Aleksandra Budzyńska

New pharmacological properties of Medicago sativa and Saponaria officinalis saponin-rich fractions addressed to Candida albicans.

The antifungal activity of the saponin-rich fractions (SFs) from Medicago sativa (aerial parts and roots) and Saponaria officinalis (used as a well-known source of plant saponins) against Candida albicans reference and clinical strains, their yeast-to-hyphal conversion, adhesion, and biofilm formation was investigated. Direct fungicidal/fungistatic properties of the tested phytochemicals used alone, as well as their synergy with azoles (probably resulting from yeast cell wall instability) were demonstrated. Here, to the best of our knowledge, we report for the first time the ability of saponin-rich extracts of M. sativa and S. officinalis to inhibit C. albicans germ tube formation, limit hyphal growth, reduce yeast adherence and biofilm formation, and eradicate mature (24 h) Candida biofilm. Moreover, M. sativa SFs (mainly obtained from aerial parts), in the range of concentrations which were active modulators of Candida virulence factors, exhibited low cytotoxicity against the mouse fibroblast line L929. These properties seem to be very promising in the context of using plant-derived SFs as potential novel antifungal therapeutics supporting classic drugs or as ingredients of disinfectants.

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.

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.

Biogenic nanosilver synthesized in Metarhizium robertsii waste mycelium extract – As a modulator of Candida albicans morphogenesis, membrane lipidome and biofilm

Due to low efficacy of classic antimicrobial drugs, finding new active preparations attracts much attention. In this study an innovative, cost-effective and environmentally friendly method was applied to produce silver nanoparticles (AgNPs) using filamentous fungi Metarhizium robertsii biomass waste. It was shown that these NPs possess prominent antifungal effects against C. albicans, C. glabrata and C. parapsilosis reference strains. Further detailed studies were performed on C. albicans ATCC 90028. AgNPs kill curve (CFU method and esterase-mediated reduction of fluorescein diacetate); fractionally inhibitory concentration index (FICI) with fluconazole (FLC); effect on fungal cell membrane permeability (propidium iodide (PI) staining), membrane lipids profile (HPLC-MS), yeast morphotypes and intracellular reactive oxygen species level (H2DCFDA probe) were investigated. Anti-adhesive and anti-biofilm properties of AgNPs (alone and in combination with FLC) were also tested. Biosafety of AgNPs use was assessed in vitro in cytotoxicity tests against L929 fibroblasts, pulmonary epithelial A549 cell line, and red blood cells. Significant reduction in the viability of yeast cells treated with AgNPs was shown within 6 h. The proportion of C. albicans PI-positive cells increased in a dose and time-dependent manner. Changes in the qualitative and quantitative profile of cell membrane lipids, including significant decline in the quantity of most phospholipid species containing C18:2 and an increase in the amount of phospholipids containing C18:1 acyl species were observed after yeast exposure to AgNPs. CLSM images showed an enhancement in ROS intracellular accumulation in C. albicans treated with biogenic nanosilver. C. albicans transformation from yeast to hyphal forms was also reduced. AgNPs decreased adhesion of yeast to abiotic surfaces, as well as acted synergistically with FLC against sessile population. At fungicidal and fungistatic concentrations, they were non-toxic to mammalian cells. Obtained results confirm suitability of our “green synthesis” method to produce AgNPs with therapeutic potential against fungal infections.

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.