Development and Evaluation of a Film Forming System Containing Myricetin and Miconazole Nitrate for Preventing Candida albicans Catheter-Related Infection.

Background: Candida albicans catheter-related infection (CRI) is a great challenge in clinic now, mainly due to the difficulty in eradicating the biofilms. Purpose: In this study, the mechanism of the antibiofilm effect of myricetin (MY) on C. albicans was illustrated. A film forming system (FFS) containing MY and miconazole nitrate (MN) was developed, optimized, and evaluated. The anti-infection effect of MY+MN@FFS against C. albicans CRI was investigated in vivo. Study Design and Methods: To clarify the mechanism of the action of MY, the influence of MY on each key process of the formation of C. albicans biofilms was evaluated. To deliver MY and MN into the skin and form a drug reservoir on the surface of the skin, the FFS was used as a carrier and MY+MN@FFS was developed, optimized, and evaluated. After preliminary confirmation of drug safety, a percutaneously inserted C. albicans CRI mouse model was established to investigate the in vivo anti-infection effect of MY+MN@FFS by fluorescence microscopy and scanning electron microscopy on the outer surface of the catheters, hematoxylin/eosin staining, and periodic acid-Schiff staining of the mice skin tissues. Results: MY was found to inhibit the morphological transition of C. albicans and the secretion of exopolysaccharides, resulting in a reduction in biofilms. MY+MN@FFS exhibited excellent properties and no irritation to mice skin. In an in vivo anti-infection study, MY+MN@FFS exhibited an excellent preventive effect against percutaneously inserted C. albicans CRI. Conclusion: MY+MN@FFS might be a potential approach for effectively preventing percutaneously inserted C. albicans CRI in clinic.

Alkylimidazolium Ionic Liquids as Antifungal Alternatives: Antibiofilm Activity Against Candida albicans and Underlying Mechanism of Action.

Candida albicans is an opportunistic pathogen causes fungal infections that range from common skin infections to persistent infections through biofilm formation on tissues, implants and life threatening systemic infections. New antifungal agents or therapeutic methods are desired due to high incidence of infections and emergence of drug-resistant strains. The present study aimed to evaluate (i) the antifungal and antibiofilm activity of 1-alklyl-3-methyl imidazolium ionic liquids ([CnMIM]+[X]-, n = 4, 12 and 16) against Candida albicans ATCC 10231 and two clinical C. albicans strains and (ii) the mechanism of action of promising antifungal ionic liquid on C. albicans. Two of the tested compounds were identified as more effective in preventing growth and biofilm formation. These ionic liquid compounds with -dodecyl and -hexadecyl alkyl groups effectively prevented biofilm formation by fluconazole resistant C. albicans 10231 and two other clinical C. albicans strains. Although both the compounds caused viability loss in mature C. albicans biofilms, an ionic liquid with -hexadecyl group ([C16MIM]+[Cl]-) was more effective in dispersing mature biofilms. This promising ionic liquid compound ([C16MIM]+[Cl]-) was chosen for determining the underlying mode of action on C. albicans cells. Light microscopy showed that ionic liquid treatment led to a significant reduction in cell volume and length. Increased cell membrane permeability in the ionic liquid treated C. albicans cells was evident in propidium iodide staining. Leakage of intracellular material was evident in terms of increased absorbance of supernatant and release of potassium and calcium ions into extracellular medium. A decrease in ergosterol content was evident when C. albicans cells were cultured in the presence of antifungal ionic liquid. 2',7'-Dichlorodihydrofluorescein acetate assay revealed reactive oxygen species generation and accumulation in C. albicans cells upon treatment with antifungal ionic liquid. The effect of antifungal ionic liquid on mitochondria was evident by decreased membrane potential (measured by Rhodamine 123 assay) and loss of metabolic activity (measured by MTT assay). This study demonstrated that imidazolium ionic liquid compound exert antifungal and antibiofilm activity by affecting various cellular processes. Thus, imidazolium ionic liquids represent a promising antifungal treatment strategy in lieu of resistance development to common antifungal drugs.

The Efficacy of Photodynamic Inactivation of the Diode Laser in Inactivation of the Candida albicans Biofilms With Exogenous Photosensitizer of Papaya Leaf Chlorophyll.

Introduction: Photodynamic inactivation has been developed to kill pathogenic microbes. In addition, some techniques have been introduced to minimize the biofilm resistance to antifungal properties in inhibiting cell growth. The principle of photodynamic inactivation different to antifungal drugs therapy which is resistant to biofilms. The presence of reactive oxygen species (ROS) that generating in photodynamic inactivation mechanisms can be damaging of biofilm cells and the principle of light transmission that could be penetrating in matrix layers of extracellular polymeric substance (EPS) until reaching the target cells at the base layers of biofilm. The present work aims to explore the potential of chlorophyll extract of papaya leaf as an exogenous photosensitizer to kill the Candida albicans biofilms after being activated by the laser. The potential of chlorophyll photosensitizer was evaluated based on the efficacy of inactivation C. albicans biofilm cell through a cell viability test and an organic compound test. Methods: The treatment of photoinactivation was administered to 12 groups of C. albicans biofilm for four days using the 445 nm laser and the 650 nm laser. The 445 nm and 650 nm lasers activated the chlorophyll extract of the papaya leaf (0.5 mg/L) at the same energy density. The energy density variation was determined as 5, 10, 20, 30 and 40 J/cm2 with the duration of exposure of each laser adjusted to the absorbance percentage of chlorophyll extract of the papaya leaf. Results: The absorbance percentage of chlorophyll extracts of the papaya leaf on wavelengths of 650 nm and 445 nm respectively were 22.26% and 60.29%, respectively. The most effective treated group was a group of the laser with the addition of chlorophyll, done by the 650 nm lasers with inactivation about 32% (P=0.001), while the 445 nm lasers only 25% (P=0.061). The maximum malondialdehyde levels by treatment of the laser 650 nm were (0.046±0.004) nmol/mg. Conclusion: The use of chlorophyll extract of the papaya leaf as a photosensitizer, resulted in the maximum spectrum of absorption at 414 nm and 668 nm, which produced a maximum reduction effect after photoinactivation up to 32% (with chlorophyll) and 25% (without chlorophyll). The utilization of chlorophyll extract of the papaya leaf would increase the antifungal effects with the activation by the diode laser in the biofilm of C. albicans.

[Mechanism of butyl alcohol extract of Baitouweng Decoction (BAEB) on Candida albicans biofilms based on pH signal pathway].

This study aimed to investigate the effect of butyl alcohol extract of Baitouweng Decoction( BAEB) on Candida albicans biofilms based on pH signal pathway. The morphology of biofilms of the pH mutants was observed by scanning electron microscope. The biofilm thickness of the pH mutants was measured by CLSM. The biofilm activity of the pH mutants was analyzed by microplate reader.The biofilm damage of the pH mutants was detected by flow cytometry. The expression of pH mutant biofilm-related genes was detected by qRT-PCR. The results showed that the deletion of PHR1 gene resulted in the defect of biofilm,but there were more substrates for PHR1 complementation. BAEB had no significant effect on the two strains. RIM101 gene deletion or complementation did not cause significant structural damage,but after BAEB treatment,the biofilms of both strains were significantly inhibited. For the biofilm thickness,PHR1 deletion or complementation caused the thickness to decrease,after BAEB treatment,the thickness of the two strains did not change significantly. However,RIM101 gene deletion or complementation had little effect on the thickness,and the thickness of the two strains became thinner after adding BAEB. For biofilm activity,PHR1 deletion or complementation and RIM101 deletion resulted in decreased activity,RIM101 complementation did not change significantly; BAEB significantly inhibited biofilm activity of PHR1 deletion,PHR1 complemetation,RIM101 deletion and RIM101 complemetation strains. For the biofilm damage,PHR1 gene deletion or complementation,RIM101 gene deletion or complementation all showed different degrees of damage; after adding BAEB,the damage rate of PHR1 deletion or complementation was not significantly different,but the damage rate of RIM101 deletion or complementation was significantly increased. Except to the up-regulation of HSP90 gene expression,ALS3,SUN41,HWP1,UME6 and PGA10 genes of PHR1 deletion,PHR1 complementation,RIM101 deletion,and RIM101 complementation strains showed a downward expression trend. In a word,this study showed that mutations in PHR1 and RIM101 genes in the pH signaling pathway could enhance the sensitivity of the strains to the antifungal drug BAEB,thus inhibiting the biofilm formation and related genes expression in C. albicans.

Mechanism of butyl alcohol extract of Baitouweng Decoction (BAEB) on Candida albicans biofilms based on pH signal pathway / 中国中药杂志

This study aimed to investigate the effect of butyl alcohol extract of Baitouweng Decoction( BAEB) on Candida albicans biofilms based on pH signal pathway. The morphology of biofilms of the pH mutants was observed by scanning electron microscope. The biofilm thickness of the pH mutants was measured by CLSM. The biofilm activity of the pH mutants was analyzed by microplate reader.The biofilm damage of the pH mutants was detected by flow cytometry. The expression of pH mutant biofilm-related genes was detected by qRT-PCR. The results showed that the deletion of PHR1 gene resulted in the defect of biofilm,but there were more substrates for PHR1 complementation. BAEB had no significant effect on the two strains. RIM101 gene deletion or complementation did not cause significant structural damage,but after BAEB treatment,the biofilms of both strains were significantly inhibited. For the biofilm thickness,PHR1 deletion or complementation caused the thickness to decrease,after BAEB treatment,the thickness of the two strains did not change significantly. However,RIM101 gene deletion or complementation had little effect on the thickness,and the thickness of the two strains became thinner after adding BAEB. For biofilm activity,PHR1 deletion or complementation and RIM101 deletion resulted in decreased activity,RIM101 complementation did not change significantly; BAEB significantly inhibited biofilm activity of PHR1 deletion,PHR1 complemetation,RIM101 deletion and RIM101 complemetation strains. For the biofilm damage,PHR1 gene deletion or complementation,RIM101 gene deletion or complementation all showed different degrees of damage; after adding BAEB,the damage rate of PHR1 deletion or complementation was not significantly different,but the damage rate of RIM101 deletion or complementation was significantly increased. Except to the up-regulation of HSP90 gene expression,ALS3,SUN41,HWP1,UME6 and PGA10 genes of PHR1 deletion,PHR1 complementation,RIM101 deletion,and RIM101 complementation strains showed a downward expression trend. In a word,this study showed that mutations in PHR1 and RIM101 genes in the pH signaling pathway could enhance the sensitivity of the strains to the antifungal drug BAEB,thus inhibiting the biofilm formation and related genes expression in C. albicans.

The possible molecular mechanisms of farnesol on the antifungal resistance of C. albicans biofilms: the regulation of CYR1 and PDE2.

BACKGROUND: Farnesol has potential antifungal activity against Candida albicans biofilms, but the molecular mechanism of this activity is still unclear. Farnesol inhibits hyphal growth by regulating the cyclic AMP (cAMP) signalling pathway in C. albicans, and CYR1 and PDE2 regulate a pair of enzymes that are directly responsible for cAMP synthesis and degradation. Here, we hypothesize that farnesol enhances the antifungal susceptibility of C. albicans biofilms by regulating CYR1 and PDE2. RESULTS: The resistance of the CYR1- and PDE2-overexpressing strains to caspofungin, itraconazole and terbinafine was increased in planktonic cells, and that to amphotericin B was increased in biofilms. Meanwhile, the biofilms of the CYR1- and PDE2-overexpressing strains were thicker (all p < 0.05) and consisted of more hyphae than that of the wild strain. The intracellular cAMP levels were higher in the biofilms of the CYR1-overexpressing strain than that in the biofilms of the wild strain (all p < 0.01), while no changes were found in the PDE2-overexpressing strain. Exogenous farnesol decreased the resistance of the CYR1- and PDE2-overexpressing strains to these four antifungals, repressed the hyphal and biofilm formation of the strains, and decreased the intracellular cAMP level in the biofilms (all p < 0.05) compared to the untreated controls. In addition, farnesol decreased the expression of the gene CYR1 and the protein CYR1 in biofilms of the CYR1-overexpressing strain (all p < 0.05) but increased the expression of the gene PDE2 and the protein PDE2 in biofilms of the PDE2-overexpressing strain (all p < 0.01). CONCLUSIONS: The results indicate that CYR1 and PDE2 regulate the resistance of C. albicans biofilms to antifungals. Farnesol suppresses the resistance of C. albicans biofilms to antifungals by regulating the expression of the gene CYR1 and PDE2, while PDE2 regulation was subordinate to CYR1 regulation.

The antioxidant activity of a prenyl flavonoid alters its antifungal toxicity on Candida albicans biofilms.

The antioxidant effect of 8PP, a prenylflavonoid from Dalea elegans on Candida albicans biofilms, was investigated. We previously reported that sensitive (SCa) and resistant C. albicans (RCa) biofilms were strongly inhibited by this compound, in a dose-depending manner (50 µM-100 µM), with a prooxidant effect leading to accumulation of endogenous oxidative metabolites and increased antioxidant defenses. In this work, the antifungal activity of high concentrations of 8PP (200-1000 µM), the cellular stress imbalance and the architecture of biofilms were evaluated. Biofilms were studied by crystal violet and confocal scanning laser microscopy (CSLM) with COMSTAT analysis. Superoxide anion radical, the activity of the superoxide dismutase and the total antioxidant capacity were measured. Intracellular ROS were detected by a DCFH-DA and visualized by CSLM; reactive nitrogen intermediates by Griess. An antioxidant effect was detected at 1000 µM and levels of oxidant metabolites remained low, with major changes in the SCa. COMSTAT analysis showed that biofilms treated with higher concentrations exhibited different diffusion distances with altered topographic surface architectures, voids, channels and pores that could change the flow inside the matrix of biofilms. We demonstrate for first time, a concentration-dependent antioxidant action of 8PP, which can alter its antifungal activity on biofilms.

Preparation, characterization and toxicity evaluation of amphotericin B loaded MPEG-PCL micelles and its application for buccal tablets.

Oral candidiasis or thrush is a fungal infection due to Candida albicans, causing discomfort in areas inside mouth or tongue. The clinical application of antifungal reagent amphotericin B (AMB), which is believed to offer a better treatment for oral candidiasis, is greatly compromised by its toxicities (mainly nephrotoxicity) and poor solubility. In order to overcome these issues, we characterized AMB-loaded MPEG-PCL micelles in vitro and in vivo. In addition, the antifungal activities of AMB/MPEG-PCL micelles-loaded buccal tablet were also evaluated in vitro. We found that micelles system could significantly improve the solubility of AMB yet reduce the overall toxicity, while the buccal tablet system is capable to suppress C. albicans biofilm formation. Furthermore, the toxicity of the buccal tablet system is also reduced compared with other standard preparations. Therefore, the prepared tablet with AMB-loaded MPEG-PCL micelles as oral topical preparations has the potential to improve current treatment of superficial oral C. albicans infections.

Heat-Polymerized Resin Containing Dimethylaminododecyl Methacrylate Inhibits Candida albicans Biofilm.

The prevalence of stomatitis, especially caused by Candida albicans, has highlighted the need of new antifungal denture materials. This study aimed to develop an antifungal heat-curing resin containing quaternary ammonium monomer (dimethylaminododecyl methacrylate, DMADDM), and evaluate its physical performance and antifungal properties. The discs were prepared by incorporating DMADDM into the polymer liquid of a methyl methacrylate-based, heat-polymerizing resin at 0% (control), 5%, 10%, and 20% (w/w). Flexure strength, bond quality, surface charge density, and surface roughness were measured to evaluate the physical properties of resin. The specimens were incubated with C. albicans solution in medium to form biofilms. Then Colony-Forming Units, XTT assay, and scanning electron microscope were used to evaluate antifungal effect of DMADDM-modified resin. DMADDM modified acrylic resin had no effect on the flexural strength, bond quality, and surface roughness, but it increased the surface charge density significantly. Meanwhile, this new resin inhibited the C. albicans biofilm significantly according to the XTT assay and CFU counting. The hyphae in C. albicans biofilm also reduced in DMADDM-containing groups observed by SEM. DMADDM modified acrylic resin was effective in the inhibition of C. albicans biofilm with good physical properties.

The role of cell density in the morphology of the Candida albicans bioflims development and the tyrosol production / 中华微生物学和免疫学杂志

Objective To study the role of cell density in the tyrosol production and morphology for Candida albicans biofilms. Methods C. albicans SC5314 and clinical isolates were propagated in yeast peptone dextrose (YPD) medium. Cells were collected by centrifugation and washed twice in sterile phosphate-buffered saline (PBS) before this study, then resuspended in RPMI 1640 supplemented with L-glutamine and adjusted to a desired concentration of 5 × 10~6 cells/ml, 5×10~5 cells/ml, 5 × 10~4 cells/ml, 5 × 10~3 cells/ml after counting with a hematocytometer. Standardized C. albicans cells were prepared as above description and 2000 μl of this standardized cell suspension was dispensed into the wells, then C. albicans biofilms were formed on the bottom of the polystyrene wells. In this study, tyrosol synthesized by SC5314 and clinical isolates of C. albicans biofilm was quantified by high performance liquid chromatography (HPLC). The effects of tyrosol on morphology of C. albicans biofilms were investigated by scanning electron microscopy (SEM). Results Tyrosol production of C. albicans biofilms was affected by cell densities. At lower inoculation size(5 μ 10~3 cells/ml), there was too less tyrosol production to be detected at the early stage of the biofilms formation. At higher inoculation size (5 μ10~6 cells/ml), tyrosol can be detectable at the early stage or at the mature stage of biofilms formation. There was a sharp increase in tyrosol concentration at 24 h, while there was a decrease in tyrosol concentration after that time from the strains when the strains were at an inoculation size of 5 × 10~6 cells/ml and 5 × 10~5 cells/ml. Cell densities affected the morphology formation of the C. albicans biofilms. At the early stage of the biofilms formation, C. albicans grew less germ tube at lower cell densities than that at the higher cell densities. With the mature of the biofilms, C. albicarts grew more hyphae at higher cell densities than that at the lower cell densities. All these above showed that cell densities played an important role in the propagation for the C. albi-cans in the biofilm formation. Conclusion Cell density play an important role in the formation of the C. albi-cans biofilms and the production of the tyrosol.