectiveness in combination with the EOs was significantly improved, giving enhancement of biofilm eradication than caused by the antibiotics alone. Moreover, dual-species biofilm formation was limited by sub-MIC of EOs, and preformed mixed biofilm was eliminated more efficiently by combined action of drugs and EOs.

New biological potential of abietane diterpenoids isolated from Salvia austriaca against microbial virulence factors.

The increasing importance of multi-resistant strains and microbial biofilms in the development of chronic infections has driven the search for more effective alternative therapy including plant-origin preparations. The present study evaluates the broadly-defined antimicrobial activity of two abietane diterpenoids isolated from Salvia austriaca transformed roots: taxodone and 15-deoxy-fuerstione. The direct biostatic/biocidal effect of these phytocompounds and their influence on Staphylococcus aureus and Candida albicans virulence factors/mechanisms (adhesion, biofilm formation, agglutination in human plasma, survival in the blood, germ tube and mycelium formation) were tested using in vitro assays. Both phytocompounds significantly inhibited microbial adhesion and biofilm formation when used at ½ and » MIC. Additionally, taxodone was able to limit staphylococcal survival in human blood, as well as C. albicans germ tube formation and hyphal growth. The tested diterpenoids express significant anti-biofilm activity against both staphylococci and yeast, and adversely affect their virulence factors/mechanisms, which are relevant in the course of the infection in vivo. Therefore, they demonstrate considerable biomedical potential as complements for classic therapy with antibiotics.

[The role of blood platelets in infections]. / Rola plytek krwi w zakazeniach.

Platelets are primarily associated with their main function, hemostasis, although it is known that these cells also exhibit biological activity in cancer progression, inflammation and infectious processes. During infection platelets, due to the expression of specific receptors - Toll-like receptors (TLRs) - which recognize molecular patterns associated with pathogens - pathogen-associated molecular patterns (PAMPs) - are activated by the presence of microorganism components and/or substances released from damaged cells/tissue. Further antimicrobial activity of platelets is based on their capacity for phagocytosis, generation of reactive oxygen species (ROS), and the synthesis, storage and release of proteins/peptides with antimicrobial activity. Another mechanism of platelet action is their immunomodulatory activity. It is based mainly on the ability to secrete chemotactic factors allowing the accumulation of professional immunocompetent cells at the site of infection, thus enhancing the effective eradication of an infectious agent. In chronic infections, platelets, due to release of numerous growth factors and various cytokines, support mechanisms of acquired immunity. They accelerate the maturation of dendritic cells, stimulate B cells to be immunoglobulin-producing plasma cells and potentiate the activity of T cells. Unfortunately, in certain situations (the existence of specific risk factors) the interaction of microorganisms with activated platelets may also be the cause of pathology within the cardiovascular system.

Saponins of Trifolium spp. aerial parts as modulators of Candida albicans virulence attributes.

The aim was to provide the insight into the biology of C. albicans influenced by undescribed yet properties of saponin-rich (80%-98%) fractions (SAPFs), isolated from extracts of Trifolium alexandrinum, T. incarnatum, T. resupinatum var. resupinatum aerial parts. Their concentrations below 0.5 mg/mL were arbitrarily considered as subMICs for C. albicans ATCC 10231 and were further used. SAPFs affected yeast enzymatic activity, lowered tolerance to the oxidative stress, to the osmotic stress and to the action of the cell wall disrupting agent. In their presence, germ tubes formation was significantly and irreversibly inhibited, as well as Candida invasive capacity. The evaluation of SAPFs interactions with anti-mycotics showed synergistic activity, mainly with azoles. Fluconazole MIC was lowered-susceptible C. albicans ATCC 10231 was more susceptible, and resistant C. glabrata (clinical strain) become more susceptible (eightfold). Moreover, the tested samples showed no hemolytic activity and at the concentrations up to 0.5 mg/mL did not reduce viability of fibroblasts L929. This study provided the original evidence that SAPFs of Trifolium spp. aerial part exhibit significant antimicrobial activity, by reduce the expression/quantity of important Candida virulence factors and have good potential for the development of novel antifungal products supporting classic drugs.

Antimicrobial/anti-biofilm activity of expired blood platelets and their released products.

INTRODUCTION: Although platelets are not part of the classical immune system, they have many features that indicate their role in the anti-infective host defense. They come into interactions with microorganisms, which results in co-aggregation and co-adhesion or destruction of the microbes due to the action of antimicrobial peptides released from platelets.The aim of this study was to evaluate the killing effect of platelets against planktonic and biofilm cultures of Staphylococcus aureus and to test their synergy with antibiotics. MATERIALS AND METHODS: S. aureus ATCC 29213; platelet rich plasma (1-3 days post shelf life). Evaluation of bactericidal activity of platelets or their lysates against planktonic cultures of S. aureus--CFU calculation after 4- and 24-hour co-incubation. Assessment of S. aureus biofilm viability under the influence of platelets--Live/Dead® BacLight™ Bacterial Viability Kit. Determination of minimum inhibitory concentrations (MICs) (oxacillin, vancomycin, linezolid) and estimation of the synergistic action of antibiotics and platelet lysates--a gradient-diffusion test strip. RESULTS: Microbicidal activity of "expired" platelets and their lysates has been shown as a significant reduction in the population of staphylococci in their planktonic cultures by 56-87% and a decrease in metabolic activity of biofilm formation by 7-38%. These activities were enhanced after activation with ADP. Platelet lysates showed a synergistic effect with ß-lactam antibiotic (oxacillin) and glycopeptide (vancomycin) but not with oxazolidinone (linezolid). CONCLUSIONS AND DISCUSSION: In summary, platelets even after the medical expiry date are still a good source of antimicrobial low molecular weight proteins (PMPs). Testing of bacterial resistance to PMPs may be advisable as a predictive indicator of susceptibility to treatment of infections such as infective endocarditis and other local infections of biofilm nature.

[In vitro efficacy analysis of absorbent dressing modified with essential oils, against Staphylococcus aureus and Candida albicans]. / Analiza in vitro skutecznosci modyfikowanych olejkami eterycznymi opatrunków absorpcyjnych wobec Staphylococcus aureus i Candida albicans.

INTRODUCTION: The widespread use of antiseptics for wound dressings, unfortunately, not always effective, prompted us to search for alternative solutions, tailored to individual patient's needs. The aim of the study was checking the validity of the idea to apply some selected essential oils in order to modify active dressings which are routinely used in the care of chronically infected wounds. Our choice is commercially available an absorptive wound dressing which does not contain antiseptics (Sorbact). METHODS: The proposed is modification of dressing by its immersion in essential oil solution and then estimation of the biocide availability and stability during storage. Evaluation of inhibition of microbial surface growth (zone inhibition) and survival of absorbed microorganisms (retentivity by CFU counting) was performed directly after modification and repeated after 7 days of their storage at 4 degrees C. RESULTS: This study indicated that the dressings containing essential oils can keep absorbed bacteria/fungi inside and efficiently limit their growth. Depending on the properties (composition of volatile fraction) of the tested essential oil, saturated dressings were more active when stored at 4 degrees C for 7 days after their modification. The differences of antimicrobial strength, duration of the effect and retentivity between essential oils used for dressing modification have been shown. CONCLUSIONS: Modification of absorbent dressings with essential oils is a good option to achieve better therapeutic effect. Using a mixture of these four essential in several different quantitative ratios can be considered and is worthy of further research.

[Alterations in tears aqueous layer during cytostatics treatment]. / Zmiany w warstwie wodnej lez podczas leczenia cytostatykami.

PURPOSE: The aim of the study was to evaluate tears secretion, pH and lysozyme activity in tears aqueous layer during chemotherapy in lung, breast and bowel cancer. MATERIAL AND METHODS: 36 patients were enrolled to the study. Depending on the type of cancer and type of chemotherapy patients were divided into three groups. Group I (12 patients) diagnosed with non-small-cell lung cancer treated with PE schema (cisplatin, etoposide), Group II (12 patients) with breast cancer treated with FAC schema (fluorouracil, doxorubicin, cyclophosphamide), Group III (12 patients) with bowel cancer treated with FU/LV schema (fluorouracil, leucovorin). In all the patients: Schirmer's I test, pH measurements and lysozyme test were performed. Patients were examined before chemotherapy, after 2nd, 4th, 6th cycle. RESULTS: In group I and II lowering of tears secretion (p < 0.001) was revealed. In group III there was higher tears secretion (p < 0.001). PH was lowered after 2nd chemotherapy course in group I and II. In further treatment pH value were in the same lower level as after the second course. In group III there was higher pH--more alkaline (p < 0.001) after 2nd cycle of treatment and it was on the same level to the end of the examination process. Lowering of lysozyme activity in the tears film in all groups (p < 0.001) was established. The higher alterations of the lysozyme activity were observed in group treated with FAC schema. CONCLUSIONS: Cytostatic treatment has major influence on tears aqueous layer causing alterations of tears secretions. PH alterations depending on type of chemotherapy was observed. Lowering of lysozyme activity in tears was observed. All the deteriorations aggravate with duration of chemotherapy. Alterations of tears film parameters during chemotherapy may influence upon eye surface homeostasis and infectious complication. tears aqueous layer, Schirmer's test, lysozyme activity, tears pH.

Synthesis and biological evaluation of α-methylidene-δ-lactones with 3,4-dihydrocoumarin skeleton.

A series of new 3-methylidenechroman-2-ones bearing various aromatic moieties and various substituents at position 4 were synthesized in a three step reaction sequence. Friedel-Crafts alkylation of phenols or naphthols using ethyl 3-methoxy-2-diethoxyphosphorylacrylate in the presence of trifluoromethanesulphonic acid gave 3-diethoxyphosphorylchromen-2-ones. These compounds were employed as Michael acceptors in the reaction with Grignard reagents to give adducts which were finally used as Horner-Wadsworth-Emmons reagents for the olefination of formaldehyde. All obtained 3-methylidenechroman-2-ones were tested against two human leukemia cell lines NALM-6 and HL-60 as well as MCF-7 breast cancer and HT-29 colon cancer adenocarcinomas. Several obtained methylidenechromanones displayed high cytotoxic activity with IC(50) values below 1 µM, mainly against leukemia and MCF-7 cell lines. Investigation of structure-activity relationships revealed that the presence of additional, ortho-fused benzene ring and n-butyl or i-propyl group in position 4 enhances the activity. Selected methylidenechromanones were also tested on normal human umbilical vein endothelial cells (HUVEC) and chromanone 14o was found to be eightfold more toxic against MCF-7 than normal cells. Furthermore, antimicrobial assays revealed that chromanone 14n is highly active and bactericidal at concentration equal to MIC or 2MIC against nosocomial and community-associated staphylococci (MRSA) which are resistant to most or all available therapeutic classes of antimicrobial drugs.

[The immunomodulatory role of plant polyphenols]. / Immunomodulacyjna rola polifenoli roslinnych.

Polyphenols, plant secondary metabolites, are present in human diet and have been widely used for medical and cosmetic purposes. They possess beneficial features such as antioxidant, immunomodulatory, anti-cancer and antibacterial activity. There is some evidence that these phytochemicals can improve wound healing. However, more and more data suggest that, under certain conditions, they can act in a different, often unpredictable way. Some investigations indicate that polyphenols, generally known as antioxidants, can exhibit pro-oxidant, and therefore cytotoxic, activity. Hence, the ability of phytochemicals to induce apoptosis of cancer cells and bacterial cell damage may be, at least partly, due to their prooxidant properties. Phytocompounds enter the body through the digestive system where they undergo metabolic processes that often change their chemical features. The gastrointestinal microbiome interacts with phytochemicals and influences their bioavailability and absorption in the gut. Except for biochemical changes of plant polyphenols in the host, the achievement of therapeutic concentration in vivo may be the main problem in the determination of their real efficacy. Ambiguous results of some studies demonstrate the need for the development of more accurate and standardized methods for the evaluation of polyphenols' properties. Better understanding of human body-polyphenol interactions is crucial for more effective use of these phytochemicals in disease prevention and therapy. 

[The estimation of systemic chemotherapy treatment administered in breast cancer on lysozyme activity in tears--preliminary report]. / Ocena wplywu systemowej chemioterapii stosowanej w leczeniu raka sutka na aktywnosc lizozymu zawartego we lzach--doniesienie wstepne.

PURPOSE: Estimation of cytostatics influence used in breast cancer treatment on lysozyme activity in human tears depend on time of treatment. MATERIAL AND METHODS: 8 women were treated at the base of chemotherapy schema: docetaxel with doxorubicin and 4 women treated with schema CMF: cyclophosphamide, methotrexate, 5-fluorouracil. Lysozyme activity in tears was assessed by measurement of diameter zone of Micrococcus lysodeicticus growth inhibition. RESULTS: It was revealed that both chemotherapy schema caused statistically significant reduction of diameter zone of M. lysodeicticus growth inhibition, after first and second course of chemotherapy treatment. After second chemotherapy course CMF schema induced loss of lysozyme activity in patient's tears (zero mm of M. lysodeicticus diameter zone growth inhibition). CONCLUSIONS: Systemic chemotherapy administered in breast cancer induce reduction of lysozyme activity in tears, that may cause higher morbidity of ocular surface infections caused by Gram-positive bacteria.

Synthetic 3-arylideneflavanones as inhibitors of the initial stages of biofilm formation by Staphylococcus aureus and Enterococcus faecalis.

The antimicrobial activity of twenty two synthetic flavonoids is reported. Among them three 3-arylideneflavanones, 2b, 2c, and 2i, were shown to be highly active against Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis reference strains, with MIC (minimal inhibitory concentration) values ranging from 4.68 microg/ml (14.3 microM) to 37.5 microg/ml (119.7 microM). The synergy of oxacillin and vancomycin with 2c, evaluated as fractional inhibitory concentration index (FICI) was shown (against planktonic culture of S. aureus A3 and E. faecium 138/09 clinical strains). The presence of 2c in the culture medium diminished the initial adhesion of bacteria to an abiotic surface. Such an effect resulted in a decrease in biofilm formation during prolonged culture. Unfortunately, 2e failed to eradicate the S. aureus mature biofilm which was already preformed, however, decreased the number of live biofilm cells. The biofilm of E. faecalis was more susceptible to the action of 3-arylideneflavanone 2c than the S. aureus biofilm. The finding that 3-arylideneflavanones are lipophilic, cause bacterial aggregation, and influence the integrity of membranes making them permeable to SYTO 9/propidium iodide dyes may implicate the cytoplasmic membrane as a target site for these compounds activity.

Antibiofilm activity of selected plant essential oils and their major components.

The aim of the study was to examine the antibiofilm activity of selected essential oils (EO): Lavandula angustifblia (LEO), Melaleuca alternifolia (TTO), Melissa officinalis (MEO) and some of their major constituents: linalool, linalyl acetate, alpha-terpineol, terpinen-4-ol. Biofilms were formed by Staphylococcus aureus ATCC 29213 and Escherichia coli NCTC 8196 on the surface of medical biomaterials (urinary catheter, infusion tube and surgical mesh). TTC reduction assay was used for the evaluation of mature biofilm eradication from these surfaces. Moreover, time-dependent eradication ofbiofilms preformed in polystyrene 96-well culture microplates was examined and expressed as minimal biofilm eradication concentration (evaluated by MTT reduction assay). TTO, alpha-terpineol and terpinen-4-ol as well as MEO, showed stronger anti-biofilm activity than LEO and linalool or linalyl acetate. Among the biomaterials tested, surgical mesh was the surface most prone to persistent colonization since biofilms formed on it, both by S. aureus and E. coli, were difficult to destroy. The killing rate studies of S. aureus biofilm treated with TTO, LEO, MEO and some of their constituents revealed that partial (50%) destruction of 24-h-old biofilms (MBEC50) was achieved by the concentration 4-8 x MIC after 1 h, whereas 2-4 x MIC was enough to obtain 90% reduction in biomass metabolic activity (MBEC90) after just 4 h of treatment. A similar dose-dependent effect was observed for E. coli biofilm which, however, was more susceptible to the action of phytochemicals than the biofilms of S. aureus. It is noteworthy that an evident decrease in biofilm cells metabolic activity does not always lead to their total destruction and eradication.

[Assessment of essential oils activity used in liquid or volatile phase against biofilm cultures of Staphylococcus aureus and Candida albicans. The methodological study]. / Ocena bójczego dzialania fazy plynnej i frakcji lotnej olejków eterycznych na hodowle biofilmowe Staphylococcus aureus oraz Candida albicans. Wlasne rozwiazania metodyczne.

Biofilm formation is a significant factor in chronic infections with fungal and bacterial pathogens. Due to the high drug resistance of biofilm populations and the frequent failures of chemotherapy i such infections, it seems necessary to take recourse to unconventional treatment methods involving e.g. the use of some phytocompounds such as essential oils or their components. In order to evaluate the effect of their action on the microbial biomass a variety of techniques are used. However, there is still a need to develop new tests or modifications of these known, for the biofilm viability assessment. They should be adapted to the physico-chemical nature of the tested compounds and should decrease the risk ofbiofilm damage during staining procedure. We described a test assessing the effect of essential oils on bacterial and fungal biofilm formed on the membrane of cell culture inserts. The proposed model provides a minimal violation of the biofilm integrity during the test. It allows easily explore the activity of essential oil volatile fraction and is useful in determination of the kinetics of their action. Using this test it is also easy to examine the relationship between antimicrobial activity and the cytotoxic effect, known as the biocompatibility index (BI, biocompatibility index). Moreover, it allows qualitative and quantitative analysis of metabolic products, released into the growth medium from biofilm's cells. In successively repeated experiments high reproducibility of results has been obtained, thus the developed methodology seems to be useful in our future studies in this field.

[The synergism of antifungals and essential oils against Candida spp. evaluated by a modified gradient-diffusion method]. / Synergizm leków przeciwgrzybiczych i olejków eterycznych wobec Candida sp., oceniany zmodyfikowana metoda gradientowo-dyfuzyjna.

The usefulness of modified method of MIC Test Strip, for determining the synergistic effect of essential oils in the liquid or volatile phase with fluconazole and voriconazole, was evaluated. Geranium oil used against C. albicans in agar dilution test, at a concentration of 1/2 MIC caused a drop in the value of fluconazole MIC from 12.0 mg/l to 0.064 mg/l and voriconazole from 0.125 mg/l to 0.006 mg/l. A similar effect of drug combinations with essential oils was obtained in the case of C. glabrata study. Volatile Clove oil and cytronelal, applied in subMIC concentrations, also caused a reduction offluconazole and voriconazole MICs. Thus, utility of this simple methods developed by us for testing the effectiveness of combinations of known drugs and new compounds with antifungal activity, has been confirmed.